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Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laserbased powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains.

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laserbased powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laser-based powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains., This work is conducted within the Additive Manufacturing for the Life Sciences Competence Center (AM4Life). The authors gratefully acknowledge financial support from Sweden's Innovation Agency VINNOVA (Grant no: 2019-00029). The authors acknowledge financial support from the Swedish Foundation for Strategic Research (RMA15-0010).

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laserbased powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains., QC 20240722

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laserbased powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains.

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laserbased powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laser-based powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains., This work is conducted within the Additive Manufacturing for the Life Sciences Competence Center (AM4Life). The authors gratefully acknowledge financial support from Sweden's Innovation Agency VINNOVA (Grant no: 2019-00029). The authors acknowledge financial support from the Swedish Foundation for Strategic Research (RMA15-0010).

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laserbased powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains.

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

The use and misuse of antibiotics have resulted in the selection of difficult-to-treat resistant bacteria. Two key parameters that influence the selection of resistant bacteria are the minimal selective concentration (MSC) and the fitness cost of resistance, both of which have been measured during planktonic growth in several studies. However, bacterial growth most often occurs in biofilms, and it is unclear if and how these parameters differ under these two growth conditions. To address this knowledge gap, we compared a selection of several types of antibiotic-resistant Escherichia coli mutants during planktonic and biofilm growth to determine the fitness costs and MSCs. Biofilm-forming Escherichia coli strains are commonly found in catheter-associated and recurrent urinary tract infections. Isogenic strains of a biofilm-forming E. coli strain, differing only in the resistance mechanisms and the fluorescent markers, were constructed, and susceptible and resistant bacteria were grown in head-to-head competitions at various concentrations of antibiotics under planktonic and biofilm conditions. Mutants with resistance to five different antibiotics were studied. The results show that during both planktonic and biofilm growth, selection for the resistant mutants occurred for all antibiotics at sub-MICs far below the MIC of the antibiotic. Even though differences were seen, the MSC values and the fitness costs did not differ systematically between planktonic and biofilm growth, implying that despite the different growth modes, the basic selection parameters are similar. These findings highlight the risk that resistant mutants may, similarly to planktonic growth, also be selected at sub-MICs of antibiotics in biofilms. IMPORTANCE Our understanding of how and where antibiotic resistance is selected in response to antibiotic exposure is still limited, and this is particularly true for selective processes when bacteria are growing in biofilms, arguably the most significant m

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

Fine manipulation of particles is essential for the analysis of complex samples such as blood or environmental water, where rare particles of interest may be masked by millions of others. Inertial focusing is amongst the most promising techniques for this task, enabling label-free manipulation of particles with sub-micron resolution at very high flow rates. However, the phenomenon still remains difficult to predict due to the focus position shifting in tortuous ways as function of the channel geometry, flow rate and particle size. Here, we present a new line of microfluidics that exploit inertial focusing in High Aspect Ratio Curved (HARC) microchannels and overcome this limitation. Consisting of a single curved channel, HARC systems provide a highly predictable, single focus position near the centre of the inner wall, largely independent of the flow rate and particle size. An explanation of the mechanism of migration and focus of particles, together with its governing equations, is provided based on simulations in COMSOL Multiphysics and experimental results. HARC microchannels built in silicon-glass were used for experimental validation, achieving a high quality, single focus position for a range of microparticles with sizes of 0.7 - 1 µm and bacterial cells (Escherichia coli). The recovery of 1 µm particles was 99.84% with a factor four in concentration. With a stable focus position, we envision that HARC systems will bring the technology closer to implementation in laboratories for analysis of complex fluids with biological particles like cells and organelles.

Fine manipulation of particles is essential for the analysis of complex samples such as blood or environmental water, where rare particles of interest may be masked by millions of others. Inertial focusing is amongst the most promising techniques for this task, enabling label-free manipulation of particles with sub-micron resolution at very high flow rates. However, the phenomenon still remains difficult to predict due to the focus position shifting in tortuous ways as function of the channel geometry, flow rate and particle size. Here, we present a new line of microfluidics that exploit inertial focusing in High Aspect Ratio Curved (HARC) microchannels and overcome this limitation. Consisting of a single curved channel, HARC systems provide a highly predictable, single focus position near the centre of the inner wall, largely independent of the flow rate and particle size. An explanation of the mechanism of migration and focus of particles, together with its governing equations, is provided based on simulations in COMSOL Multiphysics and experimental results. HARC microchannels built in silicon-glass were used for experimental validation, achieving a high quality, single focus position for a range of microparticles with sizes of 0.7 - 1 µm and bacterial cells (Escherichia coli). The recovery of 1 µm particles was 99.84% with a factor four in concentration. With a stable focus position, we envision that HARC systems will bring the technology closer to implementation in laboratories for analysis of complex fluids with biological particles like cells and organelles.

Fine manipulation of particles is essential for the analysis of complex samples such as blood or environmental water, where rare particles of interest may be masked by millions of others. Inertial focusing is amongst the most promising techniques for this task, enabling label-free manipulation of particles with sub-micron resolution at very high flow rates. However, the phenomenon still remains difficult to predict due to the focus position shifting in tortuous ways as function of the channel geometry, flow rate and particle size. Here, we present a new line of microfluidics that exploit inertial focusing in High Aspect Ratio Curved (HARC) microchannels and overcome this limitation. Consisting of a single curved channel, HARC systems provide a highly predictable, single focus position near the centre of the inner wall, largely independent of the flow rate and particle size. An explanation of the mechanism of migration and focus of particles, together with its governing equations, is provided based on simulations in COMSOL Multiphysics and experimental results. HARC microchannels built in silicon-glass were used for experimental validation, achieving a high quality, single focus position for a range of microparticles with sizes of 0.7 - 1 µm and bacterial cells (Escherichia coli). The recovery of 1 µm particles was 99.84% with a factor four in concentration. With a stable focus position, we envision that HARC systems will bring the technology closer to implementation in laboratories for analysis of complex fluids with biological particles like cells and organelles.

Bacterial populations can show heteroresistance, where an antibiotic resistant subpopulation is part of a susceptible one. Pereira et al. show in Escherichia coli and Salmonella enterica that disk diffusion, a common antibiotic susceptibility test, underestimates the occurrence of heteroresistance in clinical isolates. Many bacterial species and antibiotic classes exhibit heteroresistance, a phenomenon in which a susceptible bacterial isolate harbors a resistant subpopulation that can grow in the presence of an antibiotic and cause treatment failure. The resistant phenotype is often unstable and without antibiotic selection it reverts back to susceptibility. Here we studied the dynamics by which these resistant subpopulations are enriched in the presence of antibiotic and recede back to their baseline frequency in the absence of selection. An increasing understanding of this instability will allow more effective diagnostics and treatment of infections caused by heteroresistant bacteria. We show for clinical isolates of Escherichia coli and Salmonella enterica that different antibiotics at levels below the MIC of the susceptible main population can cause rapid enrichment of resistant subpopulations with increased copy number of genes that cause resistance. Modelling and growth rate measurements of bacteria with increased gene copy number in cultures and by microscopy of single-cells in a microfluidic chip show that the fitness cost of gene amplifications and their intrinsic instability drives their rapid loss in the absence of selection. Using a common antibiotic susceptibility test, we demonstrate that this test strongly underestimates the occurrence of heteroresistance in clinical isolates.

Bacterial populations can show heteroresistance, where an antibiotic resistant subpopulation is part of a susceptible one. Pereira et al. show in Escherichia coli and Salmonella enterica that disk diffusion, a common antibiotic susceptibility test, underestimates the occurrence of heteroresistance in clinical isolates. Many bacterial species and antibiotic classes exhibit heteroresistance, a phenomenon in which a susceptible bacterial isolate harbors a resistant subpopulation that can grow in the presence of an antibiotic and cause treatment failure. The resistant phenotype is often unstable and without antibiotic selection it reverts back to susceptibility. Here we studied the dynamics by which these resistant subpopulations are enriched in the presence of antibiotic and recede back to their baseline frequency in the absence of selection. An increasing understanding of this instability will allow more effective diagnostics and treatment of infections caused by heteroresistant bacteria. We show for clinical isolates of Escherichia coli and Salmonella enterica that different antibiotics at levels below the MIC of the susceptible main population can cause rapid enrichment of resistant subpopulations with increased copy number of genes that cause resistance. Modelling and growth rate measurements of bacteria with increased gene copy number in cultures and by microscopy of single-cells in a microfluidic chip show that the fitness cost of gene amplifications and their intrinsic instability drives their rapid loss in the absence of selection. Using a common antibiotic susceptibility test, we demonstrate that this test strongly underestimates the occurrence of heteroresistance in clinical isolates.

Bacterial populations can show heteroresistance, where an antibiotic resistant subpopulation is part of a susceptible one. Pereira et al. show in Escherichia coli and Salmonella enterica that disk diffusion, a common antibiotic susceptibility test, underestimates the occurrence of heteroresistance in clinical isolates. Many bacterial species and antibiotic classes exhibit heteroresistance, a phenomenon in which a susceptible bacterial isolate harbors a resistant subpopulation that can grow in the presence of an antibiotic and cause treatment failure. The resistant phenotype is often unstable and without antibiotic selection it reverts back to susceptibility. Here we studied the dynamics by which these resistant subpopulations are enriched in the presence of antibiotic and recede back to their baseline frequency in the absence of selection. An increasing understanding of this instability will allow more effective diagnostics and treatment of infections caused by heteroresistant bacteria. We show for clinical isolates of Escherichia coli and Salmonella enterica that different antibiotics at levels below the MIC of the susceptible main population can cause rapid enrichment of resistant subpopulations with increased copy number of genes that cause resistance. Modelling and growth rate measurements of bacteria with increased gene copy number in cultures and by microscopy of single-cells in a microfluidic chip show that the fitness cost of gene amplifications and their intrinsic instability drives their rapid loss in the absence of selection. Using a common antibiotic susceptibility test, we demonstrate that this test strongly underestimates the occurrence of heteroresistance in clinical isolates.

Alternative ways to prevent and treat infectious diseases are needed. Previously, we identified a fungal peptide, NZX, that was comparable to rifampicin in lowering M. tuberculosis load in a murine tuberculosis (TB) infection model. Here we assessed the potential synergy between this cationic host defence peptide (CHDP) and the current TB drugs and analysed its pharmacokinetics. We found additive effect of this peptide with isoniazid and ethambutol and confirmed these results with ethambutol in a murine TB-model. In vivo, the peptide remained stable in circulation and preserved lung structure better than ethambutol alone. Antibiotic resistance studies did not induce mutants with reduced susceptibility to the peptide. We further observed that this peptide was effective against nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, and several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In conclusion, the presented data supports a role for this CHDP in the treatment of drug resistant organisms.

Alternative ways to prevent and treat infectious diseases are needed. Previously, we identified a fungal peptide, NZX, that was comparable to rifampicin in lowering M. tuberculosis load in a murine tuberculosis (TB) infection model. Here we assessed the potential synergy between this cationic host defence peptide (CHDP) and the current TB drugs and analysed its pharmacokinetics. We found additive effect of this peptide with isoniazid and ethambutol and confirmed these results with ethambutol in a murine TB-model. In vivo, the peptide remained stable in circulation and preserved lung structure better than ethambutol alone. Antibiotic resistance studies did not induce mutants with reduced susceptibility to the peptide. We further observed that this peptide was effective against nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, and several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In conclusion, the presented data supports a role for this CHDP in the treatment of drug resistant organisms.

Alternative ways to prevent and treat infectious diseases are needed. Previously, we identified a fungal peptide, NZX, that was comparable to rifampicin in lowering M. tuberculosis load in a murine tuberculosis (TB) infection model. Here we assessed the potential synergy between this cationic host defence peptide (CHDP) and the current TB drugs and analysed its pharmacokinetics. We found additive effect of this peptide with isoniazid and ethambutol and confirmed these results with ethambutol in a murine TB-model. In vivo, the peptide remained stable in circulation and preserved lung structure better than ethambutol alone. Antibiotic resistance studies did not induce mutants with reduced susceptibility to the peptide. We further observed that this peptide was effective against nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, and several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In conclusion, the presented data supports a role for this CHDP in the treatment of drug resistant organisms.

Fine manipulation of particles is essential for the analysis of complex samples such as blood or environmental water, where rare particles of interest may be masked by millions of others. Inertial focusing is amongst the most promising techniques for this task, enabling label-free manipulation of particles with sub-micron resolution at very high flow rates. However, the phenomenon still remains difficult to predict due to the focus position shifting in tortuous ways as function of the channel geometry, flow rate and particle size. Here, we present a new line of microfluidics that exploit inertial focusing in High Aspect Ratio Curved (HARC) microchannels and overcome this limitation. Consisting of a single curved channel, HARC systems provide a highly predictable, single focus position near the centre of the inner wall, largely independent of the flow rate and particle size. An explanation of the mechanism of migration and focus of particles, together with its governing equations, is provided based on simulations in COMSOL Multiphysics and experimental results. HARC microchannels built in silicon-glass were used for experimental validation, achieving a high quality, single focus position for a range of microparticles with sizes of 0.7 - 1 µm and bacterial cells (Escherichia coli). The recovery of 1 µm particles was 99.84% with a factor four in concentration. With a stable focus position, we envision that HARC systems will bring the technology closer to implementation in laboratories for analysis of complex fluids with biological particles like cells and organelles.

Bacterial populations can show heteroresistance, where an antibiotic resistant subpopulation is part of a susceptible one. Pereira et al. show in Escherichia coli and Salmonella enterica that disk diffusion, a common antibiotic susceptibility test, underestimates the occurrence of heteroresistance in clinical isolates. Many bacterial species and antibiotic classes exhibit heteroresistance, a phenomenon in which a susceptible bacterial isolate harbors a resistant subpopulation that can grow in the presence of an antibiotic and cause treatment failure. The resistant phenotype is often unstable and without antibiotic selection it reverts back to susceptibility. Here we studied the dynamics by which these resistant subpopulations are enriched in the presence of antibiotic and recede back to their baseline frequency in the absence of selection. An increasing understanding of this instability will allow more effective diagnostics and treatment of infections caused by heteroresistant bacteria. We show for clinical isolates of Escherichia coli and Salmonella enterica that different antibiotics at levels below the MIC of the susceptible main population can cause rapid enrichment of resistant subpopulations with increased copy number of genes that cause resistance. Modelling and growth rate measurements of bacteria with increased gene copy number in cultures and by microscopy of single-cells in a microfluidic chip show that the fitness cost of gene amplifications and their intrinsic instability drives their rapid loss in the absence of selection. Using a common antibiotic susceptibility test, we demonstrate that this test strongly underestimates the occurrence of heteroresistance in clinical isolates.

Alternative ways to prevent and treat infectious diseases are needed. Previously, we identified a fungal peptide, NZX, that was comparable to rifampicin in lowering M. tuberculosis load in a murine tuberculosis (TB) infection model. Here we assessed the potential synergy between this cationic host defence peptide (CHDP) and the current TB drugs and analysed its pharmacokinetics. We found additive effect of this peptide with isoniazid and ethambutol and confirmed these results with ethambutol in a murine TB-model. In vivo, the peptide remained stable in circulation and preserved lung structure better than ethambutol alone. Antibiotic resistance studies did not induce mutants with reduced susceptibility to the peptide. We further observed that this peptide was effective against nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, and several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In conclusion, the presented data supports a role for this CHDP in the treatment of drug resistant organisms.

Fine manipulation of particles is essential for the analysis of complex samples such as blood or environmental water, where rare particles of interest may be masked by millions of others. Inertial focusing is amongst the most promising techniques for this task, enabling label-free manipulation of particles with sub-micron resolution at very high flow rates. However, the phenomenon still remains difficult to predict due to the focus position shifting in tortuous ways as function of the channel geometry, flow rate and particle size. Here, we present a new line of microfluidics that exploit inertial focusing in High Aspect Ratio Curved (HARC) microchannels and overcome this limitation. Consisting of a single curved channel, HARC systems provide a highly predictable, single focus position near the centre of the inner wall, largely independent of the flow rate and particle size. An explanation of the mechanism of migration and focus of particles, together with its governing equations, is provided based on simulations in COMSOL Multiphysics and experimental results. HARC microchannels built in silicon-glass were used for experimental validation, achieving a high quality, single focus position for a range of microparticles with sizes of 0.7 - 1 µm and bacterial cells (Escherichia coli). The recovery of 1 µm particles was 99.84% with a factor four in concentration. With a stable focus position, we envision that HARC systems will bring the technology closer to implementation in laboratories for analysis of complex fluids with biological particles like cells and organelles.

Bacterial populations can show heteroresistance, where an antibiotic resistant subpopulation is part of a susceptible one. Pereira et al. show in Escherichia coli and Salmonella enterica that disk diffusion, a common antibiotic susceptibility test, underestimates the occurrence of heteroresistance in clinical isolates. Many bacterial species and antibiotic classes exhibit heteroresistance, a phenomenon in which a susceptible bacterial isolate harbors a resistant subpopulation that can grow in the presence of an antibiotic and cause treatment failure. The resistant phenotype is often unstable and without antibiotic selection it reverts back to susceptibility. Here we studied the dynamics by which these resistant subpopulations are enriched in the presence of antibiotic and recede back to their baseline frequency in the absence of selection. An increasing understanding of this instability will allow more effective diagnostics and treatment of infections caused by heteroresistant bacteria. We show for clinical isolates of Escherichia coli and Salmonella enterica that different antibiotics at levels below the MIC of the susceptible main population can cause rapid enrichment of resistant subpopulations with increased copy number of genes that cause resistance. Modelling and growth rate measurements of bacteria with increased gene copy number in cultures and by microscopy of single-cells in a microfluidic chip show that the fitness cost of gene amplifications and their intrinsic instability drives their rapid loss in the absence of selection. Using a common antibiotic susceptibility test, we demonstrate that this test strongly underestimates the occurrence of heteroresistance in clinical isolates.

Alternative ways to prevent and treat infectious diseases are needed. Previously, we identified a fungal peptide, NZX, that was comparable to rifampicin in lowering M. tuberculosis load in a murine tuberculosis (TB) infection model. Here we assessed the potential synergy between this cationic host defence peptide (CHDP) and the current TB drugs and analysed its pharmacokinetics. We found additive effect of this peptide with isoniazid and ethambutol and confirmed these results with ethambutol in a murine TB-model. In vivo, the peptide remained stable in circulation and preserved lung structure better than ethambutol alone. Antibiotic resistance studies did not induce mutants with reduced susceptibility to the peptide. We further observed that this peptide was effective against nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, and several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In conclusion, the presented data supports a role for this CHDP in the treatment of drug resistant organisms.

Antimicrobial peptides (AMPs) are essential components of the innate immune system and have been proposed as promising therapeutic agents against drug-resistant microbes. AMPs possess a rapid bactericidal mode of action and can interact with different targets, but bacteria can also avoid their effect through a variety of resistance mechanisms. Apart from hampering treatment by the AMP itself, or that by other antibiotics in the case of cross-resistance, AMP resistance might also confer cross-resistance to innate human peptides and impair the anti-infective capability of the human host. A better understanding of how resistance to AMPs is acquired and the genetic mechanisms involved is needed before using these compounds as therapeutic agents. Using experimental evolution and whole-genome sequencing, we determined the genetic causes and the effect of acquired de novo resistance to three different AMPs in the opportunistic pathogen Stenotrophomonas maltophilia, a bacterium that is intrinsically resistant to a wide range of antibiotics. Our results show that AMP exposure selects for high-level resistance, generally without any reduction in bacterial fitness, conferred by mutations in different genes encoding enzymes, transporters, transcriptional regulators, and other functions. Cross-resistance to AMPs and to other antibiotic classes not used for selection, as well as collateral sensitivity, was observed for many of the evolved populations. The relative ease by which high-level AMP resistance is acquired, combined with the occurrence of cross-resistance to conventional antibiotics and the maintained bacterial fitness of the analyzed mutants, highlights the need for careful studies of S. maltophilia resistance evolution to clinically valuable AMPs. IMPORTANCE Stenotrophomonas maltophilia is an increasingly relevant multidrug-resistant (MDR) bacterium found, for example, in people with cystic fibrosis and associated with other respiratory infections and underlying pathol

A structure-activity relationship (SAR) study of NOSO-95179, a nonapeptide from the Odilorhabdin class of antibacterials, was performed by systematic variations of amino acids in positions 2 and 5 of the peptide. A series of non-proteinogenic amino acids was synthesized in high enantiomeric purity from Williams' chiral diphenyloxazinone by highly diastereoselective alkylation or by aldol-type reaction. NOSO-95179 analogues for SAR studies were prepared using solid-phase peptide synthesis. Inhibition of bacterial translation by each of the synthesized Odilorhabdin analogues was measured using an in vitro test. For the most efficient analogues, antibacterial efficacy was measured against two wild-type Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae) and against an efflux defective E. coli strain (Delta tolC) to evaluate the impact of efflux on the antibacterial activity.

Antimicrobial peptides (AMPs) are essential components of the innate immune system and have been proposed as promising therapeutic agents against drug-resistant microbes. AMPs possess a rapid bactericidal mode of action and can interact with different targets, but bacteria can also avoid their effect through a variety of resistance mechanisms. Apart from hampering treatment by the AMP itself, or that by other antibiotics in the case of cross-resistance, AMP resistance might also confer cross-resistance to innate human peptides and impair the anti-infective capability of the human host. A better understanding of how resistance to AMPs is acquired and the genetic mechanisms involved is needed before using these compounds as therapeutic agents. Using experimental evolution and whole-genome sequencing, we determined the genetic causes and the effect of acquired de novo resistance to three different AMPs in the opportunistic pathogen Stenotrophomonas maltophilia, a bacterium that is intrinsically resistant to a wide range of antibiotics. Our results show that AMP exposure selects for high-level resistance, generally without any reduction in bacterial fitness, conferred by mutations in different genes encoding enzymes, transporters, transcriptional regulators, and other functions. Cross-resistance to AMPs and to other antibiotic classes not used for selection, as well as collateral sensitivity, was observed for many of the evolved populations. The relative ease by which high-level AMP resistance is acquired, combined with the occurrence of cross-resistance to conventional antibiotics and the maintained bacterial fitness of the analyzed mutants, highlights the need for careful studies of S. maltophilia resistance evolution to clinically valuable AMPs. IMPORTANCE Stenotrophomonas maltophilia is an increasingly relevant multidrug-resistant (MDR) bacterium found, for example, in people with cystic fibrosis and associated with other respiratory infections and underlying pathol

Antimicrobial peptides (AMPs) are essential components of the innate immune system and have been proposed as promising therapeutic agents against drug-resistant microbes. AMPs possess a rapid bactericidal mode of action and can interact with different targets, but bacteria can also avoid their effect through a variety of resistance mechanisms. Apart from hampering treatment by the AMP itself, or that by other antibiotics in the case of cross-resistance, AMP resistance might also confer cross-resistance to innate human peptides and impair the anti-infective capability of the human host. A better understanding of how resistance to AMPs is acquired and the genetic mechanisms involved is needed before using these compounds as therapeutic agents. Using experimental evolution and whole-genome sequencing, we determined the genetic causes and the effect of acquired de novo resistance to three different AMPs in the opportunistic pathogen Stenotrophomonas maltophilia, a bacterium that is intrinsically resistant to a wide range of antibiotics. Our results show that AMP exposure selects for high-level resistance, generally without any reduction in bacterial fitness, conferred by mutations in different genes encoding enzymes, transporters, transcriptional regulators, and other functions. Cross-resistance to AMPs and to other antibiotic classes not used for selection, as well as collateral sensitivity, was observed for many of the evolved populations. The relative ease by which high-level AMP resistance is acquired, combined with the occurrence of cross-resistance to conventional antibiotics and the maintained bacterial fitness of the analyzed mutants, highlights the need for careful studies of S. maltophilia resistance evolution to clinically valuable AMPs. IMPORTANCE Stenotrophomonas maltophilia is an increasingly relevant multidrug-resistant (MDR) bacterium found, for example, in people with cystic fibrosis and associated with other respiratory infections and underlying pathol

Antimicrobial peptides (AMPs) are essential components of the innate immune system and have been proposed as promising therapeutic agents against drug-resistant microbes. AMPs possess a rapid bactericidal mode of action and can interact with different targets, but bacteria can also avoid their effect through a variety of resistance mechanisms. Apart from hampering treatment by the AMP itself, or that by other antibiotics in the case of cross-resistance, AMP resistance might also confer cross-resistance to innate human peptides and impair the anti-infective capability of the human host. A better understanding of how resistance to AMPs is acquired and the genetic mechanisms involved is needed before using these compounds as therapeutic agents. Using experimental evolution and whole-genome sequencing, we determined the genetic causes and the effect of acquired de novo resistance to three different AMPs in the opportunistic pathogen Stenotrophomonas maltophilia, a bacterium that is intrinsically resistant to a wide range of antibiotics. Our results show that AMP exposure selects for high-level resistance, generally without any reduction in bacterial fitness, conferred by mutations in different genes encoding enzymes, transporters, transcriptional regulators, and other functions. Cross-resistance to AMPs and to other antibiotic classes not used for selection, as well as collateral sensitivity, was observed for many of the evolved populations. The relative ease by which high-level AMP resistance is acquired, combined with the occurrence of cross-resistance to conventional antibiotics and the maintained bacterial fitness of the analyzed mutants, highlights the need for careful studies of S. maltophilia resistance evolution to clinically valuable AMPs.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Antimicrobial peptides (AMPs) are essential components of the innate immune system and have been proposed as promising therapeutic agents against drug-resistant microbes. AMPs possess a rapid bactericidal mode of action and can interact with different targets, but bacteria can also avoid their effect through a variety of resistance mechanisms. Apart from hampering treatment by the AMP itself, or that by other antibiotics in the case of cross-resistance, AMP resistance might also confer cross-resistance to innate human peptides and impair the anti-infective capability of the human host. A better understanding of how resistance to AMPs is acquired and the genetic mechanisms involved is needed before using these compounds as therapeutic agents. Using experimental evolution and whole-genome sequencing, we determined the genetic causes and the effect of acquired de novo resistance to three different AMPs in the opportunistic pathogen Stenotrophomonas maltophilia, a bacterium that is intrinsically resistant to a wide range of antibiotics. Our results show that AMP exposure selects for high-level resistance, generally without any reduction in bacterial fitness, conferred by mutations in different genes encoding enzymes, transporters, transcriptional regulators, and other functions. Cross-resistance to AMPs and to other antibiotic classes not used for selection, as well as collateral sensitivity, was observed for many of the evolved populations. The relative ease by which high-level AMP resistance is acquired, combined with the occurrence of cross-resistance to conventional antibiotics and the maintained bacterial fitness of the analyzed mutants, highlights the need for careful studies of S. maltophilia resistance evolution to clinically valuable AMPs. IMPORTANCE Stenotrophomonas maltophilia is an increasingly relevant multidrug-resistant (MDR) bacterium found, for example, in people with cystic fibrosis and associated with other respiratory infections and underlying pathol