Search Results (570)

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein–protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions. Full article

Trueperella pyogenes is a significant opportunistic pathogen that causes substantial economic losses in animal agriculture due to its ability to infect various animal tissues and organs. Limited research has been conducted on the prevalence and biological characteristics of T. pyogenes isolated from sheep and goats. This study aimed to isolate T. pyogenes from clinical samples of sheep and goats in western China, examining genetic evolutionary relationships, antibiotic resistance, and virulence genes. Between 2021 and 2023, standard bacteriological methods were used to isolate and identify T. pyogenes from 316 samples (209 from goats and 107 from sheep) collected from 39 farms. Susceptibility to 14 antibiotics was tested using broth microdilution per CLSI guidelines, and PCR detected eight virulence genes. Whole-genome sequencing analyzed genetic relationships and gene carriage status in 39 isolates. The results indicated that 86 strains of T. pyogenes were isolated from 316 samples, yielding an isolation rate of 27.2% (goats n = 47, 22.5%; sheep n = 39, 36.4%). The virulence genes plo, cbpA, nanH, nanP, fimA, fimC, and fimE were present in 100%, 66.7%, 64.1%, 71.8%, 69.2%, 59.0%, and 82.1% of isolates, respectively, with none carrying the fimG gene. The dominant virulence genotype was plo/nanH/nanP/fimA/fimC/fimE. The isolates exhibited resistance to erythromycin (44.2%, 38/86), gentamicin (38.4%, 33/86), sulfamethoxazole/trimethoprim (37.2%, 32/86), tetracycline (32.6%, 28/86), and streptomycin (32.6%, 28/86), and low resistance to chloramphenicol (14.0%, 12/86), ciprofloxacin (7.0%, 6/86), penicillin (5.8%, 5/86), and clindamycin (4.7%, 4/86). All isolates were susceptible to cefotaxime, vancomycin, and linezolid. Among the 86 isolates, 37 (43.0%) displayed multidrug resistance (MDR) characteristics. The whole genome sequencing of 39 isolates identified eight types of resistance genes, including ant(2″)-Ia, ant(3″)-Ia, cmlA1, cmx, erm(X), lnu(A), sul1, and tet(W). Except for tet(W), erm(X), and sul1, the other resistance genes were reported for the first time in T. pyogenes isolated in China. The drug susceptibility test results and resistance gene detection for the isolated strains were consistent for tetracycline, erythromycin, gentamicin, and sulfisoxazole. Similar allelic profiles and genetic evolutionary relationships were found among isolates from different farms. This study highlights the antibiotic resistance status and virulence gene-carrying rate of Trueperella pyogenes, providing a basis for clinical medication. Full article

Background: Salmonella is an important zoonotic pathogen, of which poultry products are important reservoirs. This study analyzed the prevalence, antimicrobial resistance, and characterization of Salmonella from broiler and laying hen sources in China. Methods: A total of 138 (12.27%) strains of Salmonella were isolated from 1125 samples from broiler slaughterhouses (20.66%, 44/213), broiler farms (18.21%, 55/302), and laying hen farms (6.39%, 39/610). Multiplex PCR was used to identify the serotypes. Antibiotic susceptibility testing to a set of 21 antibiotics was performed and all strains were screened by PCR for 24 selected antimicrobial resistance genes (ARGs). In addition, 24 strains of Salmonella were screened out by whole-genome sequencing together with 65 released Salmonella genomes to evaluate phylogenetic characteristics, multilocus sequence typing (MLST), and plasmid carriage percentages. Results: A total of 11 different serotypes were identified, with the dominance of S. Enteritidis (43/138, 31.16%), S. Newport (30/138, 21.74%), and S. Indiana (19/138, 13.77%). The results showed that S. Enteritidis (34.34%, 34/99) and S. Newport (51.28%, 20/39) were the dominant serotypes of isolates from broilers and laying hens, respectively. The 138 isolates showed the highest resistance to sulfisoxazole (SXZ, 100%), nalidixic acid (NAL, 54.35%), tetracycline (TET, 47.83%), streptomycin (STR, 39.86%), ampicillin (AMP, 39.13%), and chloramphenicol (CHL, 30.43%), while all the strains were sensitive to both tigacycline (TIG) and colistin (COL). A total of 45.65% (63/138) of the isolates were multidrug-resistant (MDR) strains, and most of them (61/63, 96.83%) were from broiler sources. The results of PCR assays revealed that 63.77% of the isolates were carrying the quinolone resistance gene qnrD, followed by gyrB (58.70%) and the trimethoprim resistance gene dfrA12 (52.17%). Moreover, a total of thirty-four ARGs, eighty-nine virulence genes, and eight plasmid replicons were detected in the twenty-four screened Salmonella strains, among which S. Indiana was detected to carry the most ARGs and the fewest plasmid replicons and virulence genes compared to the other serotypes. Conclusions: This study revealed a high percentage of multidrug-resistant Salmonella from poultry sources, stressing the importance of continuous monitoring of Salmonella serotypes and antimicrobial resistance in the poultry chain, and emergency strategies should be implemented to address this problem. Full article

To determine the etiological agents responsible for acute pneumonia in puppies in China, this study utilized bronchoalveolar lavage (BAL) fluid extraction to enable the isolation, culture, biochemical identification, and 16S rRNA PCR amplification of the pathogens. Following preliminary identification, the pathogens underwent analysis for antibiotic resistance phenotypes and resistance genes. Additionally, the study examined the presence of virulence genes, conducted multilocus sequence typing (MLST), and performed whole-genome sequencing (WGS). The findings revealed that all four isolated pathogens were characterized as extraintestinal pathogenic Escherichia coli (ExPEC). The examined ExPEC strains demonstrated resistance to cephalosporins, tetracyclines, and penicillins, while remaining susceptible to aminoglycosides, beta-lactamase inhibitors, carbapenems, chloramphenicols, and sulfonamides. An analysis of virulence genes identified the presence of eight genes, namely CNF-I, fyuA, fimC, papC, ompA, fimH, irp2, and iroN, which are implicated in their invasiveness and potential to inflict tissue damage. The MLST analysis revealed that all ExPEC strains were classified under either sequence type ST131 (Achtman database) or ST43 (Pasteur database). The study further determined that these strains were absent in the kennel’s drinking water source, thereby ruling out water contamination as a potential factor in the emergence of ST131-type ExPEC. This study offers a theoretical framework and empirical evidence for elucidating the potential pathogenic mechanisms and clinical therapeutic strategies of ExPEC in the etiology of acute pneumonia in puppies. Full article

In October 2023, a disease outbreak in pufferfish (Takifugu obscurus) farms in Zhongshan City, Guangdong, China, caused high mortality. Diseased fish (mean length: 15 ± 1 cm) exhibited swimming disorders, fin rot, hemorrhage, and an enlarged spleen. Histopathological observations generally revealed inflammation, necrosis, and congestion in the spleen, kidneys, and brain tissues. The most severe pathological changes included interstitial edema and tubular atrophy in the kidneys, hemosiderin deposition in the spleen, massive red blood cell infiltration, and a decrease in lymphocytes. A single strain of bacteria (Tol-1) was isolated from the diseased pufferfish and identified as a Gram-positive streptococcus strain, exhibiting α-hemolysis on sheep blood agar plates. Through biochemical characterization, 16S rDNA sequencing, morphological analysis, and specific primer-based identification, the Tol-1 strain was identified as Lactococcus garvieae, serotype I. Antimicrobial susceptibility testing indicated that Tol-1 was sensitive to Chloramphenicol, Ampicillin, Cephalexin, and Doxycycline, but resistant to Kanamycin, Gentamicin and Ciprofloxacin. In addition, 15 common virulence factors were detected in the Tol-1 strain, including adhPav, adhPsaA, adhC I–II, adh, and hly 1–3. Pufferfish (mean length: 17 ± 1 cm) subjected to artificial infection via intraperitoneal injection (IP) with the Tol-1 strain exhibited clinical symptoms and histopathological damage similar to those observed in naturally infected fish. An infection dose of 1 × 10⁵ CFU/fish resulted in 80% mortality. The study fulfilled Koch’s postulates, indicating that the disease outbreak in pufferfish was caused by L. garvieae, which exhibited a high mortality rate in pufferfish despite the subtle clinical symptoms. These results serve as a warning for pufferfish farming areas and provide a scientific basis for future prevention and control efforts. Full article

Antibiotics targeting the bacterial ribosome are essential to combating bacterial infections. These antibiotics bind to various sites on the ribosome, inhibiting different stages of protein synthesis. This review provides a comprehensive overview of the mechanisms of action of clinically relevant antibiotics that target the bacterial ribosome, including macrolides, lincosamides, oxazolidinones, aminoglycosides, tetracyclines, and chloramphenicol. The structural and functional details of antibiotic interactions with ribosomal RNA, including specific binding sites, interactions with rRNA nucleotides, and their effects on translation processes, are discussed. Focus is placed on the diversity of these mechanisms and their clinical implications in treating bacterial infections, particularly in the context of emerging resistance. Understanding these mechanisms is crucial for developing novel therapeutic agents capable of overcoming bacterial resistance. Full article

The increase in the number of hospital strains of hypervirulent and multidrug resistant (MDR) Pseudomonas aeruginosa is a major health problem that reduces medical treatment options and increases mortality. The molecular profiles of virulence and multidrug resistance of P. aeruginosa-associated hospital and community infections in Mexico have been poorly studied. In this study, we analyzed the different molecular profiles associated with the virulence genotypes related to multidrug resistance and the genotypes of multidrug efflux pumps (mex) in P. aeruginosa causing clinically critical infections isolated from Mexican patients with community- and hospital-acquired infections. Susceptibility to 12 antibiotics was determined using the Kirby–Bauer method. The identification of P. aeruginosa and the detection of virulence and efflux pump system genes were performed using conventional PCR. All strains isolated from patients with hospital-acquired (n = 67) and community-acquired infections (n = 57) were multidrug resistant, mainly to beta-lactams (ampicillin [96.7%], carbenicillin [98.3%], cefalotin [97.5%], and cefotaxime [87%]), quinolones (norfloxacin [78.2%]), phenicols (chloramphenicol [91.9%]), nitrofurans (nitrofurantoin [70.9%]), aminoglycosides (gentamicin [75%]), and sulfonamide/trimethoprim (96.7%). Most strains (95.5%) isolated from patients with hospital- and community-acquired infections carried the adhesion (pilA) and biofilm formation (ndvB) genes. Outer membrane proteins (oprI and oprL) were present in 100% of cases, elastases (lasA and lasB) in 100% and 98.3%, respectively, alkaline protease (apr) and alginate (algD) in 99.1% and 97.5%, respectively, and chaperone (groEL) and epoxide hydrolase (cif) in 100% and 97.5%, respectively. Overall, 99.1% of the strains isolated from patients with hospital- and community-acquired infections carried the efflux pump system genes mexB and mexY, while 98.3% of the strains carried mexF and mexZ. These findings show a wide distribution of the virulome related to the genotypic and phenotypic profiles of antibiotic resistance and the origin of the strains isolated from patients with hospital- and community-acquired infections, demonstrating that these molecular mechanisms may play an important role in high-pathogenicity infections caused by P. aeruginosa. Full article

This study aimed to count Enterobacteriaceae and Escherichia coli in different locations on pig carcasses (shank, loin, abdomen, shoulder, and jowl) from two slaughterhouses (A and B) between September 2019 and July 2021 during different slaughter stages (after bleeding, after passing through the epilator machine, after manual toileting in the dirty area, before and after evisceration, and after the final washing), as well as verify antimicrobial resistance and biofilm formation capacity. The main points of Enterobacteriaceae and E. coli contamination were identified in the two slaughterhouses through three collections. The stages with the highest counts were post-bleeding and evisceration in both slaughterhouses and after manual toileting in slaughterhouse B in the first collection. Most E. coli isolates were resistant to multiple antimicrobials, with higher resistance frequencies to amoxicillin, ampicillin, chloramphenicol, sulfonamides, and streptomycin. The virulence genes eae, stx1, and stx2 were also detected. Three isolates had all three genes and exhibited resistance to at least six antimicrobial classes (β-lactams, macrolides, aminoglycosides, sulfonamides, amphenicols, and quinolones). E. coli isolates also showed a high frequency of strains with moderate and strong in vitro biofilm-forming capacity. This is the first study to characterize microbial contamination by pig slaughter stage in the Federal District region, demonstrating the critical points for hygienic production. E. coli was isolated from the surface of pig carcasses, as well as the virulence genes stx1, stx2, and eae were detected. The multi-antimicrobial resistant isolates also had a moderate-to-strong biofilm formation capacity, thus demonstrating risks to public health. Full article

Background/Objectives: The objective of this study was to assess the efficacy of a cell-containing wound dressing based on fibroblasts in hydroxyethylcellulose (HEC) gel for the local treatment of deep partial-thickness and/or full-thickness skin burns in an animal model. Methods: The rats (male Wistar, n = 100) were subjected to a full-thickness thermal burn (16 cm²). Radical necrectomy was performed one day after the burn. Three days later, the rats were randomly assigned to one of four groups: group 1 (no treatment), group 2 (chloramphenicol and methyluracil ointment, a routine clinical treatment), group 3 (a gel without cells, mock treatment), and group 4 (a dermal fibroblast-impregnated HEC gel). The treatment lasted for five days. The wound-healing process was evaluated by planimetric, cytologic, histologic, and immunohistochemical methods. Results: The differences in the rate of wound healing and the characteristics of wound cytology were identified. In the group 4, a regenerative type of cytogram was revealed, characterized by a significantly increased number of fibroblastic cells in comparison to samples from non-treated and mock-treated animals. Biopsy samples of burn wounds from animals in the group 4l demonstrated the presence of mature granulation tissue and a large number of microvessels. The repair process was stimulated, as evidenced by the increased thickness of newly formed granulation tissue and epidermis in the wound zone, elevated cellularity, and enhanced re-epithelialization activity. The number of Ki-67-positive proliferating cells was significantly higher in group 4 than in the control groups). A small number of non-proliferating donor fibroblasts was observed in the wound area 3 days after the end of treatment. Conclusions: The cell product is an effective agent for promoting wound healing during the regenerative phase. The experiments demonstrated that a gel populated by dermal fibroblasts can stimulate reparative regeneration processes in deep partial- and full-thickness burn wounds. Full article

The search for synergies between natural products and commercial antibiotics is a promising strategy against bacterial resistance. This study determined the antimicrobial capacity of Nerol (NE) and Tannic Acid (TA) against 14 pathogenic bacteria, including ESKAPE pathogens. TA exhibited the lowest Minimum Inhibitory Concentrations (MICs) at 162.5 µg/mL against Pasteurella aerogenes and 187.5 µg/mL against Acinetobacter baumannii (WHO priority 1). NE showed its lowest MIC of 500 µg/mL against both Pasteurella aerogenes and Salmonella enterica. A total of 35 combinations of NE and 13 of TA with eight commercial antibiotics were analyzed. For NE, combinations with Streptomycin and Gentamicin were effective against Salmonella enterica, Bacillus subtilis, and Streptococcus agalactiae, with antibiotic MIC reductions between 75.0 and 87.5%. TA showed six synergies with Chloramphenicol, Ampicillin, Erythromycin, and Streptomycin against Acinetobacter baumannii, Streptococcus agalactiae, and Pasteurella aerogenes, with MIC reductions between 75.0 and 93.7%. Additionally, 31 additive effects with antibiotics for NE and 8 for TA were found. Kinetic studies on these synergies showed complete inhibition of bacterial growth, suggesting that natural products enhance antibiotics by facilitating their access to targets or preventing resistance. Given their safety profiles recognized by the EPA and FDA, these natural products could be promising candidates as antibiotic enhancers. Full article

Chloramphenicol (CAM), a well-known broad-spectrum antibiotic, inhibits peptide bond formation in bacterial ribosomes. It has been reported to affect ribosome assembly mainly through disrupting the balance of ribosomal proteins. The present study investigates the multifaceted effects of CAM on the maturation of the 50S ribosomal subunit in Escherichia coli (E. coli). Using label-free quantitative mass spectrometry (LFQ-MS), we observed that CAM treatment also leads to the upregulation of assembly factors. Further cryo-electron microscopy (cryo-EM) analysis of the ribosomal precursors characterized the CAM-treatment-accumulated pre-50S intermediates. Heterogeneous reconstruction identified 26 distinct pre-50S intermediates, which were categorized into nine main states based on their structural features. Our structural analysis highlighted that CAM severely impedes the formation of the central protuberance (CP), H89, and H58 during 50S ribosomal subunit maturation. The ELISA assay further demonstrated the direct binding of CAM to the ribosomal precursors, suggesting that the interference with 50S maturation occurs through a combination of direct and indirect mechanisms. These findings provide new insights into the mechanism of the action of CAM and provide a foundation for a better understanding of the assembly landscapes of the ribosome. Full article

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is considered the main cause of nosocomial and community-associated infections. Because of antimicrobial resistance, MRSA infections are difficult or impossible to treat, leading to high mortality rates and significant economic and societal costs. In view of the MRSA challenge to public health all over the world, the identification of new and effective anti-MRSA agents is a high medical priority. Objectives: A new series of tricyclic flavonoids with a methyl substituent on ring A of the flavonoid skeleton was synthesized to assess their antimicrobial properties. Methods: The structures of novel synthetic tricyclic flavonoids and their 3-dithiocarbamic flavanones were proven by X-ray structural analyses. Minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) were used to evaluate antimicrobial activity. Growth kinetic and time–kill assays were employed to confirm the antibacterial effectiveness. The mechanism of action was investigated using fluorescence microscopy. Results: Our results show that the tricyclic flavonoids exhibited important antibacterial and antifungal activities, with MIC and MBC values as low as 1.95 µg/mL and 3.90 µg/mL recorded for compound 5e against a multidrug-resistant MRSA strain. Flavonoid 5e induced a more important bacteriostatic effect compared with chloramphenicol, inhibiting the bacterial growth for up to 24 h at concentrations equivalent to 2 × MIC. Also, 5e exhibited a significant bactericidal activity, with no viable cells evidenced after 6 h of incubation in the presence of MBC and a total kill effect recorded up to 24 h. The anti-MRSA activity may be explained by the cell membrane impairment induced by 5e. Conclusions: All the data support the idea that flavonoid 5e is a reliable candidate to develop effective anti-MRSA agents, but further studies are necessary. Full article

Vancomycin-resistant enterococci (VRE) are considered one of the main nosocomial pathogens due to their increasing antibiotic resistance and ability to cause life-threatening infections in humans. This study included VRE isolates obtained from various specimens including urine, blood, faeces, wounds, sputum, and oral cavity wash. Of the 37 strains, 30 (81.1%) and 7 (18.9%) were identified by MALDI TOF as Enterococcus faecium and Enterococcus faecalis, respectively. The clinical vancomycin-resistant enterococci exhibited multi-drug resistance (MDR). Apart from vancomycin, the enterococci exhibited resistance to penicillins (89.1 to 100%), fluoroquinolones (100%), rifampicin (86.5%), tetracycline (27%), aminoglycosides (56.8 to 86.5%), quinupristin–dalfopristin (35.1%), and chloramphenicol (10.8%). Moreover, resistance to linezolid and tigecycline emerged among the tested vancomycin-resistant enterococci. The analysis of aminoglycoside modifying enzyme (AME) genes showed the presence of bifunctional aac(6′)-Ie-aph(2″)-Ia genes contributed to high-level aminoglycoside resistance (HLAR) in the E. faecalis and E. faecium isolates. The other AME gene, i.e., aph(3′)-IIIa, was also found in the VRE isolates. All strains carried the vanA gene. Enterococci from colonised gastrointestinal tracts (1/2.7%) and from infection (6/16.2%) showed cytotoxic activity against the human epithelial cell line HEp-2. Full article

Objectives: Campylobacter spp. remain a leading cause of bacterial gastroenteritis worldwide, with resistance to antibiotics posing significant challenges to treatment and public health. This study examines profiles in antimicrobial resistance (AMR) for Campylobacter isolates from human and animal sources over the past decade. Methods: We conducted a comprehensive review of resistance data from studies spanning ten years, analyzing profiles in resistance to key antibiotics, ciprofloxacin (CIP), tetracycline (TET), erythromycin (ERY), chloramphenicol (CHL), and gentamicin (GEN). Data were collated from various regions to assess global and regional patterns of resistance. Results: The analysis reveals a concerning trend of increasing resistance patterns, particularly to CIP and TET, across multiple regions. While resistance to CHL and GEN remains relatively low, the high prevalence of CIP resistance has significantly compromised treatment options for campylobacteriosis. Discrepancies in resistance patterns were observed between human and animal isolates, with variations across different continents and countries. Notably, resistance to ERY and CHL showed regional variability, reflecting potential differences in antimicrobial usage and management practices. Conclusions: The findings underscore the ongoing challenge of AMR in Campylobacter, highlighting the need for continued surveillance and research. The rising resistance prevalence, coupled with discrepancies in resistance patterns between human and animal isolates, emphasize the importance of a One Health approach to address AMR. Enhanced monitoring, novel treatment strategies, and global cooperation are crucial for mitigating the impact of resistance and ensuring the effective management of Campylobacter-related infections. Full article

A Leuconostoc mesenteroides strain (SJC113) isolated from cheese curd was found to produce large amounts of a mucoid exopolysaccharide (EPS). An analysis revealed the glucan nature of the EPS with 84.5% (1→6)-linked α-d-glucose units and 5.6% (1,3→6)-linked α-d-glucose units as branching points. The EPS showed 52% dextranase resistance and a yield of 7.4 ± 0.9 g/L from MRS medium supplemented with 10% sucrose within 48 h. Ln. mesenteroides SJC113 was also characterized and tested for the production of EPS as a fat substitute in fresh cheese. Strain SJC113 showed high tolerance to a wide range of NaCl concentrations (2, 5 and 10%), high β-galactosidase activity (2368 ± 24 Miller units), cholesterol-reducing ability (14.8 ± 4.1%), free radical scavenging activity (11.7 ± 0.7%) and hydroxyl scavenging activity (15.7 ± 0.4%). The strain had no virulence genes and was sensitive to clinically important antibiotics such as ampicillin, tetracycline and chloramphenicol. Ln. mesenteroides SJC113 produced highly viscous EPS during storage at 8 °C in skim milk with 5% sucrose. Therefore, these conditions were used for EPS production in skim milk before incorporation into fresh cheese. Four types of fresh cheese were produced: full-fat cheese (FF) made from pasteurized whole milk, non-fat cheese (NF) made from pasteurized skim milk, non-fat cheese made from skim milk fermented with Ln. mesenteroides without added sugar (NFLn0) and non-fat cheese made from skim milk fermented with Ln. mesenteroides with 5% sucrose (NFLn5). While the NF cheeses had the highest viscosity and hardness, the NFLn5 cheeses showed lower firmness and viscosity, higher water-holding capacity and lower weight loss during storage. Overall, the NFLn5 cheeses had similar rheological properties to full-fat cheeses with a low degree of syneresis. It was thus shown that the glucan-type EPS produced by Ln. mesenteroides SJC113 can successfully replace fat without altering the texture of fresh cheese. Full article