Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

A Comprehensive Guide to Quality Assessment and Data Submission for Genomic Surveillance of Enteric Pathogens.

This document outlines the steps necessary to assemble and submit the standard data package required for contributing to the global genomic surveillance of enteric pathogens. Although targeted to GenomeTrakr laboratories and collaborators, these protocols are broadly applicable for enteric pathogens collected for different purposes. There are five protocols included in this chapter: (1) quality control (QC) assessment for the genome sequence data, (2) validation for the contextual data, (3) data submission for the standard pathogen package or Pathogen Data Object Model (DOM) to the public repository, (4) viewing and querying data at NCBI, and (5) data curation for maintaining relevance of public data. The data are available through one of the International Nucleotide Sequence Database Consortium (INSDC) members, with the National Center for Biotechnology Information (NCBI) being the primary focus of this document. NCBI Pathogen Detection is a custom dashboard at NCBI that provides easy access to pathogen data plus results for a standard suite of automated cluster and genotyping analyses important for informing public health and regulatory decision-making.

Identifying Putative Biomarkers of Foodborne Pathogens Using a Metabolomic Approach.

Metabolomics is the study of low molecular weight biochemical molecules (typically <1500 Da) in a defined biological organism or system. In case of food systems, the term "food metabolomics" is often used. Food metabolomics has been widely explored and applied in various fields including food analysis, food intake, food traceability, and food safety. Food safety applications focusing on the identification of pathogen-specific biomarkers have been promising. This chapter describes a nontargeted metabolite profiling workflow using gas chromatography coupled with mass spectrometry (GC-MS) for characterizing three globally important foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica, from selective enrichment liquid culture media. The workflow involves a detailed description of food spiking experiments followed by procedures for the extraction of polar metabolites from media, the analysis of the extracts using GC-MS, and finally chemometric data analysis using univariate and multivariate statistical tools to identify potential pathogen-specific biomarkers.

An electrochemical sensor based on 2D Zn-MOFs and 2D C-Ti3C2Tx composite materials for rapid and direct detection of various foodborne pathogens.

Rapid screening for foodborne pathogens is crucial for food safety. A rapid and one-step electrochemical sensor has been developed for the detection of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhimurium). Through the construction of aptamer/two-dimensional carboxylated Ti3C2Tx (2D C-Ti3C2Tx)/two-dimensional Zn-MOF (2D Zn-MOF) composites, the recognition elements, signal tags, and signal amplifiers are integrated on the electrode surface. Pathogens are selectively captured using the aptamer, which increases the impedance of the electrode surface，leads to a decrease in the 2D Zn-MOF current. Bacteria can be rapidly quantified using a one-step detection method and the replacement of aptamers. The detection limits for E. coli, S. aureus, and S. typhimurium are 6, 5, and 5 CFU·mL-1, respectively. The sensor demonstrated reliable detection capabilities in real-sample testing. Therefore, the one-step sensor based on the 2D Zn-MOF and 2D C-Ti3C2Tx has significant application value in the detection of foodborne pathogens.

Zoonotic potential of urban wildlife faeces, assessed through metabarcoding.

Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species.

Metabolic homeostasis in fungal infections from the perspective of pathogens, immune cells, and whole-body systems.

SUMMARYThe ability to overcome metabolic stress is a major determinant of outcomes during infections. Pathogens face nutrient and oxygen deprivation in host niches and during their encounter with immune cells. Immune cells require metabolic adaptations for producing antimicrobial compounds and mounting antifungal inflammation. Infection also triggers systemic changes in organ metabolism and energy expenditure that range from an enhanced metabolism to produce energy for a robust immune response to reduced metabolism as infection progresses, which coincides with immune and organ dysfunction. Competition for energy and nutrients between hosts and pathogens means that successful survival and recovery from an infection require a balance between elimination of the pathogen by the immune systems (resistance), and doing so with minimal damage to host tissues and organs (tolerance). Here, we discuss our current knowledge of pathogen, immune cell and systemic metabolism in fungal infections, and the impact of metabolic disorders, such as obesity and diabetes. We put forward the idea that, while our knowledge of the use of metabolic regulation for fungal proliferation and antifungal immune responses (i.e., resistance) has been growing over the years, we also need to study the metabolic mechanisms that control tolerance of fungal pathogens. A comprehensive understanding of how to balance resistance and tolerance by metabolic interventions may provide insights into therapeutic strategies that could be used adjunctly with antifungal drugs to improve patient outcomes.

Deciphering Root-associated Microbial Communities in Asymptomatic Oil Palm Seedlings Exposed to Ganoderma boninense: New Insight into Disease Tolerance of Oil Palms.

Understanding the microbial communities in asymptomatic oil palm seedlings is crucial for developing disease-suppressive microbiota against basal stem rot (BSR) in oil palm. In this study, we compared the microbial communities in bulk soil, rhizosphere, and endosphere of control, asymptomatic, and symptomatic seedlings following inoculation with Ganoderma boninense. Our findings revealed significant shifts in microbial structure and interactions, particularly in asymptomatic seedlings. Both Actinobacteriota and Ascomycota were notably enriched in these samples, with Actinobacteriota identified as keystone taxa. Long-read shotgun metagenomics demonstrated that 67.4% of enriched Actinobacteriota taxa were unique to asymptomatic seedlings. Similarly, Ascomycota members showed significant enrichment, suggesting their potential role in BSR suppression. The consistent identification of these phyla across various analyses underscores their importance in disease resistance. This is the first report detailing the shifts in prokaryotic and fungal communities in asymptomatic and symptomatic seedlings, offering insights into potential disease-suppressive taxa across three compartments: bulk soil, rhizosphere, and endosphere of oil palm seedlings.

Lyme Disease Under-Ascertainment During the COVID-19 Pandemic in the United States: Retrospective Study.

Background: The COVID-19 pandemic resulted in a massive disruption in access to care and thus passive, hospital- and clinic-based surveillance programs. In 2020, the reported cases of Lyme disease were the lowest both across the United States and North Carolina in recent years. During this period, human contact patterns began to shift with higher rates of greenspace utilization and outdoor activities, putting more people into contact with potential vectors and associated vector-borne diseases. Lyme disease reporting relies on passive surveillance systems, which were likely disrupted by changes in health care-seeking behavior during the pandemic. Objective: This study aimed to quantify the likely under-ascertainment of cases of Lyme disease during the COVID-19 pandemic in the United States and North Carolina. Methods: We fitted publicly available, reported Lyme disease cases for both the United States and North Carolina prior to the year 2020 to predict the number of anticipated Lyme disease cases in the absence of the pandemic using a Bayesian modeling approach. We then compared the ratio of reported cases divided by the predicted cases to quantify the number of likely under-ascertained cases. We then fitted geospatial models to further quantify the spatial distribution of the likely under-ascertained cases and characterize spatial dynamics at local scales. Results: Reported cases of Lyme Disease were lower in 2020 in both the United States and North Carolina than prior years. Our findings suggest that roughly 14,200 cases may have gone undetected given historical trends prior to the pandemic. Furthermore, we estimate that only 40% to 80% of Lyme diseases cases were detected in North Carolina between August 2020 and February 2021, the peak months of the COVID-19 pandemic in both the United States and North Carolina, with prior ascertainment rates returning to normal levels after this period. Our models suggest both strong temporal effects with higher numbers of cases reported in the summer months as well as strong geographic effects. Conclusions: Ascertainment rates of Lyme disease were highly variable during the pandemic period both at national and subnational scales. Our findings suggest that there may have been a substantial number of unreported Lyme disease cases despite an apparent increase in greenspace utilization. The use of counterfactual modeling using spatial and historical trends can provide insight into the likely numbers of missed cases. Variable ascertainment of cases has implications for passive surveillance programs, especially in the trending of disease morbidity and outbreak detection, suggesting that other methods may be appropriate for outbreak detection during disturbances to these passive surveillance systems.

Exploring the role of levan in plant immunity to pathogens: A review.

This review article delves into the intricate relationship between levan, a versatile polysaccharide, and its role in enhancing plant resistance against pathogens. By exploring the potential applications of levan in agriculture and biotechnology, such as crop protection, stress tolerance enhancement, and biotechnological innovations, significant advancements in sustainable agriculture are uncovered. Despite challenges in optimizing application methods and addressing regulatory hurdles, understanding the mechanisms of levan-mediated plant immunity offers promising avenues for future research. This review underscores the implications of utilizing levan to develop eco-friendly solutions, reduce reliance on chemical pesticides, and promote sustainable agricultural practices. Ultimately, by unraveling the pivotal role of levan in plant-pathogen interactions, this review sets the stage for transformative innovations in agriculture and highlights the path towards a more resilient and sustainable agricultural future.

Rapid real-time quantitative colorimetric LAMP methodology for field detection of Verticillium dahliae in crude olive-plant samples.

BACKGROUND: Verticilium dahliae is the most important wilt pathogen of olive trees with a broad host range causing devastating diseases currently without any effective chemical control. Traditional detection methodologies are based on symptoms-observation or lab-detection using time consuming culturing or molecular techniques. Therefore, there is an increasing need for portable tools that can detect rapidly V. dahliae in the field. RESULTS: In this work, we report the development of a novel method for the rapid, reliable and on-site detection of V. dahliae using a newly designed isothermal LAMP assay and crude extracts of olive wood. For the detection of the fungus, LAMP primers were designed targeting the internal transcribed spacer (ITS) region of the rRNA gene. The above assay was combined with a purpose-built prototype portable device which allowed real time quantitative colorimetric detection of V. dahliae in 35 min. The limit of detection of our assay was found to be 0.8 fg/µl reaction and the specificity 100% as indicated by zero cross-reactivity to common pathogens found in olive trees. Moreover, detection of V. dahliae in purified DNA gave a sensitivity of 100% (Ct < 30) and 80% (Ct > 30) while the detection of the fungus in unpurified crude wood extracts showed a sensitivity of 80% when multisampling was implemented. The superiority of the LAMP methodology regarding robustness and sensitivity was demonstrated when only LAMP was able to detect V. dahliae in crude samples from naturally infected trees with very low infection levels, while nested PCR and SYBR qPCR failed to detect the pathogen in an unpurified form. CONCLUSIONS: This study describes the development of a new real time LAMP assay, targeting the ITS region of the rRNA gene of V. dahliae in olive trees combined with a 3D-printed portable device for field testing using a tablet. The assay is characterized by high sensitivity and specificity as well as ability to operate using directly crude samples such as woody tissue or petioles. The reported methodology is setting the basis for the development of an on-site detection methodology for V. dahliae in olive trees, but also for other plant pathogens.

Metagenomic versus targeted next-generation sequencing for detection of microorganisms in bronchoalveolar lavage fluid among renal transplantation recipients.

Background: Metagenomic next-generation sequencing (mNGS), which provides untargeted and unbiased pathogens detection, has been extensively applied to improve diagnosis of pulmonary infection. This study aimed to compare the clinical performance between mNGS and targeted NGS (tNGS) for microbial detection and identification in bronchoalveolar lavage fluid (BALF) from kidney transplantation recipients (KTRs). Methods: BALF samples with microbiological results from mNGS and conventional microbiological test (CMT) were included. For tNGS, samples were extracted, amplified by polymerase chain reaction with pathogen-specific primers, and sequenced on an Illumina Nextseq. Results: A total of 99 BALF from 99 KTRs, among which 93 were diagnosed as pulmonary infection, were analyzed. Compared with CMT, both mNGS and tNGS showed higher positive rate and sensitivity (p<0.001) for overall, bacterial and fungal detection. Although the positive rate for mNGS and tNGS was comparable, mNGS significantly outperformed tNGS in sensitivity (100% vs. 93.55%, p<0.05), particularly for bacteria and virus (p<0.001). Moreover, the true positive rate for detected microbes of mNGS was superior over that of tNGS (73.97% vs. 63.15%, p<0.05), and the difference was also significant when specific for bacteria (94.59% vs. 64.81%, p<0.001) and fungi (93.85% vs. 72.58%, p<0.01). Additionally, we found that, unlike most microbes such as SARS-CoV-2, Aspergillus, and EBV, which were predominantly detected from recipients who underwent surgery over 3 years, Torque teno virus (TTV) were principally detected from recipients within 1-year post-transplant, and as post-transplantation time increased, the percentage of TTV positivity declined. Conclusion: Although tNGS was inferior to mNGS owing to lower sensitivity and true positive rate in identifying respiratory pathogens among KTRs, both considerably outperformed CMT.

Conserved signaling modules regulate filamentous growth in fungi: a model for eukaryotic cell differentiation.

Eukaryotic organisms are composed of different cell types with defined shapes and functions. Specific cell types are produced by the process of cell differentiation, which is regulated by signal transduction pathways. Signaling pathways regulate cell differentiation by sensing cues and controlling the expression of target genes whose products generate cell types with specific attributes. In studying how cells differentiate, fungi have proved valuable models because of their ease of genetic manipulation and striking cell morphologies. Many fungal species undergo filamentous growth-a specialized growth pattern where cells produce elongated tube-like projections. Filamentous growth promotes expansion into new environments, including invasion into plant and animal hosts by fungal pathogens. The same signaling pathways that regulate filamentous growth in fungi also control cell differentiation throughout eukaryotes and include highly conserved mitogen-activated protein kinase (MAPK) pathways, which is the focus of this review. In many fungal species, mucin-type sensors regulate MAPK pathways to control filamentous growth in response to diverse stimuli. Once activated, MAPK pathways reorganize cell polarity, induce changes in cell adhesion, and promote the secretion of degradative enzymes that mediate access to new environments. However, MAPK pathway regulation is complicated because related pathways can share components with each other yet induce unique responses (i.e. signal specificity). In addition, MAPK pathways function in highly integrated networks with other regulatory pathways (i.e. signal integration). Here, we discuss signal specificity and integration in several yeast models (mainly Saccharomyces cerevisiae and Candida albicans) by focusing on the filamentation MAPK pathway. Because of the strong evolutionary ties between species, a deeper understanding of the regulation of filamentous growth in established models and increasingly diverse fungal species can reveal fundamentally new mechanisms underlying eukaryotic cell differentiation.

Wastewater Surveillance Pilot at US Military Installations: Cost Model Analysis.

Background: The COVID-19 pandemic highlighted the need for pathogen surveillance systems to augment both early warning and outbreak monitoring/control efforts. Community wastewater samples provide a rapid and accurate source of environmental surveillance data to complement direct patient sampling. Due to its global presence and critical missions, the US military is a leader in global pandemic preparedness efforts. Clinical testing for COVID-19 on US Air Force (USAF) bases (AFBs) was effective but costly with respect to direct monetary costs and indirect costs due to lost time. To remain operating at peak capacity, such bases sought a more passive surveillance option and piloted wastewater surveillance (WWS) at 17 AFBs to demonstrate feasibility, safety, utility, and cost-effectiveness from May 2021 to January 2022. Objective: We model the costs of a wastewater program for pathogens of public health concern within the specific context of US military installations using assumptions based on the results of the USAF and Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense pilot program. The objective was to determine the cost of deploying WWS to all AFBs relative to clinical swab testing surveillance regimes. Methods: A WWS cost projection model was built based on subject matter expert input and actual costs incurred during the WWS pilot program at USAF AFBs. Several SARS-CoV-2 circulation scenarios were considered, and the costs of both WWS and clinical swab testing were projected. Analysis was conducted to determine the break-even point and how a reduction in swab testing could unlock funds to enable WWS to occur in parallel. Results: Our model confirmed that WWS is complementary and highly cost-effective when compared to existing alternative forms of biosurveillance. We found that the cost of WWS was between US $10.5-$18.5 million less expensive annually in direct costs as compared to clinical swab testing surveillance. When the indirect cost of lost work was incorporated, including lost work associated with required clinical swab testing, we estimated that over two-thirds of clinical swab testing could be maintained with no additional costs upon implementation of WWS. Conclusions: Our results support the adoption of WWS across US military installations as part of a more comprehensive and early warning system that will enable adaptive monitoring during disease outbreaks in a more cost-effective manner than swab testing alone.

Dam construction alters the traits of health-related microbes along the Yangtze River.

Dams, constructed globally for energy production and water conservation, fragment rivers, and modify flow regimes, thereby altering the composition of biological communities and ecosystem functions. Despite the extensive use of dams, few studies have explored their potential health impacts, particularly concerning changes in health-related genes, such as antibiotic resistance genes (ARGs) and virulence factor genes (VFGs), and their hosts (i.e., ARB and potential pathogens). Understanding these health-related effects is crucial because they can impact human health through water quality and pathogen prevalence. In this study, we investigated the planktonic microbial community in the Three Gorges Reservoir (TGR) and adjacent upstream and downstream areas of the Yangtze River during both the dry and wet season. Our metagenomic analysis showed that dam construction significantly decreased the abundance of ARGs, but it had an insignificant effect on VFGs. The observed reduction in ARGs abundance could be mainly attributed to the decrease abundance of the major ARGs carrier - Limnohabitansin the TGR and downstream areas due to high grazing pressure and fitness cost. Conversely, the abundance of microbes carrying VFGs (potential pathogens) remained stable from upstream to the dam reservoir, which may explain the negligible impact on VFG abundance. Overall, our results provide a detailed understanding of the ecological health implications of dam construction in large river ecosystems.

Anti-biofilm mechanisms of action of essential oils by targeting genes involved in quorum sensing, motility, adhesion, and virulence: A review.

Biofilms are a critical factor for food safety, causing important economic losses. Among the novel strategies for controlling biofilms, essential oils (EOs) can represent an environmentally friendly approach, able to act both on early and mature stages of biofilm formation. This review reports the anti-biofilm mechanisms of action of EOs against five pathogenic bacterial species known for their biofilm-forming ability. These mechanisms include disturbing the expression of genes related to quorum sensing (QS), motility, adhesion, and virulence. Biofilms and QS are interconnected processes, and EOs interfere with the communication system (e.g. regulating the expression of agrBDCA, luxR, luxS, and pqsA genes), thus influencing biofilm formation. In addition, QS is an important mechanism that regulates gene expression related to bacterial survival, virulence, and pathogenicity. Similarly, EOs also influence the expression of many virulence genes. Moreover, EOs exert their effects modulating the genes associated with bacterial adhesion and motility, for example those involved in curli (csg), fimbriae (fim, lpf), and flagella (fla, fli, flh, and mot) production, as well as the ica genes responsible for synthetizing polysaccharide intercellular adhesin. This review provides a comprehensive framework on the topic for a better understanding of EOs biofilm mechanisms of action.

Multiple biological characteristics and functions of intestinal biofilm extracellular polymers: friend or foe?

The gut microbiota is vital to human health, and their biofilms significantly impact intestinal immunity and the maintenance of microbial balance. Certain pathogens, however, can employ biofilms to elude identification by the immune system and medical therapy, resulting in intestinal diseases. The biofilm is formed by extracellular polymorphic substances (EPS), which shield microbial pathogens from the host immune system and enhance its antimicrobial resistance. Therefore, investigating the impact of extracellular polysaccharides released by pathogens that form biofilms on virulence and defence mechanisms is crucial. In this review, we provide a comprehensive overview of current pathogenic biofilm research, deal with the role of extracellular polymers in the formation and maintenance of pathogenic biofilm, and elaborate different prevention and treatment strategies to provide an innovative approach to the treatment of intestinal pathogen-based diseases.

Metagenomic next-generation sequencing targeted and metagenomic next-generation sequencing for pulmonary infection in HIV-infected and non-HIV-infected individuals.

Background: When individuals infected with human immunodeficiency virus (HIV) experience pulmonary infections, they often exhibit severe symptoms and face a grim prognosis. Consequently, early, rapid, and accurate pathogen diagnosis is vital for informing effective treatment strategies. This study aimed to use metagenomic next-generation sequencing (mNGS) and targeted mNGS (tNGS) to elucidate the characteristics of pulmonary infections in HIV and non-HIV individuals. Methods: This study enrolled 90 patients with pulmonary infection at the Department of Infectious Diseases of The First Hospital of Jilin University from June 2022 to May 2023, and they were divided into HIV (n=46) and non-HIV (n=44) infection groups. Their bronchoalveolar lavage fluid (BALF) was collected for mNGS analysis to evaluate the differences in pulmonary infection pathogens, and tNGS detection was performed on BALF samples from 15 HIV-infected patients. Results: A total of 37 pathogens were identified in this study, including 21 bacteria, 5 fungi, 5 viruses, 5 mycobacteria, and 1 mycoplasma. The sensitivity of mNGS was 78.9% (71/90), which is significantly higher than that of conventional methods (CTM) (39/90, P=1.5E-8). The combination of mNGS with CTM can greatly enhance the sensitivity of pathogen detection. The prevalence of Pneumocystis jirovecii (82.6% vs. 9.1%), cytomegalovirus (CMV) (58.7% vs. 0%), and Epstein-Barr virus (EBV) (17.4% vs. 2.3%) was significantly higher in the HIV infection group than in the non-HIV infection group (P<0.05). Although no statistically significant difference was observed, the detection rate of Mycobacteria was higher in HIV-infected patients (17.4%) than in the non-HIV group (6.8%). Furthermore, the tNGS results of BALF from 15 HIV-infected patients were not entirely consistent with the mNGS results., and the concordance rate of tNGS for the detection of main pathogens reached 86.7% (13/15). Conclusion: Next-generation sequencing (NGS) can accurately detect pathogens in the BALF of patients with pulmonary infection. The sensitivity of tNGS is comparable to that of mNGS. Therefore, this technique should be promoted in the clinic for better patient outcomes.

Genome sequence of Colletotrichum karsti isolated from rose leaves exhibiting anthracnose symptoms in Potchefstroom, South Africa.

We present the genome sequence of Colletotrichum karsti isolated from rose leaves exhibiting anthracnose symptoms. The genome was assembled to 53.2 Mbp organized into 753 scaffolds having an N50 of 582,313 kbp and a GC content of 52.5%. The genome had an estimated 99.4% of the core Ascomycota genes.

Minthostachys verticillata essential oil modulates cytokine synthesis and Staphylococcus aureus internalization in MAC-T cells at least through TLR4/MyD88/NFkB pathway.

The aim of this study was to evaluate the activation pathway(s) triggered by Minthostachys verticillata essential oil (EO) in bovine mammary epithelial cells (MAC-T) challenged with a strain of bovine Staphylococcus aureus. MAC-T cells were stimulated with EO, S. aureus or pre-treated with EO and then challenged with S. aureus. Cytokine's release was measured by ELISA. The mRNA for TLR2, TLR4, NOD2, MyD88 and NFκB was quantified by RT-qPCR. S. aureus adherence and internalization was also evaluated. MAC-T cells stimulated with S. aureus synthesized high levels of IL-1ß and IL-6 were kept up to 48 h, while IL-4 levels were not altered. Cells pre-treated with EO for 2 and 6 h and then challenged with S. aureus showed a significant increase of IL-1ß and IL-6. However, in these cells, a decrease in IL-1ß and IL-6 levels and an increase of IL-4 values was observed from 24 h. No significant increase in the expression levels of TLR2 or NOD2 was detected in all stimulated cells. However, the expression of TLR4, MyD88 and NFκB was increased in cells stimulated with S. aureus at 2 and 6 h as well as in cells pre-treated with EO between 2 and 6 h and then challenged with S. aureus. The NFκB expression levels was similar to control at 24 h in all stimulated cells, although pro-inflammatory cytokine levels and TLR4 and MyD88 expression levels remained high in cells stimulated with S. aureus. This results suggested the activation of other pathways independent of MyD88 by the pathogen that involucrated the activation of others transcription factors. Pre-treatment with EO during 2, 6 and 24 h did not affect S. aureus adherence but decreased its internalization. In conclusion, pre-treatment with EO increased the IL-1ß and IL-6 synthesis during the first hours post-challenged with S. aureus up-regulating TLR4/MyD88/NFκB pathway. Furthermore, EO increased the IL-4 levels from 6 to 24 h down-regulating the NFκB and possibly other transcription factors activated by the pathogen, which decreased its internalization into MAC-T cells.