Sub-minimum Inhibitory Concentrations Research Articles

Effects of Thyme (Thymus vulgaris) Essential Oil on Bacterial Growth and Expression of Some Virulence Genes in Salmonella enterica Serovar Enteritidis.

The investigation on natural antimicrobial compounds against zoonotic pathogens has gained more attention due to the public health concerns regarding the emergence of antimicrobial resistance. The current study aimed to assess the effects of thyme essential oil at sub-minimal inhibitory concentrations (sub-MICs) on bacterial growth and expression of some virulence genes in Salmonella enteritidis. The bacterial growth rate and the expression of four virulence genes in S. enteritidis during 18-72h of exposure to the essential oil at 25%-75% MIC were evaluated via colony counting and real-time polymerase chain reaction (PCR), respectively. Sub-inhibitory concentrations of thyme essential oil significantly reduced the growth rate compared to the control. Expression of all tested virulence genes was also reduced by the essential oil in a significant dose- and time-dependent manner. As an example, decreased down-regulation of hilA, spv, sefA and invA as 1.7-, 4.14-, 2.92- and 1.04-fold in 25% MIC and 6.42-, 7.81-, 4.4- and 3.75-fold in 75% MIC was observed, respectively, after 24h of incubation. Likewise, levels of transcription for hilA, spv, sefA and invA were reduced 4.75-, 6.95-, 3.75- and 2.98-fold after 18h and 9.54-, 8.81-, 5.65- and 4.77-fold, respectively, after 72h in 75% MIC compared to the control. According to our data, aside from the growth inhibitory effect of thyme essential oil, the results of current study highlight the potential of thyme for reducing the transcriptional level of virulence genes and therefore the pathogenicity of S. enteritidis.

Subinhibitory concentrations of antibiotics affect development and parameters of Helicobacter pylori biofilm.

Helicobacter pylori causes chronic gastric diseases in nearly 50% of people around the world. It is suggested that biofilm formation has a pronounced effect on the dynamic resistance spread and recurrence of these infections. To mimic the scenario of therapeutic ineffectiveness, we investigated the impact of sub-minimal inhibitory concentrations (sub-MICs) of antibiotics on the development and parameters of biofilms produced by clinical H. pylori strains. We observed that constant exposure of planktonic forms to metronidazole or levofloxacin stimulated the speed of autoaggregation and the amount of extracellular matrix, resulting in increased dimensions of the developed biofilms. Contrary to this, continuous exposure to clarithromycin negatively affected a number of biofilm-related reactions and led to the biofilm-weakening effect. Through assessing the membrane fatty acid profiles of antibiotic-exposed cells, we confirmed that metronidazole and levofloxacin induced a biofilm-like phenotype, while clarithromycin kept bacteria in a planktonic form. Our results suggest that sub-MICs of antibiotics affect the biochemical and biophysical properties of the developing biofilm of H. pylori strains and may impact the effectiveness of antibiotic treatment.

Bactericidal and anti-quorum sensing activity of repurposing drug visomitin against staphylococcus aureus

ABSTRACT With the growing antibiotic resistance in Staphylococcus aureus, it is imperative to develop innovative therapeutic strategies against new targets to reduce selective survival pressures and incidence of resistance. In S. aureus, interbacterial communication relies on a quorum sensing system that regulates gene expression and physiological activities. Here, we identified that Visomitin, an antioxidant small molecule, exhibited bactericidal efficacy against methicillin-resistant S. aureus and its high tolerance phenotypes like intracellular bacteria and persister cells without inducing resistance. Critically, sub-minimal inhibitory concentrations (sub-MICs) of Visomitin could serve as a potent quorum-quencher reducing virulence production (such as haemolysin and staphyloxanthin), along with inhibiting biofilm formation, self-aggregation, and colony spreading of S. aureus. These effects were probably mediated by interfering with the S. aureus accessory gene regulator quorum sensing system. In summary, our findings suggest that Visomitin shows dual antimicrobial effects, including bactericidal effects at the concentrations above MIC and quorum sensing inhibition effects at sub-MICs, which holds promise for treating MRSA-related refractory infections.

Purification Of Bacteriocin Produced By Staphylococcus Aureus And Its Effect On Some Candida Spp.

Candida is the most implicated fungal pathogen in the clinical setting. Virulence factors, such as biofilms play important roles in its pathogenesis. Hence, this study aimed to investigate role of staphylococcal bacteriocin in inhibit biofilm and viability of Candida spp. Seventy-two isolates were collected from abscess, wounds and burns . All isolates were reidentified using different examinations. In addition, eight species of previously isolated clinical Candida species, from oral and vaginal cavities of patients with candidiasis, including C. albicans, C. glabrata, C. parasilosis, C. krusei, C. lusitaniae, C. kefyr, C. tropicalis and C. ciferrii, were utilized to check the antimicrobial activity of S. aureus on Candida spp. Agar well diffusion method were used to detect bacteriocin production by S. aureus, whereas only 5 isolates were selected. The antibacterial effect of selected S. aureus isolates were tested against indicators (Pseudomonas aeruginosa, Escherichia coli and Candida albicans). Isolate of S. aureus no.2 was showed the best bacteriocin production. Bacteriocin was purified using ammonium sulfate and dialysis. Different concentrations was utilized to determine sub-minimal inhibitory concentration (sub-MIC) of bacteriocin. Sub-MIC of bacteriocin was utilized to estimate its inhibitory effect on viability of Candida spp. Bacteriocin showed the maximal inhibition percentage against C. parapsilosis CV26 with 85.42%, followed by C. tropicalis CO49 with 80.49%. The viability of C. kefyr CO45, C. krusei CO41, C. glabrata CO38, C. albicans CV5 and C. lusitaniae was inhibited by 77.78, 75.41, 74.67, 71.43 and 70.18%, respectively. In addition, 69.84 and 64.62% of C. albicans CV1 and C. ciferrii. Using microtiter plate, sub-MIC of bacteriocin showed maximal inhibitory effect against C. ciferrii with 82.26%, followed by C. lusitaniae with 80.4%, the biofilm formation of C. parapsilosis CV26 and C. tropicalis CO49 was inhibited by 80.49% and 79.82%, respectively.

Desiccation tolerance and reduced antibiotic resistance: Key drivers in ST239-III to ST22-IV MRSA clonal replacement at a Malaysian teaching hospital

Molecular surveillance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from Hospital Canselor Tuanku Muhriz (HCTM), a Malaysian teaching hospital revealed clonal replacement events of SCCmec type III-SCCmercury to SCCmec type IV strains before the year 2017; however, the reasons behind this phenomenon are still unclear. This study aimed to identify factors associated with the clonal replacement using genomic sequencing and phenotypic investigations (antibiogram profiling, growth rate and desiccation tolerance determination, survival in vancomycin sub-minimum inhibitory concentration (MIC) determination) of representative HCTM MRSA strains isolated in four-year intervals from 2005 – 2017 (n = 16). HCTM Antimicrobial Stewardship (AMS) and Infection Prevention and Control (IPC) policies were also reviewed. Phylogenetic analyses revealed the presence of 3 major MRSA lineages: ST239-III, ST22-IV and ST6-IV; MRSAs with the same STs shared similar core and accessory genomes. Majority of the ST239-III strains isolated in earlier years of the surveillance (2005, 2009 and 2013) were resistant to many antibiotics and harboured multiple AMR and virulence genes compared to ST22-IV and ST6-IV strains (isolated in 2013 and 2017). Interestingly, ST22-IV and ST6-IV MRSAs grew significantly faster and were more resistant to desiccation than ST239-III (p < 0.05), even though the later clone survived better post-vancomycin exposure. Intriguingly, ST22-IV was outcompeted by ST239-III in broth co-cultures; though it survived better when desiccated together with ST239-III. Higher desiccation tolerance and fewer carriage of AMR genes by ST22-IV, together with reduction of antibiotic selection pressure in HCTM (due to AMS and IPC policies) during 2005 – 2017 may have provided the clone a competitive edge in replacing the previously dominant ST239-III in HCTM. This study highlights the importance of MRSA surveillance for a clearer picture of circulating clones and clonal changes. To our knowledge, this is the first genomic epidemiology study of MRSA in Malaysia, which will serve as baseline genomic data for future surveillance.

Evaluation of Anti-biofilm Activity of Silica nanoparticles against dental Streptococcus mutans: In-Vitro Study

Background: Dental caries is a prevalent chronic infection caused by cariogenic bacteria that cling to teeth, mainly Streptococcus mutans. These bacteria break down sugars into acid over time, demineralizing the tooth structure. Biofilm as an early colonizer and is the most important bacterium in the formation of dental caries . Materials and Methods: The present study concentrates on isolation and identification of the S.mutans pathogen from patients suffering from dental caries. On specific media, the isolates were cultivated. From human teeth, one hundred dental caries samples are gathered. Using MS-agar (Mitis Salivarius agar), only fifty samples are regarded as positive bacterial isolates to identify the S.mutans isolates, their morphological, cultural, biochemical, and VITEK features were examined. . The microtiter plate method was used to detect the development of biofilms by comparing the characteristics of the isolates. In this work, the median values of optical density (OD) at 630 nm were utilised to employ ELISA to distinguish between S. mutans isolates that developed biofilms and those that did not. To confirm the type of bacteria using molecular identification. Objectives: The purpose of this work is to determine the subminimum and minimum inhibitory concentrations of silica nanoparticles that function as anti-biofilm agents, as they have an impact on bacterial growth and details of silica nanoparticle characterization. Results: In this study considered to be S.mutans are twenty isolates the morphological, cultural, biochemical, and VITEK properties of the bacterial isolates were used to identify them . in this study molecular identification to more confirm of S.mutans the result of PCR showed that 20/20(100%) isolates were PCR positive for 16r RNA gene . in this investigation different isolates were evaluated for their capacity to create biofilm; nine (45%) were judged to be strong producers, seven (35%), to be moderate producers, and four (20%) to be weak producers. the sol-gel method was used in this study to synthesis silica nanoparticles, which were then characterised by XRD, FE-SEM, EDX, and TEM. In this study, Si-NPs were tested against isolates of S.mutans by using a resazurin mediated microtiter plate the results have shown that silica nanoparticles has inhibitory action (MIC) against S. mutans with concentration 0,78 mg/ml and sub-MIC with concentration 0.39 mg/ml. .The anti-biofilm effect of silica nanoparticles on isolates of S. mutans in the current investigation revealed that the biofilm mean of control (biofilm development without silica nanoparticles) was 0.583, whereas the findings for anti-biofilm utilizing sub-MIC of NPs, was 0.185 in microtiter plate method when the (p value) less than 0.05 this proves the NPs effected on the biofilm formation by S.murans and inhibition it . Conclusion: Silica nanoparticles' anti-biofilm action on S.mutans isolates demonstrated the importance of biofilm inhibition.

Enhancing antimicrobial efficacy against Pseudomonas aeruginosa biofilm through carbon dot-mediated photodynamic inactivation

Pseudomonas aeruginosa biofilms shield the bacteria from antibiotics and the body’s defenses, often leading to chronic infections that are challenging to treat. This study aimed to assess the impact of sub-lethal doses of antimicrobial photodynamic inactivation (sAPDI) utilizing carbon dots (CDs) derived from gentamicin and imipenem on biofilm formation and the expression of genes (pelA and pslA) associated with P. aeruginosa biofilm formation.The anti-biofilm effects of sAPDI were evaluated by exposing P. aeruginosa to sub-minimum biofilm inhibitory concentrations (sub-MBIC) of CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN, combined with sub-lethal UVA light irradiation. Biofilm formation ability was assessed by crystal violet (CV) assay and enumeration method. Additionally, the impact of sAPDI on the expression of pelF and pslA genes was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR).Compared to the control group, the sAPDI treatment with CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN resulted in a significant reduction in biofilm activity of P. aeruginosa ATCC 27853 (P < 0.0001). The CV assay method demonstrated reductions in optical density of 83.70%, 81.08%, 89.33%, and 75.71%, while the CFU counting method showed reductions of 4.03, 3.76, 4.39, and 3.21 Log10 CFU/mL. qRT-PCR analysis revealed decreased expression of the pelA and pslA genes in P. aeruginosa ATCC 27853 following sAPDI treatment compared to the control group (P < 0.05).The results indicate that sAPDI using CDs derived from gentamicin and imipenem can decrease the biofilm formation of P. aeruginosa and the expression of the pelA and pslA genes associated with its biofilm formation.

Antimicrobial Sub-MIC induces Staphylococcus aureus biofilm formation without affecting the bacterial count

BackgroundBiofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication.ObjectiveThis study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques.MethodsIn this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM).ResultsBiofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively.ConclusionExposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count.

Synergetic inhibitory effect of isopropyl methylphenol-based agents on biofilm formation by Streptococcus mutans.

Dental caries and periodontitis are the most common oral diseases in humans and the main causes of tooth loss. Streptococcus mutans is primarily responsible for dental caries and dental plaque, which are triggered by biofilm formation on the tooth surface. In this study, biofilm inhibition by 4-isopropyl-3-methylphenol (IPMP)-based agents, consisting of IPMP and polyoxyethylene-hydrogenated castor oil (POEHCO), was investigated in vitro. Notably, the use of POEHCO in addition to IPMP inhibited S. mutans biofilms more drastically than IPMP alone. Moreover, the effects of IPMP on the expression of biofilm-related genes (gtfB, gtfC, and gtfD) were examined using quantitative real-time PCR. IPMP at sub-minimum inhibitory concentrations significantly downregulated the expression of these genes. These results suggested that the inhibitory effects on biofilm formation were synergistically enhanced by the surfactant and antibiofilm activities of IPMP. Therefore, IPMP-based agents as dentifrices may be useful to prevent oral diseases originating from biofilms.

Modulatory effects of sub-MIC concentrations silver nanoparticles on virulence factor gene expression in Staphylococcus aureus

BackgroundSilver nanoparticles (AgNPs) exhibit a dose-dependent anti-bacterial effect, and it was aimed in this study to investigate the impact of sub-minimum inhibitory concentration (MIC) doses of AgNPs on the expression of virulence genes in Staphylococcus aureus (S. aureus). MethodsMinimum inhibitory concentration (MIC) values for AgNPs were determined for 183 S. aureus isolates. Gene expression was assessed in 14 isolates with sea and seb genes treated with AgNPs at a sub-MIC dose of 1 μg/ml. Accordingly, these strains were exposed to 1 μg/ml doses of AgNPs, and gene expression levels of sea, seb, and agr were assessed using quantitative RT-PCR after 4- and 12-hour post-AgNPs inoculation at 37 °C. The impact of AgNPs on the virulence factors of S. aureus was investigated over different time points, focusing on methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates. ResultsAnalysis revealed significant reductions in gene expression levels of seb and agr after 4 h post-AgNPs treatment in the MSSA group (p < 0.05), with further decreases observed at 12 h for sea, seb, and agr genes (p < 0.0001). MRSA isolates exhibited significant declines in sea and agr gene expression levels at both time points (p < 0.0001). However, no significant changes were observed in seb gene expression among MRSA isolates. Fold-change analysis indicated time-dependent effects of AgNP treatment on gene expression, highlighting substantial alterations in gene expression levels over time, particularly in seb and agr genes. ConclusionThese results show that sub-MIC levels of AgNPs greatly decrease the gene expression of important virulence factors in MSSA and MRSA strains, indicating their promise as treatments for S. aureus infections, particularly at 12 h post-treatment. The differential response between MSSA and MRSA isolates highlights the importance of strain variation in antimicrobial strategies.

Actinomycin D reduces virulence factors and biofilms against Aeromonas hydrophila.

Aeromonas hydrophila, a Gram-negative bacterium, is ubiquitously found in many aquatic habitats, causing septicemia in humans and fishes. Attributed to abuse or misuse of conventional antimicrobial drug usage, antimicrobial resistance is at an alarming rise. There is an available alternative strategy to bacterial resistance to antimicrobials, which is inhibition of virulence and pathogenicity employing quorum sensing inhibitors (QSIs). Hence, actinomycin D's effectiveness against A. hydrophila SHAe 115 as a QSI was investigated in decreasing virulence factors and preventing biofilm formation. Actinomycin D, belongs to the QSI combating Pseudomonas aeruginosa PAO1 originally isolated from an entophytic actinomycete (Streptomyces cyaneochromogenes RC1) in Areca catechu L. In the present work, further investigations were carried out to assess the effect of actinomycin D at subminimal inhibitory concentrations (sub-MICs), QS-regulated virulence factors, and biofilm inhibition strategies. Intrinsic properties encompassing inhibition of the production of protease and hemolysin and subsequent activities on biofilm formation and eradication of mature biofilm were established along with weakened swimming and swarming motilities in A. hydrophila SHAe 115. In the Tenebrio molitor survival assay, actinomycin D effectively reduced the virulence and pathogenicity of A. hydrophila, resulting in elimination of mortality. However, the hydrolysate of actinomycin D, 2-hydroxy-4,6-dimethyl-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid (HDPD), had lost the QSI activity in A. hydrophila. Actinomycin D was proved as a viable QSI in lessening A. hydrophila's the virulence and pathogenicity, as evident from our research findings.

Post-Antibiotic and Post-Antibiotic Sub-Minimum Inhibitory Concentration Effects of Carvacrol against Salmonella Typhimurium.

Carvacrol is a compound present in essential oils with proven antimicrobial activity against numerous pathogens. We firstly determine the post-antibiotic effect (PAE) of carvacrol (1×, 2×, 4× MIC) and post-antibiotic sub-minimum inhibitory concentration (MIC) effect (1× + 0.25× MIC and 2× + 0.25× MIC) for two concentrations of Salmonella Typhimurium ATCC14028 (106 and 108 CFU/mL). Prior to testing, the minimum concentration and exposure time to achieve the bacterial inhibition (MIC 0.6 mg/mL and 10 min) were determined by broth microdilution and time-kill curve methods, respectively. At the MIC, carvacrol did not generate any PAE. At twice the MIC, the PAE was 2 h with the standard inoculum (106 CFU/mL) and 1 h with the high-density inoculum (108 CFU/mL). At 4× MIC concentrations, the PAE was higher in both cases > 43.5 h. Continuous exposure of post-antibiotic phase bacteria (1× and 2× MIC) to carvacrol at 0.25× MIC (0.15 mg/mL) resulted in an increase in PAE (PA-SME) above 43.5 h with both inocula. These results suggest that the PA-SME of carvacrol for S. Typhimurium can be significantly prolonged by increasing the sub-MICs, which would allow dose spacing, reduce adverse effects and improve its efficacy in the treatment of infected animals and as a disinfectant in agri-food facilities.

Effect of subinhibitory concentrations on the spreading of the ampicillin resistance gene blaCMY-2 in an activated sludge microcosm

ABSTRACT As the problem of multi-resistant bacteria grows a better understanding of the spread of antibiotic resistance genes is of utmost importance for society. Wastewater treatment plants contain subinhibitory concentrations of antibiotics and are thought to be hotspots for antibiotic resistance gene propagation. Here we evaluate the influence of sub-minimum inhibitory concentrations of antibiotics on the spread of resistance genes within the bacterial community in activated sludge laboratory-scale sequencing batch reactors. The mixed communities were fed two different ampicillin concentrations (500 and 5000 µg/L) and the reactors were run and monitored for 30 days. During the experiment the β-lactamase resistance gene bla CMY-2 was monitored via qPCR and DNA samples were taken to monitor the effect of ampicillin on the microbial community. The relative copy number of bla CMY-2 in the reactor fed with the sub-minimum inhibitory concentration of 500 µg/L ampicillin was spread out over a wider range of values than the control and 5000 µg/L ampicillin reactors indicating more variability of gene number in the 500 µg/L reactor. This result emphasises the problem of sub-minimum inhibitory concentrations of antibiotics in wastewater. High-throughput sequencing showed that continuous exposure to ampicillin caused a shift from a Bacteroidetes to Proteobacteria in the bacterial community. The combined use of qPCR and high-throughput sequencing showed that ampicillin stimulates the spread of resistance genes and leads to the propagation of microbial populations which are resistant to it.

Sublethal Sodium Hypochlorite Exposure: Impact on Resistance-Nodulation-Cell Division Efflux Pump Overexpression and Cross-Resistance to Imipenem.

Sodium hypochlorite (NaOCl) is widely used in public healthcare facilities; this exposure can result in the development of bacterial tolerance to disinfectants, which has known links to antibiotic cross-resistance. However, the mechanism through which cross-resistance to antibiotics and disinfectants develops remains ambiguous. Therefore, this study aimed to examine the phenotypic and transcriptomic changes caused by disinfectant exposure in Gram-negative bacteria and determine the cause of cross-resistance to antibiotics. The results demonstrated that the misuse of disinfectants plays an important role in the emergence of disinfectant resistance and in the increase in antibiotic resistance. Antibiotic resistance may occur from the exposure of Gram-negative bacteria to subminimal inhibitory concentrations (MICs) of NaOCl. Ten passages of Gram-negative bacteria in increasingly higher subMICs of the NaOCl disinfectant were sufficient to increase the MIC to >2500 µg/mL NaOCl, particularly in K. pneumoniae and P. aeruginosa. To determine the development of cross-resistance to antibiotics due to NaOCl exposure, the MICs for each antibiotic before and after the exposure of each strain to sublethal concentrations of NaOCl were compared. After overnight incubation with a sublethal concentration of NaOCl, a statistically significant increase in MIC was only observed for imipenem (p < 0.01). An investigation of the mechanism of cross-resistance by means of transcriptome analysis revealed that 1250 µg/mL of NaOCl-adapted K. pneumoniae and P. aeruginosa strains increased resistance to imipenem due to the increased expression of resistance-nodulation-cell division (RND) efflux pumps, such as AcrAB-TolC and MexAB/XY-OprM. Therefore, we suggest that exposure to NaOCl can influence the expression of RND efflux pump genes, contributing to imipenem cross-resistance.

Host and antibiotic jointly select for greater virulence in Staphylococcus aureus.

Widespread antibiotic usage has resulted in the rapid evolution of drug-resistant bacterial pathogens and poses significant threats to public health. Resolving how pathogens respond to antibiotics under different contexts is critical for understanding disease emergence and evolution going forward. The impact of antibiotics has been demonstrated most directly through in vitro pathogen passaging experiments. Independent from antibiotic selection, interactions with hosts have also altered the evolutionary trajectories and fitness landscapes of pathogens, shaping infectious disease outcomes. However, it is unclear how interactions between hosts and antibiotics impact the evolution of pathogen virulence. Here, we evolved and re-sequenced Staphylococcus aureus , a major bacterial pathogen, varying exposure to host and antibiotics to tease apart the contributions of these selective pressures on pathogen adaptation. After 12 passages, S. aureus evolving in Caenorhabditis elegans nematodes exposed to a sub-minimum inhibitory concentration of antibiotic (oxacillin) became highly virulent, regardless of whether the ancestral pathogen was methicillin-resistant (MRSA) or methicillin-sensitive (MSSA). Host and antibiotic exposure selected for reduced drug susceptibility in MSSA lineages while increasing MRSA total growth outside hosts. We identified mutations in genes involved in complex regulatory networks linking virulence and metabolism, including codY , agr , and gdpP , suggesting that rapid adaptation to infect hosts may have pleiotropic effects. In particular, MSSA populations under selection from host and antibiotic accumulated mutations in the global regulator gene codY , which controls biofilm formation in S. aureus. These populations had indeed evolved more robust biofilms-a trait linked to both virulence and antibiotic resistance-suggesting evolution of one trait can confer multiple adaptive benefits. Despite evolving in similar environments, MRSA and MSSA populations proceeded on divergent evolutionary paths, with MSSA populations exhibiting more similarities across replicate populations. Our results underscore the importance of considering multiple and concurrent selective pressures as drivers of pervasive pathogen traits.

Proven anti-virulence therapies in combating methicillin- and vancomycin-resistant Staphylococcus aureus infections.

Despite years of efforts to develop new antibiotics for eradicating multidrug-resistant (MDR) and multi-virulent Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Staphylococcus aureus (VRSA) infections, treatment failures and poor prognoses in most cases have been common. Therefore, there is an urgent need for new therapeutic approaches targeting virulence arrays. Our aim is to discover new anti-virulence therapies targeting MRSA and VRSA virulence arrays. We employed phenotypic, molecular docking, and genetic studies to screen for anti-virulence activities among selected promising compounds: Coumarin, Simvastatin, and Ibuprofen. We found that nearly all detected MRSA and VRSA strains exhibited MDR and multi-virulent profiles. The molecular docking results aligned with the phenotypic and genetic assessments of virulence production. Biofilm and hemolysin productions were inhibited, and all virulence genes were downregulated upon treatment with sub-minimum inhibitory concentration (sub-MIC) of these promising compounds. Ibuprofen was the most active compound, exhibiting the highest inhibition and downregulation of virulence gene products. Moreover, in vivo and histopathological studies confirmed these results. Interestingly, we observed a significant decrease in wound area and improvements in re-epithelialization and tissue organization in the Ibuprofen and antimicrobial treated group compared with the group treated with antimicrobial alone. These findings support the idea that a combination of Ibuprofen and antimicrobial drugs may offer a promising new therapy for MRSA and VRSA infections. We hope that our findings can be implemented in clinical practice to assist physicians in making the most suitable treatment decisions.

Identification of gene targets that potentiate the action of rifampicin on Mycobacterium bovis BCG.

Tuberculosis (TB) caused by bacteria of the Mycobacterium tuberculosis complex remains one of the most important infectious diseases of mankind. Rifampicin is a first line drug used in multi-drug treatment of TB, however, the necessary duration of treatment with these drugs is long and development of resistance is an increasing impediment to treatment programmes. As a result, there is a requirement for research and development of new TB drugs, which can form the basis of new drug combinations, either due to their own anti-mycobacterial activity or by augmenting the activity of existing drugs such as rifampicin. This study describes a TnSeq analysis to identify mutants with enhanced sensitivity to sub-minimum inhibitory concentrations (MIC) of rifampicin. The rifampicin-sensitive mutants were disrupted in genes of a variety of functions and the majority fitted into three thematic groups: firstly, genes that were involved in DNA/RNA metabolism, secondly, genes involved in sensing and regulating mycobacterial cellular systems, and thirdly, genes involved in the synthesis and maintenance of the cell wall. Selection at two concentrations of rifampicin (1/250 and 1/62 MIC) demonstrated a dose response for mutants with statistically significant sensitivity to rifampicin. The dataset reveals mechanisms of how mycobacteria are innately tolerant to and initiate an adaptive response to rifampicin; providing putative targets for the development of adjunctive therapies that potentiate the action of rifampicin.

Research Note: Curbing Salmonella Enteritidis in broiler chickens with palm-free medium-chain fatty acids

Salmonellosis is still one of the most reported zoonoses worldwide and poultry meat is a major source, as chickens are often persistent carriers of Salmonella. Medium-chain fatty acids (MCFA) are known for their strong antimicrobial activity. MCFAs used today in the animal feed industry, however, mainly originate from the palm oil industry, which is notorious for its negative impact on the climate. We investigated the effect of a specific blend of palm-free MCFAs (ranging from C6 to C9) on Salmonella Enteritidis (SE) colonization in broiler chickens and in vitro SE characteristics. Fifty Ross 308 broiler chickens were randomly divided in 2 treatment groups. Chickens received either un-supplemented feed or feed supplemented with 300 ppm MCFAs from D0 onwards. On D7, all chickens were orally inoculated with 1600 CFU of SE. Cloacal swabs (D11) and samples of liver and caeca (D12) of all animals were collected and SE was enumerated. Percentage of SE-positive caecum samples was significantly (P = 0.044) reduced in birds receiving MCFAs compared to those receiving unsupplemented feed (36% vs. 64%). In vitro work performed with the same SE strain showed that preincubating the Salmonella bacteria with MCFAs at a sub-minimal inhibitory concentration significantly (p < 0.05) reduced bacterial adhesion to and invasion in Caco-2 cells, which may explain the observed reduction in intestinal SE colonization in the in vivo trial. Together, these results show that the tested eco-friendly MCFA blend could be a promising tool in the control of Salmonella in broilers.

Hla protein expression and artesunate prevented mice from further damage caused by Staphylococcus aureus pneumonia

The laboratory standard MRSA strain WHO-2 and clinical isolate S1 were used to establish a pneumonia infection model. The results showed that methicillin increased the expression of Hla and PVL protein at subminimum inhibitory concentration, while artesunate decreased the secretion of Hla and PVL protein. Artesunate alone reduced hemolysin expression and reversed methicillin-induced increases in Hla and PVL proteins. In addition, the study found that the combination of artesunate and methicillin had the best therapeutic effect, with survival rates of 70 % and 40 % at seven days, respectively (corresponding to the WHO-2 and S1 strains). The combination treatment was able to reduce cell mortality, showing a 65 % and 46 % reduction in cell mortality, respectively. The study also found that the combination therapy decreased the expression of alpha-hemolysin and pantone valentin leukin in the culture medium and significantly reduced the activation of NF-kB. This is caused by a significant decrease in the expression of inflammatory factors.

Dhvar5-chitosan nanogels and their potential to improve antibiotics activity

Infection is one of the main causes of orthopedic implants failure, with antibiotic-resistant bacteria playing a crucial role in this outcome. In this work, antimicrobial nanogels were developed to be applied in situ as implant coating to prevent orthopedic-device-related infections. To that regard, a broad-spectrum antimicrobial peptide, Dhvar5, was grafted onto chitosan via thiol–norbornene “photoclick” chemistry. Dhvar5-chitosan nanogels (Dhvar5-NG) were then produced using a microfluidic system. Dhvar5-NG (1010 nanogels (NG)/mL) with a Dhvar5 concentration of 6 μg/mL reduced the burden of the most critical bacteria in orthopedic infections - methicillin-resistant Staphylococcus aureus (MRSA) – after 24 h in medium supplemented with human plasma proteins. Transmission electron microscopy showed that Dhvar5-NG killed bacteria by membrane disruption and cytoplasm release. No signs of cytotoxicity against a pre-osteoblast cell line were verified upon incubation with Dhvar5-NG. To further explore therapeutic alternatives, the potential synergistic effect of Dhvar5-NG with antibiotics was evaluated against MRSA. Dhvar5-NG at a sub-minimal inhibitory concentration (109 NG/mL) demonstrated synergistic effect with oxacillin (4-fold reduction: from 2 to 0.5 μg/mL) and piperacillin (2-fold reduction: from 2 to 1 μg/mL). This work supports the use of Dhvar5-NG as adjuvant of antibiotics to the prevention of orthopedic devices-related infections.