The genus Aliarcobacter belongs to the Arcobacteraceae family, with nine validly published species. Among them, the species Aliarcobacter butzleri is considered the fourth most frequently found Campylobacter-like microorganism in human diarrheal stool samples and has also been included in the list of microorganisms considered a serious risk to human health by the International Commission on Microbiological Specifications for Food. Increasing rates of multidrug resistance to different antimicrobials have been described in A. butzleri isolates, with efflux pumps being one of the described resistance mechanisms. Efflux pumps of the ATP-binding cassette (ABC) family are known to export a wide variety of substances and are ubiquitous in almost all organisms. Several genes coding for efflux pumps of this family were described in the A. butzleri genome. Despite the resistance associated with this species being widely described, the research on the mechanisms involved in this process is scarce. Therefore, the objective of this work was to evaluate the role of an ABC family efflux pump system in the resistance of A. butzleri. To this end, a mutant was constructed by deletion of the ybhF gene from the ybhFSR operon, a transporter with relevance in the extrusion of antibiotics and metals in other bacterial species. After ensuring that the mutation did not modify bacterial growth, the resistance profile of the native and mutant strains to different antimicrobial agents was evaluated. This analysis included metals, disinfectants, antibiotics, germicides, and an efflux pump substrate. The evaluation was based on the determination of the minimum inhibitory concentration of the antimicrobials by the agar dilution method. The results show differences in susceptibility of the mutant strain to some compounds tested, namely heavy metals and antibiotics, compared to the native strain. It can therefore be assumed that the YbhFSR efflux pump contributes to resistance in A. butzleri.

Among the bacterial resistance mechanisms, the active efflux pumps play a role in the extrusion of different molecules, and thus contribute for antimicrobial resistance. Staphylococcus aureus is a Gram-positive bacterium that can present resistance to various antibiotics, for which NorA, a predominant efflux pump of these strains, is known to promote resistant to fluoroquinolones. Thus, the inhibition of this efflux pump may modulate resistance in S. aureus, namely to fluoroquinolones. Therefore, this study aimed to investigate the ability of a natural compound, resveratrol, to modulate fluoroquinolones resistance in S. aureus. The antimicrobial activity of resveratrol, ethidium bromide (EtBr) and norfloxacin were determined, through the minimum inhibitory concentration (MIC). Then, the modulatory effect of resveratrol was evaluated, by the determination of the MIC of the antibiotic or EtBr in presence and absence of resveratrol at a sub-MIC level. The results showed that the MIC of norfloxacin against in a wildtype S. aureus strain decreased by 2-fold and for a NorA overexpressing (norA++) strain decreased by 16-fold, when in presence of resveratrol. A similar behavior was observed for EtBr. Furthermore, EtBr accumulation assay was also performed, showing that in the presence of resveratrol, the norA++ strain had an augmented fluorescence, consequence of the accumulation of EtBr. Altogether, the results suggest that resveratrol may act by inhibiting NorA. The post-antibiotic effect (PAE) of norfloxacin alone and in combination with resveratrol was also determined, showing that the most extended PAE was observed with norfloxacin at 32 mg/L when tested in combination with resveratrol. Furthermore, there was a decrease in mutation prevention concentration of norfloxacin when combined with resveratrol. Our findings demonstrated that resveratrol could modulate the norfloxacin-resistance, by inhibition of NorA, increasing the effectiveness of this antibiotic against S. aureus.

Introduction: Approximately 30% of the population is colonized with Staphylococcus aureus on the skin, mucous membranes, and in the anterior part of the nose. Two types of carriers have been described, intermittent carriers and persistent carriers, in addition to non-carriers. In persistent carriers, the same strain of S. aureus can and often does persist for months or even years, indicating that the species has developed special mechanisms to persist in this environment, in addition to being a multifactorial process involving genetic aspects of the host, virulence factors of the pathogen and the possible interactions between the microbiota, the host and S. aureus. The objective of this work was to investigate changes in the pattern of resistance to antibiotics in persistent and intermittent S. aureus strains.

Methods. Pharyngeal and nasal exudates were performed on 98 university students once a month for three months. The exudates were incubated in Tripticasein Soy Broth at 37 °C for 24 h, followed by seeding in Salt and Mannitol Agar Petri dishes using the cross streak method and re-seeding to obtain isolated colonies. All strains that were coagulase-positive mannitol fermenters were identified as S. aureus. If a person presented three isolates of S. aureus, they were considered persistent carriers, if they presented one or two isolates in a row, they were considered intermittent carriers, and if the bacteria were never isolated, they were considered non-carriers. All strains of S. aureus underwent antibiogram against: ciprofloxacin, fosfomycin, trimethoprim-sulfamethoxazole, penicillin, vancomycin, tetracycline, erythromycin, oxacillin, clindamycin, gentamicin and cafalothin by the Kirby-Bauer method and minimum inhibitory concentration for oxacillin, following the indications of the CLSI.

Results. We analyzed 61 women (62.2%) and 37 men (37.7%) with a mean age of 21.1 (± 5.7) years, of which 18 were persistent carriers in both anatomical sites (18.3%), 19 persistent carriers exclusively in the pharynx (19.3%), no persistent carriers exclusive to the nose were found. 27 were intermittent carriers in the nose (28.5%) and 34 are intermittent carriers in the pharynx (34.7%).

For persistent strains, an average of 80% (± 21.6) resistance was found for penicillin, 14% (± 25.3) for fosfomycin, 14% (± 24.7) for trimethoprim-sulfamethoxazole, 12% (± 4.6) for clindamycin, 7 % (± 2.6) for erythromycin, 4% (± 2.2) for tetracycline, 2.7% (± 3.6) for vancomycin, the other antibiotics (ciprofloxacin, oxacillin, gentamicin and cephalothin, less than 1% of resistant strains were found).

Regarding the intermittent strains, it was found that 74% (± 15) are resistant to penicillin, 14% (± 10.9) are resistant to erythromycin, 17% (± 7) to clindamycin, 10% (± 6.4) to oxacillin, 10% (± 11.4) are resistant to trimethoprim-sulfamethoxazole, 8.6% (± 9.2) are resistant to tetracycline, 6% (± 11.22) to fosfomycin, the antibiotics ciprofloxacin, vancomycin, gentamicin and cephalothin presented resistance in less than 1% of S. aureus strains.

Discussion. The average resistance to antibiotics of the persistent and intermittent S. aureus strains are very similar, however, the variation in resistance between doses does present more variation between the intermittent strains, particularly in trimethoprim-sulfamethoxazole and oxacillin antibiotics.

Conclusions. More persistent carriers of S. aureus were found in the pharynx and nose than intermittent carriers in one or both sites. The variation in the percentage of resistance to an antibiotic was found to be more important in the intermittent strains than in the persistent ones.

Introduction. The pathophysiology of Staphylococcus aureus in nasal carriers has been extensively studied, however, it must be admitted that the clinical relevance of S. aureus carriers in the pharynx has not been extensively investigated. This omission appears to be justified, since the nose is mentioned as the primary site of S. aureus colonization. From there, other regions are colonized by manual spread In the general adult population, S. aureus can be commonly found in other body sites such as the axillae (8%), chest/abdomen (15%), perineum (22 %), intestine (17-31%), vagina (5%) (Sollid J et al; 2014) and from 4 to 64% in the pharynx. Some studies mention a higher rate of carriers in the pharynx than in the nose when samples are taken in parallel. The objective of the work was to determine if there are differences in the pattern of resistance to antibiotics of strains isolated from the nose and pharynx.

Methods. Pharyngeal and nasal exudates were performed on 98 university students once a month for three months. The exudates were incubated in Tripticasein Soy Broth at 37 °C for 24 h, followed by seeding in Salt and Mannitol Agar Petri dishes using the cross streak method and re-seeding to obtain isolated colonies. All strains that were coagulase-positive mannitol fermenters were identified as S. aureus. If a person presented three isolates of S. aureus, they were considered persistent carriers, if they presented one or two isolates in a row, they were considered intermittent carriers, and if the bacteria were never isolated, they were considered non-carriers. All strains of S. aureus underwent antibiogram against: ciprofloxacin, fosfomycin, trimethoprim-sulfamethoxazole, penicillin, vancomycin, tetracycline, erythromycin, oxacillin, clindamycin, gentamicin and cafalothin by the Kirby-Bauer method and minimum inhibitory concentration for oxacillin, following the indications of the CLSI.

Results. A total of 81 (± 9.1) strains of S. aureus were isolated from the pharynx and 43 (± 6.6) strains from the nose of the students during the three samples taken. In the case of the pharyngeal strains, 81.4% (± 4.7) were resistant to penicillin, 12.5% (± 4.1) to clindamycin, 8.6% (± 2.8) to erythromycin, 2.78% (± 1.19) they are resistant to tetracycline and oxacillin. For ciprofloxacin, fosfomycin, vancomycin, gentamicin and cephalothin, the percentage of resistant strains was less than 1%.

In the case of the strains isolated from the nose, it was found that 84.3% (± 4.6) are resistant to penicillin, 18.2% (± 4.8) to erythromycin, 12.4% (± 4.4) to clindamycin, 4.49% (± 3.8) to tetracycline, 3.6% (±0.9) were resistant to oxacillin. For ciprofloxacin, fosfomycin, trimethoprim-sulfamethoxazole, gentamicin, and cephalothin, the percentage of resistant strains was less than 1%.

Discussion. More carriers of S. aureus were found in the pharynx (75%) than in the nose (40%) in the three samples taken, which coincides with some results published in the literature (Mertz D, 2007, Hamdan et al 2018). In the case of the percentage of resistant strains, it is very similar regardless of the isolation site, and the variation in resistance during the three intakes is similar.

Conclusions. More strains of S. aureus were isolated from the pharynx than from the nose. No differences were found in resistance to antibiotics, nor changes in the percentage of resistant strains in the pharynx and nose.

The emergence and the dissemination of multi-drug resistant bacteria constitute a major public health issue. Among incriminated Gram-negative bacteria (GNB), Pseudomonas aeruginosa has been designated by the WHO as a critical priority threat. This pathogen is responsible for various nosocomial infections, usually lethal for patients suffering from cystic fibrosis. Its ability to establish biofilms reinforces its virulence and intrinsic drug resistance. Its pathogenicity is orchestrated by the Quorum Sensing (QS) that refers to bacterial communication systems. This sophisticated coordination network relies on the secretion and perception of small signalling molecules called autoinducers (AIs). The extracellular concentration of AIs acts as a population density indicator. The biomass growth provokes an increased release of AIs inducing the expression of QS-associated genes via the activation of specific transcription factors. This stimulation ensures the biosynthesis of essential proteins for the synchronisation of bacteria colonies regarding the environmental conditions and especially those implicated in the virulence pathways. Three interconnected QS circuits regulate P. aeruginosa pathogenicity. Taking into account the widespread occurrence of N-acyl-homoserine lactone (AHL)-mediated communication las and rhl systems in GNB, the third species-specific pqs network appears as a pool of promising therapeutic targets for the development of inhibitors. The main AI of this circuit is the 2-heptyl-3-hydroxy-4(1H)-quinolone named Pseudomonas quinolone signal (PQS) that activates the PqsR transcriptional regulator.⁽¹⁾

In the last decades, the interest of a quorum silencing pharmacological approach has emerged. Indeed, the selective pressure put on sensitive bacteria by conventional antimicrobial molecules causing their death promotes resistant strain survival. Non-bactericidal anti-virulence agents (AVAs) could increase pathogen sensibility to the host immune system response in monotherapy. In combination therapy, they could restore the efficiency of current ATBs by inhibiting the formation of the hermetic barrier provided by biofilms.⁽¹⁾ A benzamide-benzimidazole hybrid discovered by Starkey et al. appears as one of the most promising PqsR inhibitor in preclinical stage.⁽²⁾ It revealed the best anti-virulence activity among all reported in literature with efficient anti-pyocyanin and anti-biofilm properties (IC₅₀s of 300 nM and 1 µM, respectively). With this in mind, our team has recently developed a novel family of 2-heteroaryl-4-quinolones as AVAs. The synthesis pathways, but also the physicochemical and biological evaluations of these compounds (intrinsic antibiotic and anti-virulence properties) will be described in the presentation.

References : ⁽¹⁾ Duplantier, M.; Lohou, E.; Sonnet, P. Quorum Sensing Inhibitors to Quench P. aeruginosa Pathogenicity, Pharmaceuticals 2021, 14, 1262; doi:10.3390/ph14121262 ; ⁽²⁾ Starkey, M.; Lepine, F.; Maura, D.; Bandyopadhaya, A.; Lesic, B.; He, J.; Kitao, T.; Righi, V.; Milot, S.; Tzika, A.; et al. Identification of Anti-Virulence Compounds That Disrupt Quorum-Sensing Regulated Acute and Persistent Pathogenicity, PLoS Pathog. 2014, 10, e1004321, doi:10.1371/journal.ppat.1004321.

Antimicrobial resistance is a world health problem and with this awareness, some fluoroquinolones are classified for WHO AWaRe as “Watch”, a group with higher resistance potential. Among the known resistance mechanisms, efflux pumps have a relevant role and have been associated with multidrug resistance. Thus, the use of an efflux pump inhibitor may be an approach to restore the antibacterial activity of drugs against pathogens that cause infectious diseases. Phenothiazines have efflux-inhibiting properties and inhibit biofilm formation, which can make them a good antibacterial adjuvant. To attempt a new approach against the antimicrobial resistance of fluoroquinolones, the hybridization of ciprofloxacin and phenothiazines to afford molecules that may display antibacterial and efflux pump inhibitory activities were performed. The hybrid molecules were synthesized using nucleophilic substitution reactions and the characterization of the prepared compounds was mainly performed through nuclear magnetic resonance spectroscopy. Simultaneously, to help understand the interactions of the newly synthesized molecules and the NorA efflux pump, in silico studies were also carried out. The newly synthesized molecules were then evaluated regarding their potential antibacterial activity, by the determination of their minimum inhibitory concentration against NorA wild type Staphylococcus aureus (SA1199) and a NorA overexpressing strain (SA1199B). The results so obtained indicated that some hybrids showed antimicrobial activity even against NorA overexpressing strain. Future work will focus on the evaluation of the mechanisms underlying the antibacterial activity of these new molecules and the potential for restraining resistance development.

This work was developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES. João L. Serrano acknowledges a doctoral fellowship grant from the FCT (SFRH/BD/148028/2019).

Spiramycin is a 16-membered macrolide used in human medicine as an antibacterial and antiparasitic agent (active against Toxoplasma spp.). Spiramycin is effective against various bacterial pathogens including Gram-positive (Staphylococcus aureus, streptococci of groups A, B, C and D, and pneumococcus), Gram-negative (Neisseria, Legionella) and more (Mycoplasma, Chlamydia). In contrast, Pseudomonas aeruginosa is considered intrinsically resistant to macrolides including azithromycin and spiramycin. Despite the results of in vitro susceptibility tests, interest in macrolides in the treatment of some pseudomonal infections arose from both clinical and preclinical studies. For example, in a mouse model of P. aeruginosa bacteraemia, treatment with erythromycin led to a survival rate of 80% compared with 20% in controls. For this reason, macrolides have drawn attention as adjunct therapy against chronic and/or biofilm-mediated P. aeruginosa infections. While most of the studies on the antivirulence activity of macrolides focus on erythromycin and on its derivative azithromycin, there is almost no information on spiramycin, which differs from erythromycin for a larger macrolactone ring and a different sugar decoration. In this study, we test spiramycin as an antivirulence factor using different assays to characterize the phenotype changes in P. aeruginosa.

Our results show as spiramycin inhibits the production of pyocyanin, pyoverdine and rhamnolipids in P. aeruginosa. Moreover, the treatment of the bacterium with this antibiotic inhibits biofilm formation in an artificial biomimetic system and blocks the motility on the agar surface. Finally, to test the effect of spiramycin against P. aeruginosa in an in-vivo system we used the insect Galleria mellonella and the preliminary data obtained by this colonization model show a marked reduction in mortality. Finally, using computational modeling and docking simulation we explored the probable mechanisms of action of spiramycin against P. aeruginosa.

Abstract: Ascophyllum nodosum (Linnaeus) Le Jolis (AN) is a brown alga from the Fucaceae family and the unique species from the Ascophyllum genus. This brown alga is an edible macroalga from the North Atlantic Ocean, commonly found on the European north-western coast. High-value bioactive molecules such as pigments, polyphenols, and phlorotannin [1,2] have been found in the macroalgae composition, which turns this alga particularly interesting for exploring potential biological activities. Among sustainable extraction technologies, microwave-assisted extraction (MAE) has many advantages such as short extraction time and less solvent requirement. On the other hand, ethanol and water are eco-friendly solvents that have already been proven to be effective for obtaining bioactive compounds with antimicrobial capacity [3]. Therefore, in this work, MAE t=5 min; P=10.5 bar; using ethanol (37% ) as solvent was applied to obtain a polyphenol-rich extract from AN. The antimicrobial effect of the resulting extract against five foodborne microorganisms (Bacillus cereus, Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, Staphylococcus aureus), and the opportunistic bacteria Staphylococcus epidermidis was tested. The antimicrobial activity was performed through Kirby-Bauer disk diffusion susceptibility test protocol and the microdilution method. The analytical results indicated that the AN extract was effective against all tested bacteria except for Escherichia coli. The highest antimicrobial activity was found against Staphylococcus aureus presenting a minimal inhibitory concentration of 400µg/mL and an inhibition halo of 11,79±1,92 mm.

Acknowledgments: The research leading to these results was supported by MICINN supporting the Ramón y Cajal grant for M.A. Prieto (RYC-2017-22891) and the Juan de la Cierva Incorporación for Hui Cao (IJC2020-046055-I), Xunta de Galicia for supporting the program EXCELENCIA-ED431F 2020/12, the post-doctoral grant (ED481B-2019/096), and L. Cassani (ED481B-2021/152), and the pre-doctoral grants of P. Garcia-Oliveira (ED481A-2019/295), and M. Carpena (ED481A 2021/313). The research leading to these results was supported by the European Union through the “NextGenerationEU” program supporting the “Margarita Salas” grant awarded to P. Garcia-Perez. Authors are grateful to Ibero-American Program on Science and Technology (CYTED—AQUA-CIBUS, P317RT0003), to the Bio Based Industries Joint Undertaking (JU) under grant agreement No 888003 UP4HEALTH Project (H2020-BBI-JTI-2019) that supports the work C. Lourenço-Lopes and to AlgaMar enterprise (www.algamar.com) for the collaboration and algae material provision. The authors would like to thank the EU and FCT for funding through the programs UIDB/50006/2020; UIDP/50006/2020.

References:

1. Sardari, R.R.R.; Prothmann, J.; Gregersen, O.; Turner, C.; Karlsson, E.N. Identification of phlorotannins in the brown algae, saccharina latissima and ascophyllum nodosum by ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Molecules 2021, 26, doi:10.3390/MOLECULES26010043.
2. Garcia-Perez, P.; Lourenço-Lopes, C.; Silva, A.; Pereira, A.G.; Fraga-Corral, M.; Zhao, C.; Xiao, J.; Simal-Gandara, J.; Prieto, M.A. Pigment Composition of Nine Brown Algae from the Iberian Northwestern Coastline: Influence of the Extraction Solvent. Mar. Drugs 2022, 20, 113, doi:10.3390/md20020113.
3. Silva, A.; Rodrigues, C.; Lourenç, C.; Silva, S.A. Screening for bioactive properties on brown algae from the northwest Iberian Peninsula. 2021, 1–15.

The increased resistance of pathogenic microorganisms to a wide range of antibiotics has driven recent research efforts towards exploring and developing effective preservatives with better potential and new strategies to prevent this multi-resistance. Many of these studies have been on natural matrices such as plants (1). This is in line with consumers demand for more organic and natural products. A possible alternative could be using bioactive compounds from Camellia japonica flowers as bio-preservatives since they have been traditionally used in cosmetic products due to their biological properties (2). Among the bioactive molecules of camellias, it is worth highlighting phenolic compounds, anthocyanins, polysaccharides, polyphenols, polyunsaturated fatty acids and pigments (3). However, to incorporate these bioactive molecules into products with antimicrobial purposes, it is necessary to conduct an extraction and purification of the target compounds. Thus, in this study, the antimicrobial activity of one variety of C. japonica flowers (var. Conde de la Torre) obtained by an easy and profitable extraction technique such as maceration has been analyzed. Results from this work showed that the variety under study have a significant antimicrobial activity in terms of inhibition zones against Staphylococcus epidermidis (14.02 mm) Staphylococcus aureus (10.84 mm), Pseudomonas aeruginosa (10.36 mm), Salmonella enteritidis (7.98 mm), and Bacillus cereus (5.05 mm). However, the var. Conde de la Torre of C. japonica did not show significant activity against Escherichia coli. In conclusion, Conde de la Torre can be used as a potential antimicrobial agent. However, more studies that determinates the compounds responsible of these bioactivities are needed.

Nowadays, medicinal plants have different uses since they can play an important role in the design of modern medicines and new healthy foods. Although their consumption is not elevated, these plants are attributed pharmacological properties with beneficial properties such as antioxidant, anti-inflammatory, or antimicrobial, among others [1]. The study aimed to evaluate extracts derived from Camaemelum nobile and Arnica montana, two plants from the Asteraceae family that could be of interest for the industry, analyzing their antimicrobial capacity. After a previous bibliographic study, heat-assisted extraction (HAE) was selected and used to obtain extracts rich in bioactive compounds. Then, the antimicrobial activity was determined using three Gram-negative species: Pseudomonas aeruginosa (ATCC 10145), Escherichia coli (NCTC 9001) and Salmonella enteritidis (ATCC 13676); and two Gram-positive species: Bacillus cereus (ATCC 25923) and Staphylococcus aureus (ATCC 25923). The results obtained showed that A. montana showed a greater antimicrobial activity with inhibition halos ranging from 8.36 to 6.82 mm while the C. nobile showed inhibition halos of 7.55 and 7.91 mm against bacteria such as S. enteritidis and P. aeruginosa, respectively. None of the plants showed activity against S. aureus. These results provide scientific evidence for the evaluation of the potential of medicinal plant extracts for the development of new products with antimicrobial properties.