Activity of Epsilon-poly-L-lysine against Multidrug-Resistant Pseudomonas aeruginosa and Klebsiella pneumoniae Isolates of Urinary Tract Infections.

Pseudomonas aeruginosa and Klebsiella pneumoniae are notorious for their resistance to antibiotics and propensity for biofilm formation, posing significant threats to human health. Epsilon-poly-L-lysine (ε-PL) emerges as a naturally occurring antimicrobial poly(amino acid), which positions it as a prospective agent for addressing challenges linked to multidrug resistance. ε-PL symbolizes a promising avenue in the pursuit of efficacious therapeutic strategies and warrants earnest consideration within the realm of clinical treatment. Thus, our objective was to determine the antibiotic susceptibility profiles of 38 selected P. aeruginosa and ESBL-producing K. pneumoniae clinical isolates and determine the ability of ε-PL to inhibit biofilm formation. After PCR analysis, detection of genes related to ß-lactamases was observed among the selected isolates of P. aeruginosa [blaSPM (35.7%), blaKPC (35.7%), blaSHV (14.3%), blaCTX-M (14.3%), blaOXA (14.3%), blaTEM (7.1%), blaPER (7.1%), blaVIM (7.1%), and blaVIM-2 (7.1%)] and K. pneumoniae [blaCTX-M (91.7%), blaTEM (83.3%), blaKPC (16.7%), blaNDM (12.5%), and blaOXA (4.2%)]. The results of testing the activity of ε-PL against the clinical isolates showed relatively high minimum inhibitory concentrations (MICs) for the P. aeruginosa (range: 8-64 µg/mL) and K. pneumoniae isolates (range: 16-32 µg/mL). These results suggest the need for prior optimization of ε-PL concerning its viability as an alternative to antibiotics for treating infections caused by P. aeruginosa and K. pneumoniae of clinical origin. It is noteworthy that, in the context of a low antibiotic discovery rate, ε-PL could play a significant role in this quest, considering its low toxicity and the unlikely development of resistance. Upon exposure to ε-PL, P. aeruginosa and K. pneumoniae isolates exhibited a reduction in biofilm production, with ε-PL concentration showing an inverse relationship, particularly in isolates initially characterized as strong or moderate producers, indicating its potential as a natural antimicrobial agent with further research needed to elucidate optimal concentrations and application methods across different bacterial species. Further research is needed to optimize its use and explore its potential in various applications.

Evaluation of the Ability to Form Biofilms in KPC-Producing and ESBL-Producing Klebsiella pneumoniae Isolated from Clinical Samples.

The appearance of Klebsiella pneumoniae strains producing extended-spectrum ß-lactamase (ESBL), and carbapenemase (KPC) has turned into a significant public health issue. ESBL- and KPC-producing K. pneumoniae's ability to form biofilms is a significant concern as it can promote the spread of antibiotic resistance and prolong infections in healthcare facilities. A total of 45 K. pneumoniae strains were isolated from human infections. Antibiograms were performed for 17 antibiotics, ESBL production was tested by Etest ESBL PM/PML, a rapid test was used to detect KPC carbapenemases, and resistance genes were detected by PCR. Biofilm production was detected by the microtiter plate method. A total of 73% of multidrug resistance was found, with the highest resistance rates to ampicillin, trimethoprim-sulfamethoxazole, cefotaxime, amoxicillin-clavulanic acid, and aztreonam. Simultaneously, the most effective antibiotics were tetracycline and amikacin. blaCTX-M, blaTEM, blaSHV, aac(3)-II, aadA1, tetA, cmlA, catA, gyrA, gyrB, parC, sul1, sul2, sul3, blaKPC, blaOXA, and blaPER genes were detected. Biofilm production showed that 80% of K. pneumoniae strains were biofilm producers. Most ESBL- and KPC-producing isolates were weak biofilm producers (40.0% and 60.0%, respectively). There was no correlation between the ability to form stronger biofilms and the presence of ESBL and KPC enzymes in K. pneumoniae isolates.

The 10,000-Year Success Story of Wheat!

Wheat is one of the most important cereal crops in the world as it is used in the production of a diverse range of traditional and modern processed foods. The ancient varieties einkorn, emmer, and spelt not only played an important role as a source of food but became the ancestors of the modern varieties currently grown worldwide. Hexaploid wheat (Triticum aestivum L.) and tetraploid wheat (Triticum durum Desf.) now account for around 95% and 5% of the world production, respectively. The success of this cereal is inextricably associated with the capacity of its grain proteins, the gluten, to form a viscoelastic dough that allows the transformation of wheat flour into a wide variety of staple forms of food in the human diet. This review aims to give a holistic view of the temporal and proteogenomic evolution of wheat from its domestication to the massively produced high-yield crop of our day.

Successful Dissemination of Plasmid-Mediated Extended-Spectrum ß-Lactamases in Enterobacterales over Humans to Wild Fauna.

BACKGROUND: The emergence of multidrug-resistant bacteria remains poorly understood in the wild ecosystem and at the interface of habitats. Here, we explored the spread of Escherichia coli containing IncI1-ST3 plasmid encoding resistance gene cefotaximase-Munich-1 (blaCTX-M-1) in human-influenced habitats and wild fauna using a genomic approach. METHODS: Multilocus sequence typing (MLST), single-nucleotide polymorphism comparison, synteny-based analysis and data mining approaches were used to analyse a dataset of genomes and circularised plasmids. RESULTS: CTX-M-1 E. coli sequence types (STs) were preferentially associated with ecosystems. Few STs were shared by distinct habitats. IncI1-ST3-blaCTX-M-1 plasmids are disseminated among all E. coli phylogroups. The main divergences in plasmids were located in a shuffling zone including blaCTX-M-1 inserted in a conserved site. This insertion hot spot exhibited diverse positions and orientations in a zone-modulating conjugation, and the resulting synteny was associated with geographic and biological sources. CONCLUSIONS: The ecological success of IncI1-ST3-blaCTX-M-1 appears less linked to the spread of their bacterial recipients than to their ability to transfer in a broad spectrum of bacterial lineages. This feature is associated with the diversity of their shuffling conjugation region that contain blaCTX-M-1. These might be involved in the resistance to antimicrobials, but also in their spread.

Advances in quantification and analysis of the celiac-related immunogenic potential of gluten.

Gluten-free products have emerged in response to the increasing prevalence of gluten-related disorders, namely celiac disease. Therefore, the quantification of gluten in products intended for consumption by individuals who may suffer from these pathologies must be accurate and reproducible, in a way that allows their proper labeling and protects the health of consumers. Immunochemical methods have been the methods of choice for quantifying gluten, and several kits are commercially available. Nevertheless, they still face problems such as the initial extraction of gluten in complex matrices or the use of a standardized reference material to validate the results. Lately, other methodologies relying mostly on mass spectrometry-based techniques have been explored, and that may allow, in addition to quantitative analysis, the characterizationof gluten proteins. On the other hand, although the level of 20 mg/kg of gluten detected by these methods is sufficient for a product to be considered gluten-free, its immunogenic potential for celiac patients has not been clinically validated. In this sense, in vitro and in vivo models, such as the organoid technology applied in gut-on-chip devices and the transgenic humanized mouse models, respectively, are being developed for investigating both the gluten-induced pathogenesis and the treatment of celiac disease. Due to the ubiquitous nature of gluten in the food industry, as well as the increased prevalence of gluten-related disorders, here we intend to summarize the available methods for gluten quantification in food matrices and for the evaluation of its immunogenic potential concerning the development of novel therapies for celiac disease to highlight active research and discuss knowledge gaps and current challenges in this field.

Wheat/Gluten-Related Disorders and Gluten-Free Diet Misconceptions: A Review.

In the last 10,000 years, wheat has become one of the most important cereals in the human diet and today, it is widely consumed in many processed food products. Mostly considered a source of energy, wheat also contains other essential nutrients, including fiber, proteins, and minor components, such as phytochemicals, vitamins, lipids, and minerals, that together promote a healthy diet. Apart from its nutritional properties, wheat has a set of proteins, the gluten, which confer key technical properties, but also trigger severe immune-mediated diseases, such as celiac disease. We are currently witnessing a rise in the number of people adhering to gluten-free diets unwarranted by any medical need. In this dynamic context, this review aims to critically discuss the nutritional components of wheat, highlighting both the health benefits and wheat/gluten-related disorders, in order to address common misconceptions associated with wheat consumption.

Next-Generation Sequencing and MALDI Mass Spectrometry in the Study of Multiresistant Processed Meat Vancomycin-Resistant Enterococci (VRE).

Vancomycin-resistant enterococci (VRE), due to their intrinsic resistance to various commonly used antibiotics and their malleable genome, make the treatment of infections caused by these bacteria less effective. The aims of this work were to characterize isolates of Enterococcus spp. that originated from processed meat, through phenotypic and genotypic techniques, as well as to detect putative antibiotic resistance biomarkers. The 19 VRE identified had high resistance to teicoplanin (89%), tetracycline (94%), and erythromycin (84%) and a low resistance to kanamycin (11%), gentamicin (11%), and streptomycin (5%). Based on a Next-Generation Sequencing NGS technique, most isolates were vanA-positive. The most prevalent resistance genes detected were erm(B) and aac(6')-Ii, conferring resistance to the classes of macrolides and aminoglycosides, respectively. MALDI-TOF mass spectrometry (MS) analysis detected an exclusive peak of the Enterococcus genus at m/z (mass-to-charge-ratio) 4428 ± 3, and a peak at m/z 6048 ± 1 allowed us to distinguish Enterococcus faecium from the other species. Several statistically significant protein masses associated with resistance were detected, such as peaks at m/z 6358.27 and m/z 13237.3 in ciprofloxacin resistance isolates. These results reinforce the relevance of the combined and complementary NGS and MALDI-TOF MS techniques for bacterial characterization.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) in Purulent Subcutaneous Lesions of Farm Rabbits.

Methicillin-resistant Staphylococcus aureus (MRSA) are one of the main pathogens associated with purulent infections. MRSA clonal complex 97 (CC97) has been identified in a wide diversity of livestock animals. Therefore, we aimed to investigate the antibiotic resistance profiles of MRSA strains isolated from purulent lesions of food-producing rabbits. Samples from purulent lesions of 66 rabbits were collected in a slaughterhouse in Portugal. Samples were seeded onto ORSAB plates with 2 mg/L of oxacillin for MRSA isolation. Susceptibility to antibiotics was tested by the disk diffusion method against 14 antimicrobial agents. The presence of resistance genes, virulence factors and the immune evasion cluster (IEC) system was studied by polymerase chain reaction. All isolates were characterized by multilocus sequence typing (MLST), agr and spa typing. From the 66 samples analyzed, 16 (24.2%) MRSA were detected. All strains were classified as multidrug-resistant as they were resistant to at least three classes of antibiotics. All isolates showed resistance to penicillin, erythromycin and clindamycin. Seven isolates were resistant to gentamicin and harbored the aac(6')-Ie-aph (2'')-Ia gene. Resistance to tetracycline was detected in 10 isolates harboring the tet(K) gene. The IEC genes were detected in three isolates. MRSA strains belonged to CC97, CC1, CC5, CC15 or CC22. The isolates were assigned to six different spa types. In this study we found a moderate prevalence of multidrug-resistant MRSA strains in food-producing rabbits. This may represent concern for food safety and public health, since cross-contamination may occur, leading to the spread of MRSA and, eventually, the possibility of ingestion of contaminated meat.

Methicillin-Resistant Staphylococcus aureus CC398 in Purulent Lesions of Piglets and Fattening Pigs in Portugal.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is an important clinical problem. In 2005, a livestock-associated MRSA clone was described, named CC398, being mostly associated with pigs, and causing colonization and infection in pigs and in related humans. The prevalence of these strains in food-producing pigs raised concerns about the possibility of MRSA-CC398 being a foodborne pathogen. The objective of this study was to investigate the presence of S. aureus and MRSA in 141 carcasses of pigs at three slaughterhouses of Portugal, discarded from the food chain by signs of infection, and to characterize the recovered isolates. Methods: S. aureus isolates were identified by matrix-assisted laser desorption/ionization time-of-flight and they were typed (spa, CC398-clone, and SCCmec). The study of antibiotic resistance and virulence genes, and the detection of immune evasion cluster genes and prophages were performed by PCR and sequencing. Results: Twenty-eight S. aureus were obtained from 141 samples (one/sample, 19.9%), being 22 MRSA and 6 methicillin-susceptible S. aureus (MSSA). All MRSA strains were typed as CC398 and were ascribed to three spa types (t011, t108, and t1451). The SCCmec detected differed according to the spa types of MRSA isolates (SCCmecV: t011 and t108; SCCmecIVa: t1451). The MSSA strains were classified as spa-t1491-ST1-CC1. All the strains contained a wide range of antimicrobial resistance genes, the resistance to tetracycline being the prevalent one. In contrast, the strains contained only a few virulence genes. Among the 6 integrases of phages tested, three were detected: SΦ1, SΦ2, and SΦ7, with variations between MRSA and MSSA strains. Conclusions: MRSA-CC398 is not only a habitual pig colonizer but also an opportunistic pathogen in these animals, and must be controlled at the level of producers and slaughterhouses because of its impact on public health.