Worldwide, Mycobacterium tuberculosis (M. tb) is responsible for 10.6 million infections and 1.6 million deaths. In Europe and North America, the incidence of non-tuberculous mycobacteria (NTM) infections exceeds that of M. tb. NTM are ubiquitous and opportunistic for immunocompromised people and/or with chronic respiratory diseases. Among NTM with pulmonary pathogenicity, Mycobacterium abscessus is the most difficult to treat and Mycobacterium avium complex (MAC) is the most common, causing 80% of NTM infections. Current NTM treatments require a combination of antibiotics which can cause many side effects over a long period. For example, MAC infections should be treated with at least three drugs (macrolides, rifamycin and ethambutol) for 12 to 24 months. These drugs are not very effective with a success rate of 52 to 60%, as they were initially designed for M. tb infection treatment. In addition, they can occur many side effects, including hepatotoxicity, urine coloration, ocular disorders, … Thus, it is urgent to develop new molecules that are safer, more NTM specific and, if possible, with a novel mechanism of action. Quinoline-based pharmacophore is found in two compounds active against MAC, bedaquiline (BQ) and mefloquine (MQ). Interestingly, they target ATP synthase, an enzyme essential for mycobacteria. However, BQ and MQ can induce side effects on the liver and central nervous system, respectively. In order to reduce the toxicity of MQ, quinoline core has been pharmacomodulated. Herein, we present (i) the synthesis of piperazine-based amino-alcohol-quinolines active against NTM, (ii) their in silico physicochemical and pharmacokinetic properties and, (iii) their in vitro antimycobacterial evaluation.

Antibiotic resistance is the ability of microorganisms to resist the effect of an antibiotic to which they were once sensitive. Infections caused by bacteria resistant to first-line antibiotics and, more recently, last-line treatments, and even combination drugs could be fatal in case of minor bacterial infections or injuries. Co-trimoxazole (COT), a combination of two antifolate compounds - sulfamethoxazole and trimethoprim, is effective against Gram-negative and Gram-positive bacteria. There are multiple reports of co-trimoxazole resistance among clinical as well as environmental isolates of bacteria. We have investigated prevalence of COT resistance among bacterial isolates from urban aquatic environment and patients with urinary tract infections (UTI). Of the total bacterial cultures screened for the resistance 70-80 percent isolates were found to be positive. The transmission of ARGs among different bacterial species is a global human threat. This study aims at determining the prevalence and diversity of COT resistant bacteria in clinical and wastewater. Furthermore, this works highlights the acquisition of genetic factors viz. dfr and sul genes conferring resistance to COT among UTI causing bacterial pathogens and wastewater bacterial isolates. The study will further provide in-sights into the antibiotic resistance spread in clinical and environmental samples and how these two systems interlink.

The threat of antibiotic-resistant bacterial strains as a result of unregulated antibiotic use requires new strategies to combat microbial pathogenicity. This study aimed to investigate the potential of phenolic extracts from grape, pomegranate, and persimmon to modulate bacterial virulence factors through pseudo-targeted metabolomics. The effect of sub-inhibitory concentration of phenolic extracts on bacterial quorum sensing was assessed by monitoring the interaction of extracts and extracellular autoinducers of Pseudomonas aeruginosa and Chromobacterium violaceum. Also, the interaction of extracts, bacteria and colon cells were determined. A neutral loss pattern of the lactone ring at 102.1 m/z revealed the presence of acyl-homoserine lactones (AHLs) expressed by C. violaceum and P. aeruginosa. Multiple reaction monitoring allowed the annotation of 21 molecules of AHLs and 8 molecules of 2-alkyl-4(1H)-quinolones (AQs). MRM allowed tracking these compounds which were significantly downregulated by the extracts, indicating their potential to attenuate bacterial virulence such as biofilms, bacterial motility, and pigment production by phenolics was correlated with the downregulation of AHLs and AQs. This study highlights phenolic extracts from upcycled products as candidates to modulate bacterial pathogenicity and mitigate bacterial virulence. The use of pseudo-targeted metabolomics as a monitoring tool allows for the annotation of both virulence molecules and bioactive compounds within the extracts, shedding light on their mechanisms of action.

Our society is facing the emergence of pathogenic bacteria and fungi resistant to available antimicrobials. WHO priority and critical pathogenic fungi include Aspergillus fumigatus, an azole-resistant fungus. A. fumigatus is a globally distributed ubiquitous environmental mould with high mortality rates in those with resistant infection [1]. To combat the problem, one of the keys is the discovery of new antimicrobials compounds, as well as the improvement of their production.

Through citizen science projects such as Tiny Earth [2] and other natural sources (compost, alkaline soils, trees, insects, etc.) we have isolated a few strains of actinomycetes, mainly from the genus Streptomyces [3-4] whose genome has been sequenced. These strains have a large genome in which a high number of biosynthetic gene clusters (BGCs) can be identified, thanks to bioinformatics programmes such as AntiSMASH v.6. [5]. This genomic mining together with genome annotation programs such as Prokka [6] and Rast allow us to search for genes of interest involved in antifungal compounds.

In addition to the in silico study, we have performed bioassays of some of the isolated Streptomyces strains against A. fumigatus. These assays have been performed using cultures of the isolated strains as well as extracts obtained from them. This enabled us to select the strain with the greatest antifungal potential for a more exhaustive study of its antifungal compounds in subsequent studies. Furthermore, we have performed dual cultures against plant pathogenic fungi of commercial interest to test the antifungal potential of our strains.

References

[1] WHO fungal priority pathogens list to guide research, development and public health action. Geneva: World Health Organization; 2022.

[2] https://swiusal.wixsite.com/micromundousal

[3] Marugán Cardiel, María. TFG, Universidad de Salamanca, 2019.

[4] Morante Gómara, Helena. TFG, Universidad de Salamanca, 2020.

[5] Blin et al. Nucleic Acids Research, 2023, gkad344. https://doi.org/10.1093/nar/gkad344.

[6] Seemann, Torsten. Bioinformatics 30, n.^o 14, (2014): 2068-69. https://doi.org/10.1093/bioinformatics/btu153.

[7] Brettin et al. Scientific Reports 5, n.^o 1 (2015): 8365. https://doi.org/10.1038/srep08365.

Staphylococcus aureus and coagulase-negative staphylococci (CNS) are some of the main causes of mastitis in sheep. The ability of Staphylococcus spp. to form biofilms in vivo is considered an important virulence factor that could explain cases of mastitis refractory to antibiotic treatments.

The aim of the research was to evaluate the antibacterial and anti-biofilm-forming activity of Thyme Essential Oil (TEO) at concentrations of 9.28mg/mL, 4.64mg/mL, and 2.32mg/mL (w/v), against clinical strains of staphylococci isolated from ovine mastitis.

Two reference strains (ATCC25923 and ATCC11622) and 12 clinical isolates (6 S. aureus and 6 CNS) have been studied to assess the antimicrobial and anti-biofilm activity of TEO using the microdilution broth method.

Biochemical and molecular methods were used to identify the isolated bacteria and the disk diffusion method was employed to determine their antimicrobial resistance profile. The antibacterial efficacy was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), while the biofilm inhibition was assessed by minimum biofilm inhibitory concentration (MBIC) by measuring the absorbance of Crystal Violet at 570nm, as previously described (1). Our results showed bactericidal efficacy of TEO with MIC and MBC values of 4.64mg/mL, while a total inhibition of biofilm production was found at sublethal concentrations of 2.32mg/mL.

These preliminary results demonstrated the antimicrobial and anti-biofilm efficacy of TEO in vitro against tested bacterial strains. Further studies are needed to confirm the in vivo reproducibility of these data in order to consider TEO as a valid alternative to classical antibiotic therapies and, consequently, to counteract the onset of antimicrobial resistance.

In the struggle against multi-drug resistant bacterial infections, the opportunistic pathogen Pseudomonas aeruginosa has been identified by the WHO as a priority for the development of new treatments. This gram-negative bacterium produces a characteristic cytotoxic pigment called pyocyanin and is able to form biofilms that act as protective barriers against the immune system and antibiotics. Its pathogenicity is coordinated by the quorum sensing that is a bacterial communication network responsible for pathogenicity expression according to the population density. In the P. aeruginosa specific system pqs, the transcription factor PqsR regulates the activation of virulence-related genes via recognition of its auto-inducer PQS (Pseudomonas Quinolone Signal). This circuit stimulates the secretion of pyocyanin as well as the establishment of biofilms. Therefore, the development of quorum quenchers that disrupt connections without affecting bacterial growth appears as a promising strategy to circumvent selection pressure issues mediated by conventional antibiotherapy. These new anti-virulence agents (AVA) could restore the efficacy of antibiotics when used in bitherapy. In particular, the design of PqsR inhibitors as AVA seems like a sustainable approach to combat P. aeruginosa specifically. Bi-aromatic molecules targeting PqsR have been reported in the literature. Meanwhile, our team discovered a hit 2-heteroaryl-4-quinolone compound that displays interesting anti-biofilm and anti-pyocyanin activities. By structural analogy, we have recently developed a new family of 2-heteroaryl-4-aminoquinolines with promising anti-virulence properties. The synthesis of those new AVA as well as their physicochemical and biological evaluation is described in the poster.

The rapid emergence and dissemination of multi- and extensively drug-resistant bacteria pose a significant public health concern. Antibiotic-resistant bacteria are projected to kill 10 million people by 2050. Moreover, there has been a lack of introduction of new antibiotic classes for over five decades and the once successful Waksman platform for the discovering novel antibiotics has been largely depleted. Consequently, this all have resulted in a limited array of therapeutic options. Hence, there is an urgent need for the development of new discovery platforms to identify novel antibacterials and replenish the antibiotic portfolio.

Lysins, or bacteriophage-encoded peptidoglycan hydrolases, represent a promising and alternative class of antibiotics. They are highly specific at the species level, resulting in a narrow spectrum of activity. Yet, we and other experts claim that more novel lysin candidates must be discovered and engineered to feed the (pre)clinical pipeline.

Several promising approaches have emerged within the field of metagenomics. Here I will elucidate two concepts that have been the focus of my research. Their main idea rests upon the premise that the largely unexplored metagenome derived from uncultivable bacteriophages represents an essentially infinite reservoir of potentially potent lysins. The aim is to build an efficient discovery platform specifically tailored for novel lysin-based antibiotics, employing functional and sequence-based metagenomic methodologies.

Ramoplanin (Rml) is a non-ribosomally produced lipoglycodepsipeptide antibiotic from Actinoplanes ramoplaninifer ATCC 33076. Many biosynthetic and regulatory aspects of its production remain unclear, partially due to the challenges with cultivation and gene-engineering manipulations of the producer strain. This work aims to address some of these issues via investigating the Rml synthesis laboratory conditions and morphological properties of the wild-type strain.

As the first step, we investigated cultural properties of ATCC 33076 while cultivated on different solid media. We have revealed that on ISP4 agar the culture exhibited heterogeneity, which led to the purification of two morphotypes, further denoted as Lt (a light orange colony color), and Bt (a bright orange colony color).

Next, during separate cultivation under the Rml production conditions in liquid media Lt exhibited notably higher productivity of Rml when compared to Bt morphotype grown under the same conditions. During co-cultivation of Lt and Bt in productive liquid media, Bt gradually outcompeted Lt over time, resulting in the dominance of Bt as the sole morphotype present in the culture. These results correlated with the decreased level of Rml synthesis in co-culture, corresponding to the reduced abundance of the Lt morphotype in it.

We established separate cultures for Lt and Bt morphotypes presented in wild-type strain, and conducted a thorough examination of their respective characteristics and properties. The work to correlate morphological differences with the genomic properties of both strains by sequencing and comparing the genomes of Lt and Bt is underway in our laboratories.

The emerging bacterial resistance to conventional antibiotics has led to the search for new therapeutic alternatives. Antimicrobial cationic peptides are promising candidates, since they act on bacterial membranes causing their rapid destruction, with a low tendency to generate resistance. However, these compounds present low stability against proteases. This study aimed to design a retro inverse analog containing D amino acids of TA4R (RLFR)3 to evaluate the effect of substitution by D amino acids and inverted sequence on their biological properties and enzymatic stability.

For this, RI-dTA4R was synthesized by Fmoc chemistry. The Minimal inhibitory concentration (MIC) was determined against the following bacterial strains: Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25929, Staphylococcus aureus Methicillin Resistant SAMR1. Hemolytic activity and enzymatic stability were determined against human erythrocytes and digestive and serum proteases, respectively. DC analyses were performed in aqueous media and membrane mimetic vesicles.

RI-dTA4R showed antimicrobial activity against all bacteria strains tested (MIC=4.6 to 2.3 µM), increasing the inhibitory activity of TA4R, and improving their enzymatic stability against digestive and serum proteases. Nevertheless, this analog was hemolytic (~50% of hemolysis at 25 µM), but at MIC concentration presented less than 20% of hemolysis. CD analysis showed that the peptide is strongly structured in DPPG vesicles, adopting a levorotatory helical structure.

Based on these results, RI-dTA4R may be considered a potential therapeutic compound for treating infections produced by gram (+) and (-) bacteria with improved enzymatic stability.

In swine farming, E. coli plays a critical role in transmitting multi-drug resistance (MDR). Antibiotic use in swine management promotes antibiotic-resistant bacteria emergence, limiting treatment options and spread through food-chain. The aim of this study is investigating the role of E. coli, focusing on MDR, genotypic analysis, Multi-Locus Sequence Typing (MLST), and phylogenetic analysis from different pig farms.

Fifty-nine swine fecal samples were obtained from 12 pig farms, yielding 47 E. coli isolates. Of these, 44 were from breeding pigs, distributed as different compartments. Eight samples from fattening pigs yielded two E. coli strains. Antibiotic resistance was observed in all 46 E. coli isolates tested, with high resistance to tetracycline (100%), ampicillin (97.8%), tobramycin (97.8%), and trimethoprim-sulfamethoxazole (95.7%). MDR was common, with 9 isolates resistant to four antibiotic classes, 18 to five, 13 to six, and 6 to seven classes. In genotypic analysis, ampC gene was predominant (86.95%), followed by blaTEM (60.86%), sul3 (60.86%). For MLST, we selected 7 strains resulting in the identification of 6 STs: ST101, ST5229, ST48, ST5757, ST10, and ST1147. Phylogroup A dominated at 60.8%, followed by B1 at 30.4%, and D at 8.69%.

High rates of resistance were observed and MDR was widespread, with resistant to multiple antibiotic classes. Overall, this study highlights the urgent need for surveillance and intervention strategies to mitigate the transmission of antibiotic-resistant E. coli from swine to humans, protecting both animal and public health in the context of swine farming practices.