Feruloyl esterases (FAEs) are a crucial component of the hemicellulose-degrading enzyme family that facilitates the degradation of lignocellulose while releasing hydroxycinnamic acids such as ferulic acid with high added value. Currently, the low enzyme yield of FAEs is one of the primary factors limiting its application. Therefore, in this paper, we optimized the fermentation conditions for the expression of FAE BpFaeT132C-D143C with excellent thermal stability in Escherichia coli by experimental design. Firstly, we explored the effects of 11 factors such as medium type, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, and inoculum size on BpFaeT132C-D143C activity separately by the single factor design. Then, the significance of the effects of seven factors, such as post-induction temperature, shaker rotational speed, and inoculum size on BpFaeT132C-D143C activity, was analyzed by Plackett-Burman design. We identified the main factors affecting the fermentation conditions of E. coli expressing BpFaeT132C-D143C as post-induction temperature, pre-induction period, and post-induction period. Finally, we used the steepest ascent path design and response surface method to optimize the levels of these three factors further. Under the optimal conditions, the activity of BpFaeT132C-D143C was 3.58 U/ml, which was a significant 6.6-fold increase compared to the pre-optimization (0.47 U/ml), demonstrating the effectiveness of this optimization process. Moreover, BpFaeT132C-D143C activity was 1.52 U/ml in a 3-l fermenter under the abovementioned optimal conditions. It was determined that the expression of BpFaeT132C-D143C in E. coli was predominantly intracellular in the cytoplasm. This study lays the foundation for further research on BpFaeT132C-D143C in degrading agricultural waste transformation applications.

• MeSH
• Escherichia coli * genetika   metabolismus   enzymologie   MeSH
• fermentace * MeSH
• isopropylthiogalaktosid metabolismus   MeSH
• karboxylesterhydrolasy * genetika   metabolismus   chemie   biosyntéza   MeSH
• kultivační média chemie   MeSH
• kyseliny kumarové metabolismus   MeSH
• lignin MeSH
• rekombinantní proteiny genetika   metabolismus   biosyntéza   chemie   MeSH
• stabilita enzymů MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH

The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation.

• MeSH
• bakteriální RNA * metabolismus   genetika   chemie   MeSH
• dusík metabolismus   MeSH
• Escherichia coli * genetika   metabolismus   MeSH
• hydrofobní a hydrofilní interakce * MeSH
• konzervovaná sekvence MeSH
• malá nekódující RNA * metabolismus   genetika   chemie   MeSH
• mutace MeSH
• protein hostitelského faktoru 1 * metabolismus   genetika   chemie   MeSH
• proteiny z Escherichia coli * metabolismus   genetika   chemie   MeSH
• regulace genové exprese u bakterií MeSH
• stabilita RNA * genetika   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom genetika   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Helicobacter pylori colonizes the human gastric mucosa of more than half of the human population and has a unique lipopolysaccharide (LPS) structure. LPS is the most dominant and suitable pathogen-associated molecular pattern that is detected via pattern recognition receptors. Although the priming effect of H. pylori LPS on reactive oxygen species (ROS) production of PMNs is lower than that of Escherichia coli O111:B4 LPS, LPS released from H. pylori associated with antibiotics eradication therapy may activate PMNs and increase ROS production. In addition, we describe the effects of H. pylori and E. coli O111:B4 LPSs on gene expression and the anti-inflammatory effect of lansoprazole (LPZ) in human polymorphonuclear leukocytes. LPS isolated from H. pylori and E. coli O111:B4 alters toll-like receptor 2 (TLR) and TLR4 expressions similarly. However, LPS from E. coli O111:B4 and H. pylori caused a 1.8-fold and 1.5-fold increase, respectively, in CD14 expression. All LPS subtypes upregulated TNFα and IL6 expression in a concentration-dependent manner. Although E. coli O111:B4 LPS upregulated IL8R mRNA levels, H. pylori LPS did not (≦ 100 ng/mL). Gene expression levels of ITGAM demonstrated no significant change on using both LPSs. These different effects on the gene expression in PMNs may depend on variations in LPS structural modifications related to the acquired immunomodulatory properties of H. pylori LPS. Proton pump inhibitors, i.e., LPZ, are used in combination with antibiotics for the eradication therapy of H. pylori. LPZ and its acid-activated sulphenamide form AG-2000 suppress ROS production of PMNs in a dose-dependent manner. These results suggest that LPZ combination with antibiotics for H. pylori eradication reduces gastric inflammation by suppressing ROS release from PMNs.

• MeSH
• cytokiny metabolismus   genetika   MeSH
• Escherichia coli účinky léků   genetika   MeSH
• Helicobacter pylori * účinky léků   genetika   MeSH
• inhibitory protonové pumpy * farmakologie   chemie   MeSH
• lanzoprazol * farmakologie   chemie   MeSH
• lidé MeSH
• lipopolysacharidy * metabolismus   farmakologie   MeSH
• neutrofily * účinky léků   imunologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• toll-like receptor 4 metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Despite numerous studies on Escherichia coli (E. coli) from sheep, there have been few reports on the characterization of E. coli isolates from various organs of individual sheep until now. The present study conducted molecular typing, antibiotics resistance, biofilm formation, and virulence genes on E. coli isolated from 57 freshly slaughtered apparently healthy sheep carcasses, gallbladders, fecal samples, and mesenteric lymph nodes (MLNs). The results demonstrated that the detection rate of R1 LPS core type in E. coli isolated from fecal samples (70.83%) was higher than that from other organs, but the detection rate of antibiotic resistance genes was lower (P < 0.05). The predominant phylogenetic group of E. coli isolated from the carcasses was group B1 (93.33%), and the detection rate of multidrug-resistance phenotype (80%) and the resistance rate of E. coli was higher than that from other organs (P < 0.05). Interestingly, the intensity of biofilm formation of E. coli isolated from MLNs was higher than that from other organs (P < 0.05). However, except for ibeB, the detection rates of virulence genes did not differ in E.coli isolated from different organs. In conclusion, differences were noted in these parameters of E. coli isolated from different organs of individual sheep. Therefore, the data may contain considerable mistakes concerning the actual situation in the host if we only analyze the data of E. coli isolated from feces or carcasses.

• MeSH
• antibakteriální látky * farmakologie   MeSH
• bakteriální léková rezistence genetika   MeSH
• biofilmy * růst a vývoj   účinky léků   MeSH
• Escherichia coli * genetika   účinky léků   izolace a purifikace   klasifikace   fyziologie   MeSH
• faktory virulence * genetika   MeSH
• feces mikrobiologie   MeSH
• fylogeneze MeSH
• infekce vyvolané Escherichia coli mikrobiologie   veterinární   MeSH
• molekulární typizace MeSH
• nemoci ovcí mikrobiologie   MeSH
• ovce MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Fosfomycin-resistant FosA8-producing Enterobacterales are uncommon strains with extremely low incidence in Europe, based on only three reports in the literature. We detected FosA8-producing Escherichia coli ST131 in clinical isolates from two patients admitted in February 2023 to a rehabilitation unit in Italy. The occurrence of rare fosA-like genes in the high-risk clone ST131 is of clinical relevance. The dissemination of FosA-producing E. coli, although still at low levels, should be continuously monitored.

• MeSH
• antibakteriální látky * farmakologie   terapeutické užití   MeSH
• bakteriální léková rezistence MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• Escherichia coli * izolace a purifikace   genetika   účinky léků   MeSH
• fosfomycin farmakologie   terapeutické užití   MeSH
• infekce vyvolané Escherichia coli * mikrobiologie   epidemiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• multilokusová sekvenční typizace MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Geografické názvy
• Itálie MeSH

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

• MeSH
• antibakteriální látky * farmakologie   MeSH
• azithromycin * farmakologie   MeSH
• bakteriální geny MeSH
• bakteriální léková rezistence * genetika   MeSH
• epidemiologické monitorování MeSH
• Escherichia coli * účinky léků   genetika   MeSH
• genotyp MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• makrolidy farmakologie   MeSH
• maso * mikrobiologie   MeSH
• mikrobiální testy citlivosti * MeSH
• plazmidy genetika   MeSH
• prasata MeSH
• Salmonella * účinky léků   genetika   izolace a purifikace   MeSH
• sekvenování celého genomu MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

Enteroaggregative Escherichia coli (EAEC) strains including those of serogroup O111 are important causes of diarrhea in children. In the Czech Republic, no information is available on the etiological role of EAEC in pediatric diarrhea due to the lack of their targeted surveillance. To fill this gap, we determined the proportion of EAEC among E. coli O111 isolates from children with gastrointestinal disorders ≤ 2 years of age submitted to the National Reference Laboratory for E. coli and Shigella during 2013-2022. EAEC accounted for 177 of 384 (46.1 %) E. coli O111 isolates, being the second most frequent E. coli O111 pathotype. Most of them (75.7 %) were typical EAEC that carried aggR, usually with aaiC and aatA marker genes; the remaining 24.3 % were atypical EAEC that lacked aggR but carried aaiC and/or aatA. Whole genome sequencing of 11 typical and two atypical EAEC O111 strains demonstrated differences in serotypes, sequence types (ST), virulence gene profiles, and the core genomes between these two groups. Typical EAEC O111:H21/ST40 strains resembled by their virulence profiles including the presence of the aggregative adherence fimbriae V (AAF/V)-encoding cluster to such strains from other countries and clustered with them in the core genome multilocus sequence typing (cgMLST). Atypical EAEC O111:H12/ST10 strains lacked virulence genes of typical EAEC and differed from them in cgMLST. All tested EAEC O111 strains displayed stacked-brick aggregative adherence to human intestinal epithelial cells. The AAF/V-encoding cluster was located on a plasmid of 95,749 bp or 93,286 bp (pAAO111) which also carried aggR, aap, aar, sepA, and aat cluster. EAEC O111 strains were resistant to antibiotics, in particular to aminopenicillins and cephalosporins; 88.3 % produced AmpC β-lactamase, and 4.1 % extended spectrum β-lactamase. We conclude that EAEC are frequent among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic. To reliably assess the etiological role of EAEC in pediatric diarrhea, a serotype-independent, PCR-based pathotype surveillance system needs to be implemented in the future.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Escherichia coli * genetika   izolace a purifikace   patogenita   klasifikace   MeSH
• faktory virulence genetika   MeSH
• gastrointestinální nemoci mikrobiologie   MeSH
• genom bakteriální MeSH
• infekce vyvolané Escherichia coli * mikrobiologie   epidemiologie   MeSH
• kojenec MeSH
• lidé MeSH
• multilokusová sekvenční typizace MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• proteiny z Escherichia coli genetika   MeSH
• průjem * mikrobiologie   MeSH
• sekvenování celého genomu * MeSH
• séroskupina MeSH
• trans-aktivátory MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

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

• MeSH
• antibakteriální látky * farmakologie   MeSH
• azabicyklické sloučeniny * farmakologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• beta-laktamasy * genetika   metabolismus   MeSH
• ceftazidim * farmakologie   MeSH
• centra terciární péče MeSH
• Enterobacteriaceae rezistentní na karbapenemy genetika   účinky léků   izolace a purifikace   MeSH
• Escherichia coli * genetika   účinky léků   MeSH
• fixní kombinace léků * MeSH
• genom bakteriální MeSH
• karbapenemy * farmakologie   MeSH
• Klebsiella pneumoniae * genetika   účinky léků   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence * genetika   MeSH
• plazmidy genetika   MeSH
• přenos genů horizontální MeSH
• sekvenování celého genomu * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Libanon MeSH

A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917 bp, encoding 638 amino acids with a predicted molecular mass of 62.3 kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50 °C, respectively, and it was stable within pH 4.5‒7.0 and up to 45 °C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4 °C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products.

• MeSH
• bakteriální proteiny genetika   metabolismus   chemie   MeSH
• beta-galaktosidasa * genetika   metabolismus   chemie   izolace a purifikace   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• glykosylace MeSH
• klonování DNA * MeSH
• koncentrace vodíkových iontů MeSH
• laktosa * metabolismus   MeSH
• mléko mikrobiologie   MeSH
• molekulová hmotnost MeSH
• oligosacharidy metabolismus   MeSH
• Pedobacter * enzymologie   genetika   MeSH
• rekombinantní proteiny genetika   metabolismus   chemie   izolace a purifikace   MeSH
• sekvence aminokyselin MeSH
• stabilita enzymů * MeSH
• substrátová specifita MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH

A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-β-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

• MeSH
• antibakteriální látky farmakologie   MeSH
• beta-laktamasy * genetika   MeSH
• biofilmy růst a vývoj   MeSH
• epidemický výskyt choroby * MeSH
• Escherichia coli * genetika   účinky léků   izolace a purifikace   enzymologie   MeSH
• faktory virulence genetika   MeSH
• fylogeneze MeSH
• genom bakteriální MeSH
• genomika metody   MeSH
• infekce vyvolané Escherichia coli * mikrobiologie   epidemiologie   MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH