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Background: Tranexamic acid (TXA) is an antifibrinolytic agent applied in orthopedic surgery and has been proven to reduce post-surgery infection rates. We previously showed that TXA also had an additional direct antimicrobial effect against planktonic bacteria. Therefore, we aimed to evaluate whether it has a synergistic effect if in combination with antibiotics. Materials and Methods: Three ATCC and seven clinical strains of staphylococci were tested against serial dilutions of vancomycin and gentamicin alone and in combination with TXA at 10 and 50 mg/ml. The standardized microtiter plate method was used. Minimal inhibitory concentrations (MICs) were calculated by standard visualization of well turbidity (the lowest concentration at which complete absence of well bacterial growth was observed by the researcher) and using the automated method (the lowest concentration at which ≥80% reduction in well bacterial growth was measured using a spectrophotometer). Results: Tranexamic acid-10 mg/ml reduced the MIC of vancomycin and gentamicin with both the standard method (V: 1-fold dilution, G: 4-fold dilutions) and the automated turbidity method (vancomycin: 8-fold dilutions, gentamicin: 8-fold dilutions). TXA-50 mg/ml reduced the MIC of gentamicin with both the standard turbidity method (6-fold dilutions) and the automated turbidity method (1-fold dilutions). In contrast, for vancomycin, the MIC remained the same using the standard method, and only a 1-fold dilution was reduced using the automated method. Conclusion: Ours was a proof-of-concept study in which we suggest that TXA may have a synergistic effect when combined with both vancomycin and gentamicin, especially at 10 mg/ml, which is the concentration generally used in clinical practice. [ABSTRACT FROM AUTHOR]

Introduction: Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, has been a global threat to human beings for several decades. Treating tuberculosis has become more difficult as the prevalence of drug-resistant tuberculosis has increased globally. Evidence suggests that the comprehensive landscape of resistance mechanisms in MTB is ambiguous. More importantly, little is known regarding the series of events connected to resistance mechanisms in MTB before exposure to anti-TB drugs, during exposure to the drugs, and finally, when the MTB becomes resistant after exposure, upon analyses of its genome. Methods: We used the wild-type strain of MTB (H37Rv) in an in vitro model for generating induced resistance using a sub-inhibitory concentration of isoniazid, and the generated resistance-associated variants (RAVs) were identified using the whole genome sequencing method. Results: The detection of an inhA promoter mutation (fabG1-15C>T), which results in increased production of InhA protein, was found to be a major mechanism for developing resistance to isoniazid in the first place. We observed adaptation of MTB resistance mechanisms in high isoniazid stress by alteration and abolishment of KatG due to the detection of katG S315N, the common region of mutation that confers isoniazid resistance, along with katG K414N, katG N138S, and katG A162E. Furthermore, we detected the ahpC-72C>T and ahpC 21C>A mutations, but further investigation is needed to determine their role in compensating for the loss of KatG activity. Discussion: This suggests that increased InhA production is themainmechanism where there are low levels of isoniazid, whereas the alteration of KatG was found to be utilized in mycobacterium with a high concentration of isoniazid. Our work demonstrates that this in vitro approach of generating induced resistance could provide clinically relevant information after the fabG1-15C>T mutation, which is the common mutation found in clinical isolates. Moreover, other mutations detected in this work can also be found in clinical isolates. These findingsmay shed light on the impact of isoniazid in generating RAV and the resistance mechanism scenario that mycobacterium used under various isoniazid-pressuring conditions. More research is needed to understand better the role of RAV and mechanical resistance events within the mycobacterium genome in promoting a promising drug prediction platform that could lead to the right treatment for patients with MDR-TB and XDR-TB. [ABSTRACT FROM AUTHOR]

The human gastrointestinal tract consists of different regions, each characterized by a distinct physiology, anatomy, and microbial community. While the colonic microbiota has received a lot of attention in recent research projects, little is known about the small intestinal microbiota and its interactions with ingested compounds, primarily due to the inaccessibility of this region in vivo. This study therefore aimed to develop and validate a dynamic, long-term simulation of the ileal microbiota using the SHIME®- technology. Essential parameters were identified and optimized from a screening experiment testing different inoculation strategies, nutritional media, and environmental parameters over an 18-day period. Subjecting a synthetic bacterial consortium to the selected conditions resulted in a stable microbiota that was representative in terms of abundance [8.81 ± 0.12 log (cells/ml)], composition and function. Indeed, the observed community mainly consisted of the genera Streptococcus, Veillonella, Enterococcus, Lactobacillus, and Clostridium (qPCR and 16S rRNA gene targeted Illumina sequencing), while nutrient administration boosted lactate production followed by cross-feeding interactions towards acetate and propionate. Furthermore, similarly as in vivo, bile salts were only partially deconjugated and only marginally converted into secondary bile salts. After confirming reproducibility of the small intestinal microbiota model, it was integrated into the established M-SHIME® where it further increased the compositional relevance of the colonic community. This long-term in vitro model provides a representative simulation of the ileal bacterial community, facilitating research of the ileum microbiota dynamics and activity when, for example, supplemented with microbial or diet components. Furthermore, integration of this present in vitro simulation increases the biological relevance of the current M-SHIME® technology. [ABSTRACT FROM AUTHOR]

IntroductionMycobacterium tuberculosis (MTB), the causative agent of tuberculosis, has been a global threat to human beings for several decades. Treating tuberculosis has become more difficult as the prevalence of drug-resistant tuberculosis has increased globally. Evidence suggests that the comprehensive landscape of resistance mechanisms in MTB is ambiguous. More importantly, little is known regarding the series of events connected to resistance mechanisms in MTB before exposure to anti-TB drugs, during exposure to the drugs, and finally, when the MTB becomes resistant after exposure, upon analyses of its genome.MethodsWe used the wild-type strain of MTB (H37Rv) in an in vitro model for generating induced resistance using a sub-inhibitory concentration of isoniazid, and the generated resistance-associated variants (RAVs) were identified using the whole genome sequencing method.ResultsThe detection of an inhA promoter mutation (fabG1−15C>T), which results in increased production of InhA protein, was found to be a major mechanism for developing resistance to isoniazid in the first place. We observed adaptation of MTB resistance mechanisms in high isoniazid stress by alteration and abolishment of KatG due to the detection of katG S315N, the common region of mutation that confers isoniazid resistance, along with katG K414N, katG N138S, and katG A162E. Furthermore, we detected the ahpC−72C>T and ahpC 21C>A mutations, but further investigation is needed to determine their role in compensating for the loss of KatG activity.DiscussionThis suggests that increased InhA production is the main mechanism where there are low levels of isoniazid, whereas the alteration of KatG was found to be utilized in mycobacterium with a high concentration of isoniazid. Our work demonstrates that this in vitro approach of generating induced resistance could provide clinically relevant information after the fabG1−15C>T mutation, which is the common mutation found in clinical isolates. Moreover, other mutations detected in this work can also be found in clinical isolates. These findings may shed light on the impact of isoniazid in generating RAV and the resistance mechanism scenario that mycobacterium used under various isoniazid-pressuring conditions. More research is needed to understand better the role of RAV and mechanical resistance events within the mycobacterium genome in promoting a promising drug prediction platform that could lead to the right treatment for patients with MDR-TB and XDR-TB.

The human gastrointestinal tract consists of different regions, each characterized by a distinct physiology, anatomy, and microbial community. While the colonic microbiota has received a lot of attention in recent research projects, little is known about the small intestinal microbiota and its interactions with ingested compounds, primarily due to the inaccessibility of this region in vivo. This study therefore aimed to develop and validate a dynamic, long-term simulation of the ileal microbiota using the SHIME®-technology. Essential parameters were identified and optimized from a screening experiment testing different inoculation strategies, nutritional media, and environmental parameters over an 18-day period. Subjecting a synthetic bacterial consortium to the selected conditions resulted in a stable microbiota that was representative in terms of abundance [8.81 ± 0.12 log (cells/ml)], composition and function. Indeed, the observed community mainly consisted of the genera Streptococcus, Veillonella, Enterococcus, Lactobacillus, and Clostridium (qPCR and 16S rRNA gene targeted Illumina sequencing), while nutrient administration boosted lactate production followed by cross-feeding interactions towards acetate and propionate. Furthermore, similarly as in vivo, bile salts were only partially deconjugated and only marginally converted into secondary bile salts. After confirming reproducibility of the small intestinal microbiota model, it was integrated into the established M-SHIME® where it further increased the compositional relevance of the colonic community. This long-term in vitro model provides a representative simulation of the ileal bacterial community, facilitating research of the ileum microbiota dynamics and activity when, for example, supplemented with microbial or diet components. Furthermore, integration of this present in vitro simulation increases the biological relevance of the current M-SHIME® technology.

Background: Tranexamic acid (TXA) is an antifibrinolytic agent applied in orthopedic surgery and has been proven to reduce post-surgery infection rates. We previously showed that TXA also had an additional direct antimicrobial effect against planktonic bacteria. Therefore, we aimed to evaluate whether it has a synergistic effect if in combination with antibiotics. Materials and Methods: Three ATCC and seven clinical strains of staphylococci were tested against serial dilutions of vancomycin and gentamicin alone and in combination with TXA at 10 and 50 mg/ml. The standardized microtiter plate method was used. Minimal inhibitory concentrations (MICs) were calculated by standard visualization of well turbidity (the lowest concentration at which complete absence of well bacterial growth was observed by the researcher) and using the automated method (the lowest concentration at which ≥80% reduction in well bacterial growth was measured using a spectrophotometer). Results: Tranexamic acid-10 mg/ml reduced the MIC of vancomycin and gentamicin with both the standard method (V: 1-fold dilution, G: 4-fold dilutions) and the automated turbidity method (vancomycin: 8-fold dilutions, gentamicin: 8-fold dilutions). TXA-50 mg/ml reduced the MIC of gentamicin with both the standard turbidity method (6-fold dilutions) and the automated turbidity method (1-fold dilutions). In contrast, for vancomycin, the MIC remained the same using the standard method, and only a 1-fold dilution was reduced using the automated method. Conclusion: Ours was a proof-of-concept study in which we suggest that TXA may have a synergistic effect when combined with both vancomycin and gentamicin, especially at 10 mg/ml, which is the concentration generally used in clinical practice. [ABSTRACT FROM AUTHOR]

Treatment for moderate-to-severe acne vulgaris relies on prolonged use of oral tetracycline-class antibiotics; however, these broad-spectrum antibiotics are often associated with off-target effects and negative gastrointestinal sequelae. Sarecycline is a narrow-spectrum antibiotic treatment option. Here, we investigated the effect of prolonged sarecycline exposure, compared with broad-spectrum tetracyclines (doxycycline and minocycline) upon the colonic microbiota. Three in vitro models of the human colon were instilled with either minocycline, doxycycline or sarecycline, and we measured microbiota abundance and diversity changes during and after antibiotic exposure. Significant reductions in microbial diversity were observed following minocycline and doxycycline exposure, which failed to recover post antibiotic withdrawal. Specifically, minocycline caused a ~10% decline in Lactobacillaceae and Bifidobacteriaceae abundances, while doxycycline caused a ~7% decline in Lactobacillaceae and Bacteroidaceae abundances. Both minocycline and doxycycline were associated with a large expansion (>10%) of Enterobacteriaceae. Sarecycline caused a slight decline in bacterial diversity at the start of treatment, but abundances of most families remained stable during treatment. Ruminococcaceae and Desulfovibrionaceae decreased 9% and 4%, respectively, and a transient increased in Enterobacteriaceae abundance was observed during sarecycline administration. All populations recovered to pre-antibiotic levels after sarecycline exposure. Overall, sarecycline had minimal and transient impact on the gut microbiota composition and diversity, when compared to minocycline and doxycycline. [ABSTRACT FROM AUTHOR]

BackgroundTranexamic acid (TXA) is an antifibrinolytic agent applied in orthopedic surgery and has been proven to reduce post-surgery infection rates. We previously showed that TXA also had an additional direct antimicrobial effect against planktonic bacteria. Therefore, we aimed to evaluate whether it has a synergistic effect if in combination with antibiotics.Materials and MethodsThree ATCC and seven clinical strains of staphylococci were tested against serial dilutions of vancomycin and gentamicin alone and in combination with TXA at 10 and 50 mg/ml. The standardized microtiter plate method was used. Minimal inhibitory concentrations (MICs) were calculated by standard visualization of well turbidity (the lowest concentration at which complete absence of well bacterial growth was observed by the researcher) and using the automated method (the lowest concentration at which ≥80% reduction in well bacterial growth was measured using a spectrophotometer).ResultsTranexamic acid-10 mg/ml reduced the MIC of vancomycin and gentamicin with both the standard method (V: 1-fold dilution, G: 4-fold dilutions) and the automated turbidity method (vancomycin: 8-fold dilutions, gentamicin: 8-fold dilutions). TXA-50 mg/ml reduced the MIC of gentamicin with both the standard turbidity method (6-fold dilutions) and the automated turbidity method (1-fold dilutions). In contrast, for vancomycin, the MIC remained the same using the standard method, and only a 1-fold dilution was reduced using the automated method.ConclusionOurs was a proof-of-concept study in which we suggest that TXA may have a synergistic effect when combined with both vancomycin and gentamicin, especially at 10 mg/ml, which is the concentration generally used in clinical practice.

Treatment for moderate-to-severe acne vulgaris relies on prolonged use of oral tetracycline-class antibiotics; however, these broad-spectrum antibiotics are often associated with off-target effects and negative gastrointestinal sequelae. Sarecycline is a narrow-spectrum antibiotic treatment option. Here, we investigated the effect of prolonged sarecycline exposure, compared with broad-spectrum tetracyclines (doxycycline and minocycline) upon the colonic microbiota. Three in vitro models of the human colon were instilled with either minocycline, doxycycline or sarecycline, and we measured microbiota abundance and diversity changes during and after antibiotic exposure. Significant reductions in microbial diversity were observed following minocycline and doxycycline exposure, which failed to recover post antibiotic withdrawal. Specifically, minocycline caused a ~10% decline in Lactobacillaceae and Bifidobacteriaceae abundances, while doxycycline caused a ~7% decline in Lactobacillaceae and Bacteroidaceae abundances. Both minocycline and doxycycline were associated with a large expansion (>10%) of Enterobacteriaceae. Sarecycline caused a slight decline in bacterial diversity at the start of treatment, but abundances of most families remained stable during treatment. Ruminococcaceae and Desulfovibrionaceae decreased 9% and 4%, respectively, and a transient increased in Enterobacteriaceae abundance was observed during sarecycline administration. All populations recovered to pre-antibiotic levels after sarecycline exposure. Overall, sarecycline had minimal and transient impact on the gut microbiota composition and diversity, when compared to minocycline and doxycycline.

Background: In the practice of breast augmentation and reconstruction, implant irrigation with various solutions has been widely used to prevent infection and capsular contracture, but to date, there is no consensus on the optimal protocol to use. Recently, application of povidone iodine (PI) for 30 min has shown in vitro to be the most effective irrigating formula in reducing contamination in smooth breast implants. However, as 30 min is not feasible intraoperatively, it is necessary to determine whether shorter times could be equally effective as well as to test it in both smooth and textured implants. Methods: We tested the efficacy of 10% PI at 1′, 3′, and 5′ against biofilms of 8 strains (2 ATCC and 6 clinical) of Staphylococcus spp. on silicone disks obtained from Mentor® and Polytech® implants of different textures. We analyzed the percentage reduction of cfu counts, cell viability and bacterial density between treatment (PI) and control (sterile saline, SS) groups for each time of application. We consider clinical significance when > 25% reduction was observed in cell viability or bacterial density. Results: All textured implants treated with PI at any of the 3 exposure times reduced 100% bacterial load by culture. However, none of the implants reached enough clinical significance in percentage reduction of living cells. Regarding bacterial density, only 25–50 μm Polytxt® Polytech® implants showed significant reduction at the three PI exposure times. Conclusion: PI is able to inhibit bacterial growth applied on the surface of breast implants regardless of the exposure time. However, no significant reduction on living cells or bacterial density was observed. This lack of correlation may be caused by differences in texture that directly affect PI absorption. [ABSTRACT FROM AUTHOR]

BackgroundIn the practice of breast augmentation and reconstruction, implant irrigation with various solutions has been widely used to prevent infection and capsular contracture, but to date, there is no consensus on the optimal protocol to use. Recently, application of povidone iodine (PI) for 30 min has shown in vitro to be the most effective irrigating formula in reducing contamination in smooth breast implants. However, as 30 min is not feasible intraoperatively, it is necessary to determine whether shorter times could be equally effective as well as to test it in both smooth and textured implants.MethodsWe tested the efficacy of 10% PI at 1′, 3′, and 5′ against biofilms of 8 strains (2 ATCC and 6 clinical) of Staphylococcus spp. on silicone disks obtained from Mentor® and Polytech® implants of different textures. We analyzed the percentage reduction of cfu counts, cell viability and bacterial density between treatment (PI) and control (sterile saline, SS) groups for each time of application. We consider clinical significance when > 25% reduction was observed in cell viability or bacterial density.ResultsAll textured implants treated with PI at any of the 3 exposure times reduced 100% bacterial load by culture. However, none of the implants reached enough clinical significance in percentage reduction of living cells. Regarding bacterial density, only 25–50 μm Polytxt® Polytech® implants showed significant reduction at the three PI exposure times.ConclusionPI is able to inhibit bacterial growth applied on the surface of breast implants regardless of the exposure time. However, no significant reduction on living cells or bacterial density was observed. This lack of correlation may be caused by differences in texture that directly affect PI absorption.

The small intestine is a digestive organ that has a complex and dynamic ecosystem, which is vulnerable to the risk of pathogen infections and disorders or imbalances. Many studies have focused attention on intestinal mechanisms, such as host–microbiome interactions and pathways, which are associated with its healthy and diseased conditions. This review highlights the intestine models currently used for simulating such normal and diseased states. We introduce the typical models used to simulate the intestine along with its cell composition, structure, cellular functions, and external environment and review the current state of the art for in vitro cell-based models of the small intestine system to replace animal models, including ex vivo , 2D culture, organoid, lab-on-a-chip, and 3D culture models. These models are described in terms of their structure, composition, and co-culture availability with microbiomes. Furthermore, we discuss the potential application for the aforementioned techniques to these in vitro models. The review concludes with a summary of intestine models from the viewpoint of current techniques as well as their main features, highlighting potential future developments and applications. [ABSTRACT FROM AUTHOR]

Continuous in vitro fermentation models provide a useful tool for a fast, reproducible, and direct assessment of treatment-related changes in microbiota metabolism and composition independent of the host. In this study, we used the PolyFermS model to mimic the conditions of the chicken cecum and evaluated three nutritive media for in vitro modeling of the chicken cecal microbiota ecology and metabolism. We observed that our model inoculated with immobilized cecal microbiota and fed with a modified Viande Levure medium (mVL-3) reached a high bacterial cell density of up to approximately 10.5 log cells per mL and stable microbiota composition, akin to the host, during 82 days of continuous operation. Relevant bacterial functional groups containing primary fibrolytic (Bacteroides , Bifidobacteriaceae , Ruminococcaceae), glycolytic (Enterococcus), mucolytic (Bacteroides), proteolytic (Bacteroides), and secondary acetate-utilizing butyrate-producing and propionate-producing (Lachnospiraceae) taxa were preserved in vitro. Besides, conserved metabolic and functional Kyoto Encyclopedia of Genes and Genomes pathways were observed between in vitro microbiota and cecal inoculum microbiota as predicted by functional metagenomics analysis. Furthermore, we demonstrated that the continuous inoculation provided by the inoculum reactor generated reproducible metabolic profiles in second-stage reactors comparable to the chicken cecum, allowing for the simultaneous investigation and direct comparison of different treatments with a control. In conclusion, we showed that PolyFermS is a suitable model for mimicking chicken cecal microbiota fermentation allowing ethical and ex vivo screening of environmental factors, such as dietary additives, on chicken cecal fermentation. We report here for the first time a fermentation medium (mVL-3) that closely mimics the substrate conditions in the chicken cecum and supports the growth and metabolic activity of the cecal bacterial akin to the host. Our PolyFermS chicken cecum model is a useful tool to study microbiota functionality and structure ex vivo. [ABSTRACT FROM AUTHOR]

The small intestine is a digestive organ that has a complex and dynamic ecosystem, which is vulnerable to the risk of pathogen infections and disorders or imbalances. Many studies have focused attention on intestinal mechanisms, such as host–microbiome interactions and pathways, which are associated with its healthy and diseased conditions. This review highlights the intestine models currently used for simulating such normal and diseased states. We introduce the typical models used to simulate the intestine along with its cell composition, structure, cellular functions, and external environment and review the current state of the art for in vitro cell-based models of the small intestine system to replace animal models, including ex vivo, 2D culture, organoid, lab-on-a-chip, and 3D culture models. These models are described in terms of their structure, composition, and co-culture availability with microbiomes. Furthermore, we discuss the potential application for the aforementioned techniques to these in vitro models. The review concludes with a summary of intestine models from the viewpoint of current techniques as well as their main features, highlighting potential future developments and applications.

Continuous in vitro fermentation models provide a useful tool for a fast, reproducible, and direct assessment of treatment-related changes in microbiota metabolism and composition independent of the host. In this study, we used the PolyFermS model to mimic the conditions of the chicken cecum and evaluated three nutritive media for in vitro modeling of the chicken cecal microbiota ecology and metabolism. We observed that our model inoculated with immobilized cecal microbiota and fed with a modified Viande Levure medium (mVL-3) reached a high bacterial cell density of up to approximately 10.5 log cells per mL and stable microbiota composition, akin to the host, during 82 days of continuous operation. Relevant bacterial functional groups containing primary fibrolytic (Bacteroides, Bifidobacteriaceae, Ruminococcaceae), glycolytic (Enterococcus), mucolytic (Bacteroides), proteolytic (Bacteroides), and secondary acetate-utilizing butyrate-producing and propionate-producing (Lachnospiraceae) taxa were preserved in vitro. Besides, conserved metabolic and functional Kyoto Encyclopedia of Genes and Genomes pathways were observed between in vitro microbiota and cecal inoculum microbiota as predicted by functional metagenomics analysis. Furthermore, we demonstrated that the continuous inoculation provided by the inoculum reactor generated reproducible metabolic profiles in second-stage reactors comparable to the chicken cecum, allowing for the simultaneous investigation and direct comparison of different treatments with a control. In conclusion, we showed that PolyFermS is a suitable model for mimicking chicken cecal microbiota fermentation allowing ethical and ex vivo screening of environmental factors, such as dietary additives, on chicken cecal fermentation. We report here for the first time a fermentation medium (mVL-3) that closely mimics the substrate conditions in the chicken cecum and supports the growth and metabolic activity of the cecal bacterial akin to the host. Our PolyFermS chicken cecum model is a useful tool to study microbiota functionality and structure ex vivo.

The intestinal microbiota plays important roles in the maintenance of health. Strategies aiming at its modulation, such as probiotics, have received a deal of attention. Several strains have been studied in different in vitro models; however, the correlation of results obtained with the in vivo data has been limited. This questions the usefulness of such in vitro selection models, traditionally relying on over-simplified tests, not considering the influence of the accompanying microbiota or focusing on microbiota composition without considering functional traits. Here we assess the potential of six Bifidobacterium, Lactobacillus and Lacticaseibacillus strains in an in vitro model to determine their impact on the microbiota not just in terms of composition but also of functionality. Moreover, we compared the responses obtained in two different population groups: normal-weight and severely obese subjects. Fecal cultures were conducted to evaluate the impact of the strains on specific intestinal microbial groups, on the production of short-chain fatty acids, and on two functional responses: the production of gas and the interaction with human intestinal epithelial cells. The response to the different probiotics differed between both human groups. The addition of the probiotic strains did not induce major changes on the microbiota composition, with significant increases detected almost exclusively for the species added. Higher levels of gas production were observed in cultures from normal-weight subjects than in the obese population, with some strains being able to significantly reduce gas production in the latter group. Moreover, in obese subjects all the Bifidobacterium strains tested and Lacticaseibacillus rhamnosus GG were able to modify the response of the intestinal cells, restoring values similar to those obtained with the microbiotas of normal-weight subjects. Our results underline the need for the screening and selection of probiotics in a target-population specific manner by using appropriate in vitro models before enrolling in clinical intervention trials.

The intestinal microbiota plays important roles in the maintenance of health. Strategies aiming at its modulation, such as probiotics, have received a deal of attention. Several strains have been studied in different in vitro models; however, the correlation of results obtained with the in vivo data has been limited. This questions the usefulness of such in vitro selection models, traditionally relying on over-simplified tests, not considering the influence of the accompanying microbiota or focusing on microbiota composition without considering functional traits. Here we assess the potential of six Bifidobacterium , Lactobacillus and Lacticaseibacillus strains in an in vitro model to determine their impact on the microbiota not just in terms of composition but also of functionality. Moreover, we compared the responses obtained in two different population groups: normal-weight and severely obese subjects. Fecal cultures were conducted to evaluate the impact of the strains on specific intestinal microbial groups, on the production of short-chain fatty acids, and on two functional responses: the production of gas and the interaction with human intestinal epithelial cells. The response to the different probiotics differed between both human groups. The addition of the probiotic strains did not induce major changes on the microbiota composition, with significant increases detected almost exclusively for the species added. Higher levels of gas production were observed in cultures from normal-weight subjects than in the obese population, with some strains being able to significantly reduce gas production in the latter group. Moreover, in obese subjects all the Bifidobacterium strains tested and Lacticaseibacillus rhamnosus GG were able to modify the response of the intestinal cells, restoring values similar to those obtained with the microbiotas of normal-weight subjects. Our results underline the need for the screening and selection of probiotics in a target-population specific manner by using appropriate in vitro models before enrolling in clinical intervention trials. [ABSTRACT FROM AUTHOR]

Murine models are valuable tools to study the role of gut microbiota in health or disease. However, murine and human microbiota differ in species composition, so further investigation of the murine gut microbiota is important to gain a better mechanistic understanding. Continuous in vitro fermentation models are powerful tools to investigate microbe-microbe interactions while circumventing animal testing and host confounding factors, but are lacking for murine gut microbiota. We therefore developed a novel continuous fermentation model based on the PolyFermS platform adapted to the murine caecum and inoculated with immobilized caecal microbiota. We followed a stepwise model development approach by adjusting parameters [pH, retention time (RT), growth medium] to reach fermentation metabolite profiles and marker bacterial levels similar to the inoculum. The final model had a stable and inoculum-alike fermentation profile during continuous operation. A lower pH during startup and continuous operation stimulated bacterial fermentation (115 mM short-chain fatty acids at pH 7 to 159 mM at pH 6.5). Adjustments to nutritive medium, a decreased pH and increased RT helped control the in vitro Enterobacteriaceae levels, which often bloom in fermentation models, to 6.6 log gene copies/mL in final model. In parallel, the Lactobacillus, Lachnospiraceae, and Ruminococcaceae levels were better maintained in vitro with concentrations of 8.5 log gene copies/mL, 8.8 log gene copies/mL and 7.5 log gene copies/mL, respectively, in the final model. An independent repetition with final model parameters showed reproducible results in maintaining the inoculum fermentation metabolite profile and its marker bacterial levels. Microbiota community analysis of the final model showed a decreased bacterial diversity and compositional differences compared to caecal inoculum microbiota. Most of the caecal bacterial families were represented in vitro, but taxa of the Muribaculaceae family were not maintained. Functional metagenomics prediction showed conserved metabolic and functional KEGG pathways between in vitro and caecal inoculum microbiota. To conclude, we showed that a rational and stepwise approach allowed us to model in vitro the murine caecal microbiota and functions. Our model is a first step to develop murine microbiota model systems and offers the potential to study microbiota functionality and structure ex vivo.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3–5% and early development of P. falciparum exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.

Murine models are valuable tools to study the role of gut microbiota in health or disease. However, murine and human microbiota differ in species composition, so further investigation of the murine gut microbiota is important to gain a better mechanistic understanding. Continuous in vitro fermentation models are powerful tools to investigate microbe-microbe interactions while circumventing animal testing and host confounding factors, but are lacking for murine gut microbiota. We therefore developed a novel continuous fermentation model based on the PolyFermS platform adapted to the murine caecum and inoculated with immobilized caecal microbiota. We followed a stepwise model development approach by adjusting parameters [pH, retention time (RT), growth medium] to reach fermentation metabolite profiles and marker bacterial levels similar to the inoculum. The final model had a stable and inoculum-alike fermentation profile during continuous operation. A lower pH during startup and continuous operation stimulated bacterial fermentation (115 mM short-chain fatty acids at pH 7 to 159 mM at pH 6.5). Adjustments to nutritive medium, a decreased pH and increased RT helped control the in vitro Enterobacteriaceae levels, which often bloom in fermentation models, to 6.6 log gene copies/mL in final model. In parallel, the Lactobacillus , Lachnospiraceae , and Ruminococcaceae levels were better maintained in vitro with concentrations of 8.5 log gene copies/mL, 8.8 log gene copies/mL and 7.5 log gene copies/mL, respectively, in the final model. An independent repetition with final model parameters showed reproducible results in maintaining the inoculum fermentation metabolite profile and its marker bacterial levels. Microbiota community analysis of the final model showed a decreased bacterial diversity and compositional differences compared to caecal inoculum microbiota. Most of the caecal bacterial families were represented in vitro , but taxa of the Muribaculaceae family were not maintained. Functional metagenomics prediction showed conserved metabolic and functional KEGG pathways between in vitro and caecal inoculum microbiota. To conclude, we showed that a rational and stepwise approach allowed us to model in vitro the murine caecal microbiota and functions. Our model is a first step to develop murine microbiota model systems and offers the potential to study microbiota functionality and structure ex vivo. [ABSTRACT FROM AUTHOR]

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3–5% and early development of P. falciparum exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV) has a wide host range. Its pathogenesis varies among hosts and types of viruses. Most investigations of pathogenesis have been performed on cattle and swine. However, FMDV research in cattle is hampered by the lack of a stable in vitro infection model. In this study, the stable bovine thyroid (BTY) cell line hTERT-BTY from primary BTY cells was established by telomerase reverse transcriptase over expression. The results of karyotype analysis and experiments on morphological and biological characteristics indicated that this cell line possessed the qualities of primary BTY cells, which could be extended indefinitely with stable morphology and steady growth rates. The hTERT-BTY cell line, has 60 chromosomes including 29 pairs of autosomes and 1 pair of sex chromosomes without structure aberrations. It can express thyroid-specific function genes thyroid-stimulating hormone receptor and sodium/iodide symporter in high abundance ratios. The cell line is sensitive to FMDV strains and is expected to be used as a powerful tool for FMDV clinical diagnosis, separation, detection and culture. Also, the different mRNA expression levels in infected and uninfected hTERT-BTY cells were analyzed in this study to identify the pathways of immunity using RNA-seq. The results suggested that the hTERT-BTY cell line could be regarded as an effective tool for the immune response exploration of FMDV. In conclusion, this study provided a useful tool for FMDV clinical diagnosis, separation, detection, and culture. The cell line also could serve as an in vitro model to study the mechanism underlying FMDV pathogenicity and host–virus interaction.

Foot-and-mouth disease virus (FMDV) has a wide host range. Its pathogenesis varies among hosts and types of viruses. Most investigations of pathogenesis have been performed on cattle and swine. However, FMDV research in cattle is hampered by the lack of a stable in vitro infection model. In this study, the stable bovine thyroid (BTY) cell line hTERT-BTY from primary BTY cells was established by telomerase reverse transcriptase over expression. The results of karyotype analysis and experiments on morphological and biological characteristics indicated that this cell line possessed the qualities of primary BTY cells, which could be extended indefinitely with stable morphology and steady growth rates. The hTERT-BTY cell line, has 60 chromosomes including 29 pairs of autosomes and 1 pair of sex chromosomes without structure aberrations. It can express thyroid-specific function genes thyroid-stimulating hormone receptor and sodium/iodide symporter in high abundance ratios. The cell line is sensitive to FMDV strains and is expected to be used as a powerful tool for FMDV clinical diagnosis, separation, detection and culture. Also, the different mRNA expression levels in infected and uninfected hTERT-BTY cells were analyzed in this study to identify the pathways of immunity using RNA-seq. The results suggested that the hTERT-BTY cell line could be regarded as an effective tool for the immune response exploration of FMDV. In conclusion, this study provided a useful tool for FMDV clinical diagnosis, separation, detection, and culture. The cell line also could serve as an in vitro model to study the mechanism underlying FMDV pathogenicity and host–virus interaction. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a major cause of bacterial keratitis (BK) worldwide. Inappropriate or non-optimal antibiotic chemotherapy can lead to corneal perforation and rapid sight loss. In this study, we tested the hypothesis that P. aeruginosa strain PAO1 invades primary human corneal fibroblasts (hCFs) in vitro and persists intracellularly, despite chemotherapy with antibiotics used commonly to treat BK. In rank order, ciprofloxacin, levofloxacin and polymyxin B showed the highest activity against planktonic PAO1 growth (100% inhibitory concentration ≤10 μg/mL; 50% inhibitory concentration ≤1 μg/mL), followed by gentamicin and ofloxacin (100% inhibitory concentration ≤50 μg/mL; 50% inhibitory concentration ≤10 μg/mL). These bactericidal antibiotics (50–200 μg/mL concentrations) all killed PAO1 in the extracellular environment of infected hCF monolayers. By contrast, the bactericidal antibiotic cefuroxime and the bacteriostatic antibiotic chloramphenicol failed to sterilize both PAO1 broth cultures, even at a concentration of ≥200 μg/mL) and infected hCF monolayers. Statistically, all antibiotics were able to prevent LDH release from PAO1-infected hCF monolayers at both concentrations tested. Intracellular Pseudomonas were significantly reduced (>99%, P < 0.05) following treatment with ciprofloxacin, levofloxacin and ofloxacin, whereas gentamicin, polymyxin B and cefuroxime failed to clear intracellular bacteria over 24 h. Intracellular Pseudomonas infection was resistant to chloramphenicol, with hCF death observed by 9 h. Eventual growth of remaining intracellular Pseudomonas was observed in hCF after removal of all antibiotics, resulting in re-infection cycles and cell death by 48 h. All of the antibiotics reduced significantly (P < 0.05) IL-1β secretion by hCF infected with a Multiplicity Of Infection (MOI) = 1 of PAO1. With higher MOI, no pro-inflammatory effects were observed with antibiotic treatment, expect with polymyxin B and ofloxacin, which induced significant increased IL-1β secretion (P < 0.001). The findings from our study demonstrated that bactericidal and bacteriostatic antibiotics, routinely used to treat BK, failed to eradicate Pseudomonas infection of hCFs in vitro and that their bactericidal efficacies were influenced by the cellular location of the organism.

Pseudomonas aeruginosa is a major cause of bacterial keratitis (BK) worldwide. Inappropriate or non-optimal antibiotic chemotherapy can lead to corneal perforation and rapid sight loss. In this study, we tested the hypothesis that P. aeruginosa strain PAO1 invades primary human corneal fibroblasts (hCFs) in vitro and persists intracellularly, despite chemotherapy with antibiotics used commonly to treat BK. In rank order, ciprofloxacin, levofloxacin and polymyxin B showed the highest activity against planktonic PAO1 growth (100% inhibitory concentration ≤10 μg/mL; 50% inhibitory concentration ≤1 μg/mL), followed by gentamicin and ofloxacin (100% inhibitory concentration ≤50 μg/mL; 50% inhibitory concentration ≤10 μg/mL). These bactericidal antibiotics (50-200 μg/mL concentrations) all killed PAO1 in the extracellular environment of infected hCF monolayers. By contrast, the bactericidal antibiotic cefuroxime and the bacteriostatic antibiotic chloramphenicol failed to sterilize both PAO1 broth cultures, even at a concentration of ≤200 mg/mL) and infected hCF monolayers. Statistically, all antibiotics were able to prevent LDH release from PAO1-infected hCF monolayers at both concentrations tested. Intracellular Pseudomonas were significantly reduced (>99%, P < 0.05) following treatment with ciprofloxacin, levofloxacin and ofloxacin, whereas gentamicin, polymyxin B and cefuroxime failed to clear intracellular bacteria over 24 h. Intracellular Pseudomonas infection was resistant to chloramphenicol, with hCF death observed by 9 h. Eventual growth of remaining intracellular Pseudomonas was observed in hCF after removal of all antibiotics, resulting in re-infection cycles and cell death by 48 h. All of the antibiotics reduced significantly (P < 0.05) IL-1b secretion by hCF infected with a Multiplicity Of Infection (MOI) D 1 of PAO1. With higher MOI, no pro-inflammatory effects were observed with antibiotic treatment, expect with polymyxin B and ofloxacin, which induced significant increased IL-1b secretion (P < 0.001). The findings from our study demonstrated that bactericidal and bacteriostatic antibiotics, routinely used to treat BK, failed to eradicate Pseudomonas infection of hCFs in vitro and that their bactericidal efficacies were influenced by the cellular location of the organism. [ABSTRACT FROM AUTHOR]

Cefquinome is a fourth-generation Cephalosporin approved for use in animals exclusively. The objective of this study was to explore the relationship of cefquinome pharmacokinetic/pharmacodynamic (PK/PD) indices with resistance selection of Staphylococcus aureus ATCC25923 in an in vitro model. Six dosing regiments of cefquinome at an interval of 24 h for three consecutive times were simulated, resulting in maximum concentrations (Cmax) from 1/2 MIC to 16 MIC and half-lives (t1/2β) of 3 and 6 h, respectively. The in vitro sensitivity of S. aureus was monitored by bacterial susceptibility and dynamic time-kill curve experiments over the six cefquinome concentrations. The correlation between changes in bacterial susceptibility (MIC72/MIC0) and the percentage of time within mutant selection window (MSW) versus dosing interval (TMSW %) was subjected to Gaussian function and regression analysis. The results favored the consensus that time above MIC (T>MIC) was recognized as an important PK/PD parameter of cephalosporins for antibacterial efficiency. Cefquinome reached the maximum killing effect when T>MIC% attained approximately 40%~60%. The subsequent correlation analysis demonstrated that resistant S. aureus ATCC25923 was easy to occur when TMSW% attained an index of about 20% with t1/2β of 3 h after multiple dosing, and 40% with t1/2β of 6 h after multiple dosing.

The human gastrointestinal tract consists of different regions, each characterized by a distinct physiology, anatomy, and microbial community. While the colonic microbiota has received a lot of attention in recent research projects, little is known about the small intestinal microbiota and its interactions with ingested compounds, primarily due to the inaccessibility of this region in vivo . This study therefore aimed to develop and validate a dynamic, long-term simulation of the ileal microbiota using the SHIME ® -technology. Essential parameters were identified and optimized from a screening experiment testing different inoculation strategies, nutritional media, and environmental parameters over an 18-day period. Subjecting a synthetic bacterial consortium to the selected conditions resulted in a stable microbiota that was representative in terms of abundance [8.81 ± 0.12 log (cells/ml)], composition and function. Indeed, the observed community mainly consisted of the genera Streptococcus, Veillonella, Enterococcus, Lactobacillus , and Clostridium (qPCR and 16S rRNA gene targeted Illumina sequencing), while nutrient administration boosted lactate production followed by cross-feeding interactions towards acetate and propionate. Furthermore, similarly as in vivo , bile salts were only partially deconjugated and only marginally converted into secondary bile salts. After confirming reproducibility of the small intestinal microbiota model, it was integrated into the established M-SHIME® where it further increased the compositional relevance of the colonic community. This long-term in vitro model provides a representative simulation of the ileal bacterial community, facilitating research of the ileum microbiota dynamics and activity when, for example, supplemented with microbial or diet components. Furthermore, integration of this present in vitro simulation increases the biological relevance of the current M-SHIME® technology., Competing Interests: FM, MM, and TW are employees of ProDigest, that provides services to other companies using the model developed in this study. SD and PA were employees of ProDigest when this project was conducted. During the current project WP, Bv, and EK were employed by BaseClear, the company providing NGS services throughout the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deyaert, Moens, Pirovano, van den Bogert, Klaassens, Marzorati, Van de Wiele, Kleerebezem and Van den Abbeele.)

Mycoplasma gallisepticum is one of the most important pathogens that cause chronic respiratory disease in chicken. This study investigated the antibacterial activity of doxycycline against M. gallisepticum strain S6. In static time-killing studies with constant antibiotic concentrations [0-64 minimum inhibitory concentration (MIC)], M. gallisepticum colonies were quantified and kill rates were calculated to estimate the drug effect. The half-life of doxycycline in chicken was 6.51 ± 0.63 h. An in vitro dynamic model (the drug concentrations are fluctuant) was also established and two half-lives of 6.51 and 12 h were simulated. The samples were collected for drug concentration determination and viable counting of M. gallisepticum. In static time-killing studies, doxycycline produced a maximum antimycoplasmal effect of 5.62 10 (CFU/mL) reduction and the maximum kill rate was 0.11 h-1. In the in vitro dynamic model, doxycycline had a mycoplasmacidal activity in the two regimens, and the maximum antimycoplasmal effects were 4.1 and 4.75log10 (CFU/mL) reduction, respectively. Furthermore, the cumulative percentage of time over a 48-h period that the drug concentration exceeds the MIC (%T > MIC) was the pharmacokinetic-pharmacodynamic index that best correlated with antimicrobial efficacy (R2 = 0.986, compared with 0.897 for the peak level divided by the MIC and 0.953 for the area under the concentration-time curve over 48 h divided by the MIC). The estimated %T > MIC values for 0log10 (CFU/mL) reduction, 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction were 32.48, 45.68, and 54.36%, respectively, during 48 h treatment period of doxycycline. In conclusion, doxycycline shows excellent effectiveness and time-dependent characteristics against M. gallisepticum strain S6 in vitro. Additionally, these results will guide optimal dosing strategies of doxycycline in M. gallisepticum infection. [ABSTRACT FROM AUTHOR]

Over the past decades the food industry has been revolutionized toward the production of functional foods due to an increasing awareness of the consumers on the positive role of food inwellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer's health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut-brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell ® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus , and Cunninghamella bertholletiae . Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell ® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.

Pseudomonas aeruginosa is a major cause of bacterial keratitis (BK) worldwide. Inappropriate or non-optimal antibiotic chemotherapy can lead to corneal perforation and rapid sight loss. In this study, we tested the hypothesis that P. aeruginosa strain PAO1 invades primary human corneal fibroblasts (hCFs) in vitro and persists intracellularly, despite chemotherapy with antibiotics used commonly to treat BK. In rank order, ciprofloxacin, levofloxacin and polymyxin B showed the highest activity against planktonic PAO1 growth (100% inhibitory concentration ≤10 μg/mL; 50% inhibitory concentration ≤1 μg/mL), followed by gentamicin and ofloxacin (100% inhibitory concentration ≤50 μg/mL; 50% inhibitory concentration ≤10 μg/mL). These bactericidal antibiotics (50-200 μg/mL concentrations) all killed PAO1 in the extracellular environment of infected hCF monolayers. By contrast, the bactericidal antibiotic cefuroxime and the bacteriostatic antibiotic chloramphenicol failed to sterilize both PAO1 broth cultures, even at a concentration of ≥200 μg/mL) and infected hCF monolayers. Statistically, all antibiotics were able to prevent LDH release from PAO1-infected hCF monolayers at both concentrations tested. Intracellular Pseudomonas were significantly reduced (>99%, P < 0.05) following treatment with ciprofloxacin, levofloxacin and ofloxacin, whereas gentamicin, polymyxin B and cefuroxime failed to clear intracellular bacteria over 24 h. Intracellular Pseudomonas infection was resistant to chloramphenicol, with hCF death observed by 9 h. Eventual growth of remaining intracellular Pseudomonas was observed in hCF after removal of all antibiotics, resulting in re-infection cycles and cell death by 48 h. All of the antibiotics reduced significantly ( P < 0.05) IL-1β secretion by hCF infected with a Multiplicity Of Infection (MOI) = 1 of PAO1. With higher MOI, no pro-inflammatory effects were observed with antibiotic treatment, expect with polymyxin B and ofloxacin, which induced significant increased IL-1β secretion ( P < 0.001). The findings from our study demonstrated that bactericidal and bacteriostatic antibiotics, routinely used to treat BK, failed to eradicate Pseudomonas infection of hCFs in vitro and that their bactericidal efficacies were influenced by the cellular location of the organism.

Mycoplasma gallisepticum is one of the most important pathogens that cause chronic respiratory disease in chicken. This study investigated the antibacterial activity of doxycycline against M. gallisepticum strain S6. In static time-killing studies with constant antibiotic concentrations [0-64 minimum inhibitory concentration (MIC)], M. gallisepticum colonies were quantified and kill rates were calculated to estimate the drug effect. The half-life of doxycycline in chicken was 6.51 ± 0.63 h. An in vitro dynamic model (the drug concentrations are fluctuant) was also established and two half-lives of 6.51 and 12 h were simulated. The samples were collected for drug concentration determination and viable counting of M. gallisepticum. In static time-killing studies, doxycycline produced a maximum antimycoplasmal effect of 5.62log10 (CFU/mL) reduction and the maximum kill rate was 0.11 h(-1). In the in vitro dynamic model, doxycycline had a mycoplasmacidal activity in the two regimens, and the maximum antimycoplasmal effects were 4.1 and 4.75log10 (CFU/mL) reduction, respectively. Furthermore, the cumulative percentage of time over a 48-h period that the drug concentration exceeds the MIC (%T > MIC) was the pharmacokinetic-pharmacodynamic index that best correlated with antimicrobial efficacy (R (2) = 0.986, compared with 0.897 for the peak level divided by the MIC and 0.953 for the area under the concentration-time curve over 48 h divided by the MIC). The estimated %T > MIC values for 0log10 (CFU/mL) reduction, 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction were 32.48, 45.68, and 54.36%, respectively, during 48 h treatment period of doxycycline. In conclusion, doxycycline shows excellent effectiveness and time-dependent characteristics against M. gallisepticum strain S6 in vitro. Additionally, these results will guide optimal dosing strategies of doxycycline in M. gallisepticum infection.