Search Results (129)

Background: Antibiotics at sub-inhibitory concentrations can rewire bacterial regulatory networks, impacting virulence. Objective: The way that exposure to selected antibiotics (ciprofloxacin, amikacin, azithromycin, ceftazidime, and meropenem) below their minimum inhibitory concentration (sub-MIC) modulates the physiology of Pseudomonas aeruginosa is examined in this study using growth-phase-resolved analysis. Methods: Standard P. aeruginosa strain cultures were exposed to ¼ and ½ MIC to determine the growth kinetics under antibiotic stress. The study measured protease and pyocyanin production and the expression level of important quorum sensing and virulence genes (lasI/R, rhlI/R, pqsR/A, and phzA) at different growth phases. Results: Meropenem produced the most noticeable growth suppression at ½ MIC. Sub-MIC antibiotics did not completely stop growth, but caused distinct, dose-dependent changes. Azithromycin eliminated protease activity in all phases and had a biphasic effect on pyocyanin. Ciprofloxacin consistently inhibited both pyocyanin and protease in all phases. The effects of amikacin varied by phase and dose, while β-lactams markedly increased pyocyanin production during the log phase. In contrast to the plateau phase, when expression was often downregulated or unchanged, most quorum-sensing- and virulence-associated genes showed significant upregulation during the death phase under sub-MIC exposure. Conclusions: These findings indicate that sub-MIC antibiotics act as biochemical signal modulators, preserving stress-adapted sub-populations that, in late growth phases, activate quorum sensing and stress tolerance pathways. Full article

The repurposing potential of Efavirenz (EFV), a clinically established non-nucleoside reverse transcriptase inhibitor, was comprehensively evaluated for its in vitro antibacterial effect either alone or in combination with other antibacterial agents on several Gram-positive clinical strains showing different antibiotic resistance profiles. The binding potential assessed by an in silico study included Penicillin-binding proteins (PBPs) and WalK membrane kinase. Despite the relatively high minimum inhibitory concentration (MIC) limiting the use of EFV as a single antibacterial agent, it exhibits significant synergistic activity at sub-MIC levels when paired with various antibiotics against Enterococcus species and Staphylococcus aureus. EFV showed restored sensitivity of β-lactams against Methicillin-resistant S. aureus (MRSA). It increased the effectiveness of antibiotics tested against Methicillin-sensitive S. aureus (MSSA). It also helped to overcome the intrinsic resistance barrier for several antibiotics in Enterococcus spp. In silico binding studies aligned remarkably with experimental antimicrobial testing results and highlighted the potential of EFV to direct the engagement of PBPs with moderate to strong binding affinities (pK_a 5.2–6.1). The dual-site PBP2 binding mechanism emerged as a novel inhibition strategy, potentially circumventing resistance mutations. Special attention should be paid to WalK binding predictions (pK_a = 4.94), referring to the potential of EFV to interfere with essential regulatory pathways controlling cell wall metabolism and virulence factor expression. These findings, in general, suggest the possibility of EFV as a promising lead for the development of new antibacterial agents. Full article

Pseudomonas aeruginosa is a major opportunistic pathogen with high levels of antibiotic resistance. Phage therapy represents a promising alternative for the treatment of difficult infections both alone and in combination with antibiotics. Here, we isolated and characterized three novel lytic myoviruses, Cisa, Nello, and Moonstruck. Genomic analysis revealed that Cisa and Nello belong to the Pbunavirus genus, while Moonstruck is a novel Pakpunavirus species. All lacked lysogeny, virulence, or resistance-associated genes, supporting their therapeutic suitability. Phage Nello and Moonstruck were active against P. aeruginosa Pa3GrPv, isolated from a patient with lung infection candidate for phage therapy. Moonstruck exhibited superior lytic activity with ciprofloxacin sub-MIC value (0.125 µg/mL), achieving bacterial suppression for 48 h. However, to improve the lytic efficacy of the phages on the clinical isolate, phage adaptation via serial passage was investigated. The killing efficacy of Nello was enhanced, whereas Moonstruck showed a less consistent improvement, suggesting phage-specific differences in evolutionary dynamics. Sequencing of the evolved phages revealed point mutations in tail-associated genes, potentially linked to a better phage–host interaction. These results support the use of phage–antibiotic combinations and directed evolution as strategies to enhance phage efficacy against drug-resistant infections. Overall, these findings support the therapeutic potential of the newly isolated phages in treating P. aeruginosa lung infections. Full article

Background: The opportunistic pathogen Staphylococcus aureus causes skin and soft tissue infections that are associated with biofilm formation, and in immunocompromised patients can progress to surgical site infections, pneumonia, bacteremia, sepsis, and even death. Most antibiotics actively damage living, dividing cells on the surface of the biofilm, where there is a high concentration of nutrients and oxygen, while in the depths, where these factors are scarce, slowly growing cells remain. Objectives: The aim of our study was to evaluate the antibiofilm potential of ethyl acetate roots (EtOAcR) and aerial parts (EtOAcAP) extracts from the perennial Bulgarian plant Geum urbanum L. against methicillin-resistant S. aureus (MRSA) NBIMCC 8327. Methods: The effects of both extracts on the expression of biofilm-related genes, icaA and icaD, were investigated. The cytotoxicity of EtOAcR and EtOAcAP on A-375 (human melanoma), A-431 (epidermoid skin cancer) and HaCaT (normal keratinocytes) cell lines, and the induction of apoptosis were determined. Finally, the in vivo skin irritation potential of the most active extract was studied. Results: Both tested extracts inhibited biofilm formation at concentrations that did not affect bacterial growth. Interestingly, the expression of icaA and icaD was upregulated, although the biofilm development was inhibited 72.4–90.5% by EtOAcAP and 18.9–20.4% by EtOAcR at sub-MICs. EtOAcAP extract showed a more favorable cytotoxic profile on non-tumorigenic cells and stronger antineoplastic activity (IC₅₀ = 6.7–14.68 µg/mL) as compared to EtOAcR extract (IC₅₀ = 8.73–23.67 µg/mL). Therefore, a skin irritation test was performed with the EtOAcAP extract at ten-times higher concentrations than the minimum inhibitory one, and, resultantly, the primary irritation index was equal to zero (no skin irritation observed). Conclusions: The EtOAcAP extract was proven to be an effective antistaphylococcal agent with favorable skin tolerance. The extract showed strong antineoplastic activity and antibiofilm effect at sub-MICs, which outlines new prospects for its development as a natural product for specific skin applications in medical practice. Full article

With the increasing occurrence of bacterial resistance, it is now essential to look for new alternatives to protect the curative utilization of antibiotics within the One Health concept. Here, we adapt and optimize a broth microdilution method and compare it against the broth macrodilution method for evaluating the antibacterial activity of a complex essential oils mix (EO mix) against four livestock pathogens: Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Staphylococcus aureus. Microdilution method performance (final volume well: 300 µL; inoculum: 1.0 × 10⁶ CFU/mL) was evaluated following CLSI recommendations, by comparing the MIC of each of the four strains with the MICs obtained with the macrodilution method (final volume tube: 2 mL; inoculum 1.0 × 10⁶ CFU/mL). Microdilution analysis was performed with an automated plate reader (Bioscreen C), and three bacterial growth parameters (OD max, lag phase, and growth rate) were calculated (DMFit curve-fitting software (v2.1; courtesy of the Institute of Food Research, Norwich, UK)). EO mix MICs were determined for E. coli, S. aureus, and B. cereus. Our results emphasize the importance of ensuring the accuracy of MIC results by performing three technical and three biological replicates, and combining OD max, lag phase, and growth rate to assess the impact of an EO mix at sub-MIC levels. Full article

The effect of sub-inhibitory doses of three disinfectants, sodium hypochlorite (SHY), peracetic acid (PAA), and benzalkonium chloride (BZK), on the resistance to biocides and antibiotics of five strains of Listeria monocytogenes was determined. The minimum inhibitory concentration (MIC) of these chemicals ranged (ppm) between 3533.3 ± 28.9 and 3783.3 ± 28.9 for SHY, between 1000.0 ± 25.0 and 1050.0 ± 25.0 for PAA, and between 1.3 ± 0.6 and 4.3 ± 0.6 for BZK. The minimum bactericidal concentration (ppm) was between 3683.3 ± 57.7 and 3983.3 ± 28.9 for SHY, between 1050.0 ± 25.0 and 1250.0 ± 25.0 for PAA, and between 1.7 ± 1.2 and 4.7 ± 1.2 for BZK. Exposure of the strains to increasing sub-inhibitory concentrations of the biocides caused adaptation and cross-adaptation to these substances, markedly so in the case of BZK, relative to which some strains saw their MIC value increase up to 5.2 times after being exposed to low doses of this disinfectant. After exposure to biocides, changes in the resistance to antibiotics of the strains were also observed. In some cases, strains moved from a category of susceptible or of reduced susceptibility to resistant, especially when exposure was to SHY. These findings suggest a need to avoid the application of sublethal concentrations of disinfectants in both the food industry and the healthcare system. Full article

Aquatic products play a crucial role in meeting the increasing global demands for high-quality proteins. However, the occurrence of bacterial diseases results in significant economic losses worldwide. Aeromonas hydrophila (A. hydrophila) is the pathogen of several fish diseases. Antibiotics were widely used in combating bacterial diseases in aquaculture. The increasing occurrences of antibiotic resistance necessitate the restricted use of antibiotics. Consequently, developing drugs that avoid antibiotic resistance is important for the future of aquaculture. Quorum sensing (QS) is critical for bacterial pathogens in regulating bacterial virulence and is a promising target for developing anti-infective agents. Here, we found that turmeric oil with a MIC of 256 μg/mL could dose-dependently reduce the virulence phenotypes regulated by QS, ranging from 8 to 64 μg/mL, suggesting that sub-inhibitory concentrations of turmeric oil could inhibit bacterial virulence. Further qPCR findings demonstrated that turmeric oil could significantly inhibit the transcription of aerA, ahyI, and ahyR by a 54-fold, 36-fold, and 56-fold change reduction, respectively. Cell live/dead staining and animal study results showed that turmeric oil could inhibit the pathogenicity of A. hydrophila. Fish treated with turmeric oil showed a reduced mortality rate of 60%, whereas all fish in the positive control group died. Moreover, treatment with turmeric oil could alleviate the renal injury. Collectively, the results suggested that targeting bacterial virulence might be a useful approach to combating bacterial infections, and turmeric oil could serve as a potential agent for combating A. hydrophila infections. Full article

The increasing prevalence of antibiotic-resistant bacteria has necessitated the exploration of alternative antimicrobial agents, particularly natural products like essential oils. This study investigated the antibacterial potential of a unique blend of four essential oils (EOB) across a gradient of concentrations (0.1 to 50%) against Gram-positive and Gram-negative bacteria using an adapted broth microdilution method, minimum inhibitory concentrations (MICs), and 24-h growth assays. The Gram-positive bacteria were Staphylococcus epidermidis and Bacillus subtilis, while the Gram-negative bacteria were Escherichia coli and Klebsiella aerogenes. The results demonstrated that the EOB exerted a concentration-dependent inhibitory effect on bacterial growth, with MICs determined at 25% for all the species tested. Growth curve analysis revealed that lower concentrations of the EOB (0.1 to 0.78%) allowed for normal bacterial proliferation, while at intermediate concentrations (1.56 to 3.13%), inconsistent trends in growth were exhibited. At higher concentrations (25 and 50%), the EOB effectively halted bacterial growth, as indicated by flat growth curves. The increase in the lag phase and the decrease in the growth rate at a sub-MIC concentration (12.5%) suggest a significant effect on bacterial adaptation and survival. Relative fitness analyses further highlighted the inhibitory effects of higher essential oil concentrations. S. epidermidis and E. coli had a significant (p < 0.05) reduction in fitness starting from the 6.25% concentration, while the other two species experienced a significant (p < 0.001) reduction in relative fitness from a concentration of 12.5%. These findings underscore the potential of this EOB as an effective antimicrobial agent, particularly in the context of rising antibiotic resistance. Furthermore, the study suggests that the EOB used in the present study could be integrated into therapeutic strategies as a natural alternative or adjunct to traditional antibiotics, offering a promising avenue for combating resistant bacterial strains. Full article

Background. Extended-spectrum β-lactamases (ESBLs) hydrolyze nearly all β-lactam antibiotics, affecting one of the most important groups of antimicrobials used in Gram-negative infections. Among them, CTX-M is the most widespread type of ESBL. This study aimed to evaluate the hydrolytic activity of CTX-M-type ESBLs following exposure to the antimicrobial peptide Melittin. Methods. Melittin was purified from Apis mellifera venom through ultrafiltration and characterized by SDS-PAGE. The minimum inhibitory concentration (MIC) of Melittin against ESBL-producing E. coli was determined by the broth microdilution method. The inhibition of ESBL’s hydrolytic activity following exposure to sub-MIC doses of Melittin was quantified using a kinetic assay based on hydrolyzed nitrocefin. Additionally, the effect of Melittin on the expression of the bla_CTX-M gene was evaluated via RT-PCR. Results. The peptide fraction of Apitoxin smaller than 10 kDa exhibited a protein band corresponding to Melittin, devoid of higher molecular weight proteins. The MIC of Melittin ranged from 50 to 80 µg/mL. Exposure to Melittin at sub-MIC doses significantly inhibited ESBL hydrolytic activity, reducing it by up to 67%. However, the transcription of the bla_CTX-M gene in the presence of Melittin revealed no significant changes. Conclusions. Melittin is able to inhibit ESBL’s hydrolytic activity but not bla_CTX-M transcription possibly indicating an effect at the translational or post-translational level. Full article

This study examines the impact of inhaled tobramycin therapy on the within-host changes in P. aeruginosa strains isolated from Bulgarian patients with CF prior to and post treatment. Genotypic comparison by RAPD-PCR indicated that most of the pre-treatment isolates had a high similarity and were genetically comparatively close to strains from other countries with known increased morbidity or treatment requirements. Most of the post-treatment isolates were, however, genetically distant from their pre-treatment counterparts, showing genotypic diversification after the treatment. Phenotypic comparisons showed a lower ODmax reached during groswth and an increased lag-time in the post-treatment isolates. All strains were capable of invasion and intracellular reproduction within A549 cultured cells. The addition of sub-inhibitory amounts (1/4 or 1/2 MIC) of tobramycin during growth showed the higher relative fitness (as a percentage of the untreated control) of the post-treatment strains. The effects of sub-MICs on biofilm growth did not show such a pronounced trend. However, when a resazurin-based viability test was applied, the advantage of the post-treatment strains was confirmed for both broth and biofilm cultures. In spite of that, according to the determined MIC values, all isolates were tobramycin-sensitive, and the data from this study imply the development of tolerance to the antibiotic in the strains that survived the treatment. Full article

Antimicrobial resistance is spreading rapidly throughout the world. The food chain can be one of the routes of transmission for microorganisms containing drug-resistance genes and thus serve as a channel for their transmission. Environmental stress and methods of preventing the spread of microorganisms trigger adaptive responses in bacterial cells. The aim of the present study was to determine the effect of the stress induced by sub-inhibitory concentrations (SICs) of cadmium chloride and benzalkonium chloride on antibiotic resistance and the expression of selected virulence factors in Staphylococcus isolates from food. The study was conducted on strains of the species S. epidermidis, S. heamolyticus, S. saprophyticus, and S. aureus. The values of the minimum inhibitory concentration against erythromycin, tetracycline, and oxacillin were determined before and after the incubation of the tested strains under stress conditions. The ability to form biofilm and slime production was also investigated. The expression levels of the genes responsible for antibiotic resistance (blaZ, tetK, tetM, ermB, and mecA) and virulence (eno) were conducted using Real-Time PCR. The MIC values of the antibiotics tested against the strains analyzed were found to be elevated in the presence of SICs of benzalkonium chloride and cadmium chloride. Furthermore, the intensity of biofilm production was also increased. SICs of benzalkonium chloride induced the expression of the tetM, tetK, mecA, and blaZ genes in 75%, 66.6%, 33.3%, and 40% of the isolates tested, respectively. Similar treatment with cadmium chloride induced the expression of the same genes in 75%, 100%, 66.6%, and 40% of the strains. In both cases, the expression of the ermB gene was reduced in 100% of the isolates. The eno gene was found to be overexpressed in 66.6% of the strains following benzalkonium chloride stress, and in 100% of the strains following cadmium chloride stress. These findings suggest that in Staphylococcus spp. strains, changes in the expression of the genes encoding antibiotic resistance and virulence factors may occur in response to the applied stress factors. The results indicate the possibility of selecting more resistant and virulent strains due to the use of too low concentrations of disinfectants, which emphasizes the need to use appropriate inhibitory doses of disinfectants in the food industry. Full article

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections of the urinary tract, upper respiratory tract, gastrointestinal tract, central nervous system, etc. It is possible to develop bacteremia and sepsis in immunocompromised patients. A major problem in treatment is the development of antibiotic resistance. Therefore, new preparations of natural origin are sought, such as plant extracts, which are phytocomplexes and to which it is practically impossible to develop resistance. Geum urbanum L. (Rosacea) is a perennial herb known for many biological properties. This study aimed to investigate the redox-modulating capacity and effect of ethyl acetate (EtOAc) extracts from roots (EtOAcR) and aerial parts (EtOAcAP) of the Bulgarian plant on the phenotype inhibition of the P. aeruginosa Las/RhI quorum sensing (QS) system, which primarily determines drug resistance in pathogenic bacteria, including biofilm formation, motility, and pigment production. We performed QS assays to account for the effects of the two EtOAc extracts. At sub-minimal inhibitory concentrations (sub-MICs) ranging from 1.56 to 6.25 mg/mL, the biofilm formation was inhibited 85% and 84% by EtOAcR and 62% and 39% by EtOAcAP extracts, respectively. At the same sub-MICs, the pyocyanin synthesis was inhibited by 17–27% after treatment with EtOAcAP and 26–30% with EtOAcR extracts. The motility was fully inhibited at 3.12 mg/mL and 6.25 mg/mL (sub-MICs). We investigated the inhibitory potential of lasI, lasR, rhiI, and rhiR gene expression in biofilm and pyocyanin probes with the PCR method. Interestingly, the genes were inhibited by two extracts at 3.12 mg/mL and 6.25 mg/mL. Antiradical studies, assessed by DPPH, CUPRAC, and ABTS radical scavenging methods and superoxide anion inhibition showed that EtOAcAP extract has effective antioxidant capacity. These results could help in the development of new phytocomplexes that could be applied as biocontrol agents to inhibit the phenotype of the P. aeruginosa QS system and other antibiotic-resistant pathogens. Full article

Background: The burden of prosthetic joint infection in combination with antibiotic-resistant bacterial strains is a rising dilemma for patients experiencing total joint replacements. Around 0.8–2% of patients experience prosthetic joint infections, while up to 21% of patients are considered fatal cases after 5 years. Staphylococcus aureus is one of the main reasons for prosthetic joint infections. Its capability of forming biofilms and developing mechanisms against antibiotics is one of the most dangerous clinical topics being currently discussed. Previous studies have shown the promising results of omega-3 fatty acids as an antimicrobial agent against Staphylococcus aureus. Though an antimicrobial effect has been examined, the influence of polyunsaturated fatty acids on Staphylococcus aureus in the presence of human osteoblasts has not been reported yet. In this study, we aimed to investigate the influence of omega-3 fatty acids on the biofilm formation of Staphylococcus aureus ATCC 29213 in the presence of hFOB 1.19 cells. The co-culture setup helped to examine the influence of omega-3 fatty acids on the race for surface to simulate prosthetic joint infections. Methods: In this study, we tested Staphylococcus aureus ATCC 29213 co-cultured with human fetal osteoblasts hFOB 1.19 in the presence of sub-MIC and MIC concentrations of docosahexaenoic acid (1.25 mg/L, 2.5 mg/L) and eicosapentaenoic acid (0.15 mg/L, 0.3 mg/L) after 1, 6 and 24 h of incubation. After establishing the co-culture, cell culture and biofilm, we performed colony-forming unit counting and cell counting to examine cell survivability. In addition, we carried out scanning electron microscopy to study the race for surface behaviour of the cells. Results: We found a protective influence of omega-3 fatty acids on osteoblasts when present in co-culture with Staphylococcus aureus after 6 h of incubation. Omega-3 fatty acids increase the cell survival of osteoblasts after 6 h in a co-culture with bacteria and are able to influence the race for surface. In this study, the strain of Staphylcoccus aureus ATCC 29213 showed signs of growth inhibition within the first 6 h. Conclusions: Omega-3 fatty acids can be a valuable antimicrobial agent in terms of decreasing the risk of on-site infection during surgery. Omega-3 fatty acids were shown to decrease the bacterial load within the first 6 h of incubation and increase the survivability of osteoblasts. Full article

Bacterial biofilms are closely associated with the rising threat of antimicrobial resistance, which is becoming a global concern. Recently, there has been increased interest in natural extracts as potential antimicrobial agents. One such extract is Dead Sea mud. While there is some evidence of its antimicrobial properties, it has not been extensively studied. Therefore, we designed a study to evaluate the potential of Dead Sea soil as an antimicrobial agent. For this purpose, three bacterial species (Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) were isolated from the ulcerated foot of a patient in a hospital in Tabuk. P. aeruginosa exhibited significant antibiotic resistance, particularly to Levofloxacin (90%) and Tobramycin (80%), while S. aureus showed 70% resistance to Levofloxacin but no vancomycin resistance. Biofilm activity varied among bacterial strains, with P. aeruginosa showing 30% strong biofilm production. MIC values indicated resistance levels, with P. aeruginosa strain PA8 having the highest MIC at 650 µL/mL. All strains showed significant differences in exopolysaccharide (EPS) production at 0.25 × MIC (p ≤ 0.05) and 0.5 × MIC (p ≤ 0.005). Similarly, alginate production was significantly reduced at 0.25 × MIC (p ≤ 0.05), with even greater inhibition at 0.5 × MIC for combinations such as EC7 + SA5 (p ≤ 0.001). Hydrophobicity significantly changed at 0.25 × MIC (p ≤ 0.05), and combinations revealed highly significant reductions at 0.5 × MIC (p ≤ 0.001). Additionally, significant differences in outer membrane disruption were observed (p ≤ 0.05) with greater effects at 0.5 × MIC (p ≤ 0.005). Swarming motility was notably reduced for SA5 at 0.25 × MIC (p ≤ 0.05) and for PA2 at 0.5 × MIC (p ≤ 0.001). Chitinase activity showed greater reductions at 0.5 × MIC, with EC7 exhibiting the highest decrease. Lastly, sub-MIC concentrations enhanced reactive oxygen species (ROS) production, particularly for strains PA2 and SA5. Our results demonstrate the excellent potential of Dead Sea soil extract as an antimicrobial compound. Future studies should incorporate in vivo models to validate these findings clinically. Full article

Background: MSSA and MRSA strains are challenging human pathogens that can develop resistance to antibiotics, highlighting the need for alternative antimicrobial agents. Plant metabolites, particularly volatile phytochemicals, may offer promising antimicrobial properties. The aim was to evaluate the antimicrobial and antibiofilm efficacy of various commercial volatile phytochemicals from the terpene and terpenoid groups against reference MSSA and MRSA strains, focusing on synergistic effects in both binary combinations and combinations with antibiotics. Methods: The microdilution method was used to determine the minimum inhibitory concentrations (MICs) for antibiotics and phytochemicals. The checkerboard method assessed synergistic interactions between phytochemicals and between phytochemicals and antibiotics, while the time-kill method was used to confirm these results. Biofilm quantification was performed using the microtiter plate method to evaluate the effects of phytochemicals, antibiotics, and their binary combinations on the eradication of 48-h-old biofilms. Results: Carvacrol and thymol demonstrated the strongest anti-staphylococcal activity, while other terpene compounds showed weaker effects. In binary combinations, carvacrol and thymol exhibited synergy against one MSSA strain (FICI = 0.50) and with tetracycline and chloramphenicol (FICI = 0.28–0.50). Synergy was also noted with streptomycin sulfate against one MRSA strain (FICI = 0.31–0.50) and with other antibiotics, including gentamicin (FICI = 0.25–0.50) and oxacillin (FICI = 0.44). Additionally, effective combinations achieved over 50% biofilm removal at both minimum inhibitory and sub-inhibitory concentrations. Conclusions: Results showed that synergy varies based on strain sensitivity to chemical agents, highlighting their potential for personalized therapy. Despite the difficulty in removing preformed biofilms, the findings highlight the importance of combined treatments to enhance antibiotic effectiveness. Full article