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Intracellular delivery is critical for a plethora of biomedical applications, including mRNA transfection and gene editing. High transfection efficiency and low cytotoxicity, however, are often beyond the capabilities of bulk techniques and synonymous with extensive empirical optimization. Moreover, bulk techniques are not amenable to large screening applications. Here, we propose an expeditious workflow for achieving optimal electroporation-based intracellular delivery. Using the multiplexing ability of a high-definition microelectrode array (MEA) chip, we performed a sequence of carefully designed experiments, multiple linear regression modelling and validation to obtain optimal conditions for on-chip electroporation of primary fibroblasts. Five electric pulse parameters were varied to generate 32 different electroporation conditions. The effect of the parameters on cytotoxicity and intracellular delivery could be evaluated with just two experiments. Most successful electroporation conditions resulted in no cell death, highlighting the low cytotoxicity of on-chip electroporation. The resulting delivery models were then used to achieve dosage-controlled delivery of small molecules, delivery of Cas9-GFP single-guide RNA complexes and transfection with an mCherry-encoding mRNA, resulting in previously unreported high-efficiency, single-cell transfection on MEAs: cells expressed mCherry on 81% of the actuated electrodes, underscoring the vast potential of CMOS MEA technology for the transfection of primary cells.

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections. ispartof: BIOSENSORS & BIOELECTRONICS vol:217 ispartof: location:England status: published

Partitions in digital PCR (dPCR) assays do not reach the detection threshold at the same time. This heterogeneity in amplification results in intermediate endpoint fluorescence values (i.e., rain) and misclassification of partitions, which has a major impact on the accuracy of nucleic acid quantification. Rain most often results from a reduced amplification efficiency or template inaccessibility; however, exactly how these contribute to rain has not been described. We developed and experimentally validated an analytical model that mechanistically explains the relationship between amplification efficiency, template accessibility, and rain. Using Monte Carlo simulations, we show that a reduced amplification efficiency leads to broader threshold cycle (

Advances in gene editing and cell therapies have recently led to outstanding clinical successes. However, the lack of a cost-effective manufacturing process prevents the democratization of these innovative medical tools. Due to the common use of viral vectors, the step of transfection in which cells are engineered to gain new functions, is a major bottleneck in making safe and affordable cell products. A promising opportunity lies in Single-Cell Transfection Technologies (SCTTs). SCTTs have demonstrated higher efficiency, safety and scalability than conventional transfection methods. They can also feature unique abilities such as substantial dosage control over the cargo delivery, single-cell addressability and integration in microdevices comprising multiple monitoring modalities. Unfortunately, the potential of SCTTs is not fully appreciated: they are most often restricted to research settings with little adoption in clinical settings. To encourage their adoption, we review and compare recent developments in SCTTs, and how they can enable selected clinical applications. To help bridge the gap between fundamental research and its translation to the clinic, we also describe how Good Manufacturing Practices (GMP) can be integrated in the design of SCTTs. ispartof: JOURNAL OF CONTROLLED RELEASE vol:330 pages:963-975 ispartof: location:Netherlands status: published

Forensic Short Tandem Repeat (STR) genotyping is almost exclusively performed by capillary electrophoresis (CE) in specialized laboratories. As an alternative to CE, and to enable miniaturized lab-on-a-chip STR profiling, we developed the QueSTR probes, a hybridization-based genotyping assay that relies on the recognition and cleavage of an RNA:DNA duplex by the RNase H2 enzyme. For each STR allele to be genotyped, a matching DNA probe containing one RNA moiety is designed. After performing asymmetric STR PCR, a hybridization curve analysis indicates the matching probe(s), and thus indicates the allele(s) present in the sample. Accurate genotyping of 13 samples was obtained using the QueSTR probes for three CODIS core loci (D165539, D75820, and TH01). A probe corresponding to the TH01 9.3 allele was included to demonstrate accurate genotyping, even in the presence of a partial repeat. The QueSTR probes are a valuable option to miniaturize STR genotyping in lab-on-a-chip devices that cannot harbor a CE analysis.

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, a cornerstone for any disease-control strategy, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It potentially measures contagiousness directly, the sample is easy to obtain, its production can be standardized between patients, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an in-situ RT-qPCR molecular assay to detect SARS-CoV-2 shedding. We demonstrate that sampling subjects using a one-minute breathing protocol, yields sufficient viral RNA to detect infections with a sensitivity comparable to standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test is the first to consistently report a negative result, putatively signaling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

The demand for forensic DNA profiling at the crime scene or at police stations is increasing. DNA profiling is currently performed in specialized laboratories by PCR amplification of Short Tandem Repeats (STR) followed by amplicon sizing using capillary electrophoresis. The need for bulky equipment to identify alleles after PCR presents a challenge for shifting to a decentralized workflow. We devised a novel hybridization-based STR-genotyping method, using Short Tandem Repeat Identification (STRide) probes, which could help tackle this issue. STRide probes are fluorescently labeled oligonucleotides that rely on the quenching properties of guanine on fluorescein derivatives. Mismatches between STRide probes and amplicons can be detected by melting curve analysis after asymmetric PCR. The functionality of the STRide probes was demonstrated by analyzing synthetic DNA samples for the D16S539 locus. Next, STRide probes were developed for five different CODIS core loci (D16S539, TH01, TPOX, FGA, and D7S820). These probes were validated by analyzing 13 human DNA samples. Successful genotyping was obtained using inputs as low as 31 pg of DNA, demonstrating high sensitivity. The STRide probes are ideally suited to be implemented in a microarray and present an important step towards a portable device for fast on-site forensic DNA fingerprinting. ispartof: BIOSENSORS & BIOELECTRONICS vol:180 ispartof: location:England status: published

In addition to the well-known strategies of antibiotic resistance and biofilm formation, bacterial populations possess an additional survival strategy to endure hostile environments or antibiotic exposure. A small fraction of transiently antibiotic-tolerant phenotypical variants, called persister cells, is capable of surviving treatment with high doses of antibiotics. When antibiotic pressure drops, persisters that switch back to a normal phenotype can resume growth, ensuring survival of the bacterial population. Persister cells have been identified in every major pathogen, contribute to the antibiotic tolerance observed in biofilms, and are responsible for the recalcitrant nature of chronic infections. Also, evidence is accumulating that persister cells can contribute to the emergence of antibiotic resistance. Consequently, effective treatment of persister cells could greatly improve patient outcome. The small number of persisters and the redundancy in mechanisms of persister formation impede target-based development of anti-persister therapies. Nonetheless, the armory of anti-persister molecules is increasing. This review presents a comprehensive overview of anti-persister molecules and strategies described in literature to date and offers perspectives on potential anti-persistence targets and methods for the development of future therapies. Furthermore, we highlight in vivo model systems for pre-clinical testing and summarize ongoing clinical trials of candidate anti-persister therapeutics.

Bacterial proliferation depends on the cells' capability to proceed through consecutive rounds of the cell cycle. The cell cycle consists of a series of events during which cells grow, copy their genome, partition the duplicated DNA into different cell halves and, ultimately, divide to produce two newly formed daughter cells. Cell cycle control is of the utmost importance to maintain the correct order of events and safeguard the integrity of the cell and its genomic information. This review covers insights into the regulation of individual key cell cycle events in Escherichia coli. The control of initiation of DNA replication, chromosome segregation and cell division is discussed. Furthermore, we highlight connections between these processes. Although detailed mechanistic insight into these connections is largely still emerging, it is clear that the different processes of the bacterial cell cycle are coordinated to one another. This careful coordination of events ensures that every daughter cell ends up with one complete and intact copy of the genome, which is vital for bacterial survival.

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*&ndash, GTP, but not ObgE*&ndash, GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.

Obg is a versatile GTPase that plays a pivotal role in bacterial persistence. We previously showed that the Escherichia coli homolog ObgE exerts this activity through transcriptional activation of a toxin-antitoxin module and subsequent membrane depolarization. Here, we assessed the role of G-domain functionality in ObgE-mediated persistence. Through screening of a mutant library, we identified five obgE alleles (with substitutions G166V, D246G, S270I, N283I and I313N) that have lost their persistence function and no longer activate hokB expression. These alleles support viability of a strain otherwise deprived of ObgE, indicating that ObgE's persistence function can be uncoupled from its essential role. Based on the ObgE crystal structure, we designed two additional mutant proteins (T193A and D286Y), one of which (D286Y) no longer affects persistence. Using isothermal titration calorimetry, stopped-flow experiments and kinetics, we subsequently assessed nucleotide binding and GTPase activity in all mutants. With the exception of the S270I mutant that is possibly affected in protein-protein interactions, all mutants that have lost their persistence function display severely reduced binding to GDP or the alarmone ppGpp. However, we find no clear relation between persistence and GTP or pppGpp binding nor with GTP hydrolysis. Combined, our results signify an important step toward understanding biochemical determinants underlying persistence. ispartof: MOLECULAR MICROBIOLOGY vol:112 issue:5 pages:1593-1608 ispartof: location:England status: Published online

For decades, mankind has dominated the battle against bacteria, yet the tide is slowly turning. Our antibacterial strategies are becoming less effective, allowing bacteria to get the upper hand. The alarming rise in antibiotic resistance is an important cause of anti-infective therapy failure. However, other factors are at play as well. It is widely recognized that bacterial populations display high levels of heterogeneity. Population heterogeneity generates phenotypes specialized in surviving antibiotic attacks. Nonetheless, the presence of antibiotic-insensitive subpopulations is not considered when initiating treatment. It is therefore time to reevaluate how we combat bacterial infections. We here focus on antibiotic persistence and heteroresistance, phenomena in which small fractions of the population are tolerant (persisters) and resistant to antibiotics, respectively. We discuss molecular mechanisms involved, their clinical importance, and possible therapeutic strategies. Moving forward, we argue that these heterogeneous phenotypes should no longer be ignored in clinical practice and that better diagnostic and therapeutic approaches are urgently needed. ispartof: Molecular Cell vol:76 issue:2 pages:255-267 ispartof: location:United States status: published

Antibiotic resistance poses an alarming and ever-increasing threat to modern health care. Although the current antibiotic crisis is widely acknowledged, actions taken so far have proved insufficient to slow down the rampant spread of resistant pathogens. Problematically, routine screening methods and strategies to restrict therapy failure almost exclusively focus on genetic resistance, while evidence for dangers posed by other bacterial survival strategies is mounting., Antibiotic resistance poses an alarming and ever-increasing threat to modern health care. Although the current antibiotic crisis is widely acknowledged, actions taken so far have proved insufficient to slow down the rampant spread of resistant pathogens. Problematically, routine screening methods and strategies to restrict therapy failure almost exclusively focus on genetic resistance, while evidence for dangers posed by other bacterial survival strategies is mounting. Antibiotic tolerance, occurring either population-wide or in a subpopulation of cells, allows bacteria to transiently overcome antibiotic treatment and is overlooked in clinical practice. In addition to prolonging treatment and causing relapsing infections, recent studies have revealed that tolerance also accelerates the emergence of resistance. These critical findings emphasize the need for strategies to combat tolerance, not only to improve treatment of recurrent infections but also to effectively address the problem of antibiotic resistance at the root.

Image-based high-throughput screening strategies for quantifying morphological phenotypes have proven widely successful. Here we describe a combined experimental and multivariate image analysis approach for systematic large-scale phenotyping of morphological dynamics in bacteria. Using off-the-shelf components and software, we established a workflow for high-throughput time-resolved microscopy. We then screened the single‐gene deletion collection of Escherichia coli for antibiotic-induced morphological changes. Using single-cell quantitative descriptors and supervised classification methods, we measured how different cell morphologies developed over time for all strains in response to the β-lactam antibiotic cefsulodin. 191 strains exhibit significant variations under antibiotic treatment. Phenotypic clustering provided insights into processes that alter the antibiotic response. Mutants with stable bulges show delayed lysis, contributing to antibiotic tolerance. Lipopolysaccharides play a crucial role in bulge stability. This study demonstrates how multiparametric phenotyping by high-throughput time-resolved imaging and computer-aided cell classification can be used for comprehensively studying dynamic morphological transitions in bacteria., In a high-throughput time-resolved microscopy screen, Taiyeb Zahir et al identify bacterial genes mediating morphological changes to antibiotics. An image analysis workflow enables the classification of single cells and deletion strains according to morphological changes.

The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis. ispartof: SCIENTIFIC REPORTS vol:9 issue:1 ispartof: location:England status: published

Persisters are transiently tolerant variants that allow populations to avoid eradication by antibiotic treatment. Their antibiotic tolerance is non-genetic, not inheritable and results from a phenotypic switch from the normal, sensitive cell type to the tolerant, persister state. Here we give a comprehensive overview on bacterial persistence. We first define persistence, summarize the various aspects of persister physiology and show their heterogeneous nature. We then focus on the role of key cellular processes and mechanisms controlling the formation of a subpopulation of tolerant cells. Being a prime example of a risk-spreading strategy, we next discuss the eco-evolutionary aspects of persistence, e.g. how persistence evolves in the face of treatment with antibiotics. Finally, we illustrate the clinical importance of persisters, as persistence is worsening the worldwide antibiotic crisis by prolonging antibiotic treatment, causing therapy failure or catalyzing the development of genetically encoded antibiotic resistance. A better understanding of this phenotype is critical in our fight against pathogenic bacteria and to obtain a better outlook on future therapies. ispartof: FEMS Microbiology Reviews vol:41 issue:3 pages:219-251 ispartof: location:England status: published

peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=ierz20 ispartof: EXPERT REVIEW OF ANTI-INFECTIVE THERAPY vol:14 issue:7 pages:613-616 ispartof: location:England status: published

Sulfite is often added to beverages as an antioxidant and antimicrobial agent. In fermented beverages, sulfite is also naturally produced by yeast cells. However, sulfite causes adverse health effects in asthmatic patients and accurate measurement of the sulfite concentration is therefore very important. Current sulfite analysis methods are time- and reagent-consuming and often require costly equipment. Here, we present a system allowing sensitive, ultralow-volume sulfite measurements based on a reusable glass-silicon microdroplet platform on which microdroplet generation, addition of enzymes through chemical-induced emulsion destabilization and pillar-induced droplet merging, emulsion restabilization, droplet incubation, and fluorescence measurements are integrated. In a first step, we developed and verified a fluorescence-based enzymatic assay for sulfite by measuring its analytical performance (LOD, LOQ, the dynamic working range, and the influence of salts, colorant, and sugars) and comparing fluorescent microplate readouts of fermentation samples with standard colorimetric measurements using the 5,5′-dithiobis-(2-nitrobenzoic acid) assay of the standard Gallery Plus Beermaster analysis platform. Next, samples were analyzed on the microdroplet platform, which also showed good correlation with the standard colorimetric analysis. Although the presented platform does not allow stable reinjection of droplets due to the presence of a tight array of micropillars at the fluidics entrances to prevent channel clogging by dust, removing the pillars, and integrating miniaturized pumps and optics in a future design would allow to use this platform for high-throughput, automated, and portable screening of microbes, plant, or mammalian cells. Graphical abstractᅟ Electronic supplementary material The online version of this article (10.1007/s00216-018-1516-6) contains supplementary material, which is available to authorized users.

In experimental evolution, laboratory-controlled conditions select for the adaptation of species, which can be monitored in real time. Despite the current popularity of such experiments, nature's most pervasive biological force was long believed to be observable only on time scales that transcend a researcher's life-span, and studying evolution by natural selection was therefore carried out solely by comparative means. Eventually, microorganisms' propensity for fast evolutionary changes proved us wrong, displaying strong evolutionary adaptations over a limited time, nowadays massively exploited in laboratory evolution experiments. Here, we formulate a guide to experimental evolution with microorganisms, explaining experimental design and discussing evolutionary dynamics and outcomes and how it is used to assess ecoevolutionary theories, improve industrially important traits, and untangle complex phenotypes. Specifically, we give a comprehensive overview of the setups used in experimental evolution. Additionally, we address population dynamics and genetic or phenotypic diversity during evolution experiments and expand upon contributing factors, such as epistasis and the consequences of (a)sexual reproduction. Dynamics and outcomes of evolution are most profoundly affected by the spatiotemporal nature of the selective environment, where changing environments might lead to generalists and structured environments could foster diversity, aided by, for example, clonal interference and negative frequency-dependent selection. We conclude with future perspectives, with an emphasis on possibilities offered by fast-paced technological progress. This work is meant to serve as an introduction to those new to the field of experimental evolution, as a guide to the budding experimentalist, and as a reference work to the seasoned expert. ispartof: MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS vol:82 issue:3 ispartof: location:United States status: published

Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1-3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5-7 fold) and mexX (3.6-5.4 fold) in the parental strain but to a lower extent (3-4-fold for gyrA and 1.8-2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target. ispartof: FRONTIERS IN MICROBIOLOGY vol:9 issue:JUL ispartof: location:Switzerland status: published

Antibiotic-tolerant persister cells are difficult to eradicate by conventional classes of antibiotics. Kim and colleagues have discovered a new class of synthetic retinoid antibiotics that kill Staphylococcus aureus persisters by disrupting their cytoplasmic membrane. ispartof: MOLECULAR CELL vol:70 issue:5 pages:763-764 ispartof: location:United States status: published

Clinical laboratory-based nucleic acid amplification tests (NAT) play an important role in diagnosing viral infections. However, laboratory infrastructure requirements and their failure to diagnose at the point-of-need (PON) limit their clinical utility in both resource-rich and -limited clinical settings. The development of fast and sensitive PON viral NAT may overcome these limitations. The scalability of silicon microchip manufacturing combined with advances in silicon microfluidics present an opportunity for development of rapid and sensitive PON NAT on silicon microchips. In the present study, we present rapid and sensitive NAT for a number of RNA and DNA viruses on the same silicon microchip platform. We first developed sensitive (4 copies per reaction) one-step RT-qPCR and qPCR assays detecting HCV, HIV, Zika, HPV 16, and HPV 18 on a benchtop real-time PCR instrument. A silicon microchip was designed with an etched 1.3 μL meandering microreactor, integrated aluminum heaters, thermal insulation trenches and microfluidic channels; this chip was used in all on-chip experiments. Melting curve analysis confirmed precise and localized heating of the microreactor. Following minimal optimization of reaction conditions, the bench-scale assays were successfully transferred to 1.3 μL silicon microreactors with reaction times of 25 min with no reduction in sensitivity, reproducibility, or reaction efficiencies. Taken together, these results demonstrate that rapid and sensitive detection of multiple viruses on the same silicon microchip platform is feasible. Further development of this technology, coupled with silicon microchip-based nucleic acid extraction solutions, could potentially shift viral nucleic acid detection and diagnosis from centralized clinical laboratories to the PON.

PCR enables sensitive and specific detection of infectious disease agents, but application in point-of-care diagnostic testing remains scarce. A compact tool that runs PCR assays in less than a few minutes and that relies on mass-producible, disposable reactors could revolutionize while-you-wait molecular testing. We here exploit well-established semiconductor manufacturing processes to produce silicon ultra-fast quantitative PCR (UF-qPCR) chips that can run PCR protocols with limited assay optimization. A total of 110 clinical samples were analyzed for the detection of group B streptococci using both a validated benchtop and an on-chip qPCR assay. For the on-chip assay, the total reaction time was reduced after optimization to less than 5 min. The standard curve, spanning a concentration range of 5 log units, yielded a PCR efficiency of 94%. The sensitivity obtained was 96% (96/100; CI: 90-98%) and the specificity 70% (7/10; CI: 40-90%). We show that if melting analyses would be integrated, the obtained sensitivity would drop slightly to 93% (CI: 86-96%), while the specificity would increase to 100% (CI: 72% - 100%). In comparison to the benchtop reference qPCR assay performed on a LightCycler©96, the on-chip assay demonstrated a highly significant qualitative (Spearman's rank correlation) and quantitative (linear regression) correlation. Using a mass-producible qPCR chip and limited assay optimization, we were able to develop a validated qPCR protocol that can be carried out in less than five minutes. The analytical performance of the microchip-based UF-qPCR system was shown to match that of a benchtop assay. This is the first report to provide UF-qPCR validation using clinical samples. We demonstrate that qPCR-based while-you-wait testing is feasible without jeopardizing assay performance. ispartof: TALANTA vol:192 pages:220-225 ispartof: location:Netherlands status: published

Persisters are transiently antibiotic-tolerant cells that complicate the treatment of bacterial infections. Both theory and experiments have suggested that persisters facilitate genetic resistance by constituting an evolutionary reservoir of viable cells. Here, we provide evidence for a strong positive correlation between persistence and the likelihood to become genetically resistant in natural and lab strains of E. coli. This correlation can be partly attributed to the increased availability of viable cells associated with persistence. However, our data additionally show that persistence is pleiotropically linked with mutation rates. Our theoretical model further demonstrates that increased survival and mutation rates jointly affect the likelihood of evolving clinical resistance. Overall, these results suggest that the battle against antibiotic resistance will benefit from incorporating anti-persister therapies.

The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (µPCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 µl each. Diluted reference DNA samples (1 ng-31 pg) were amplified on the µPCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both µPCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis. ispartof: SCIENTIFIC REPORTS vol:8 issue:1 ispartof: location:England status: published

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies. ispartof: Frontiers in Microbiology vol:9 issue:FEB ispartof: location:Switzerland status: published

Antibiotics typically fail to completely eradicate a bacterial population, leaving a small fraction of transiently antibiotic-tolerant persister cells intact. Persisters are therefore seen to be a major cause of treatment failure and greatly contribute to the recalcitrant nature of chronic infections. The current study focused on Pseudomonas aeruginosa , a Gram-negative pathogen belonging to the notorious ESKAPE group of pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species) and, due to increasing resistance against most conventional antibiotics, posing a serious threat to human health. Greatly contributing to the difficult treatment of P. aeruginosa infections is the presence of persister cells, and elimination of these cells would therefore significantly improve patient outcomes. In this study, a small-molecule library was screened for compounds that, in combination with the fluoroquinolone antibiotic ofloxacin, reduced the number of P. aeruginosa persisters compared to the number achieved with treatment with the antibiotic alone. Based on the early structure-activity relationship, 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009) was selected for further characterization. Combination of SPI009 with mechanistically distinct classes of antibiotics reduced the number of persisters up to 10 6 -fold in both lab strains and clinical isolates of P. aeruginosa . Further characterization of the compound revealed a direct and efficient killing of persister cells. SPI009 caused no erythrocyte damage and demonstrated minor cytotoxicity. In conclusion, we identified a novel antipersister compound active against P. aeruginosa with promising applications for the design of novel, case-specific combination therapies in the fight against chronic infections.

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.22939.001, eLife digest A cell’s DNA can acquire errors over the course of its lifetime. These errors, known as mutations, are often harmful and can cripple the cell. However, some mutations are needed to enable a cell or organism to adapt to changes in its environment. Since there is a trade-off between acquiring beneficial mutations versus harmful ones, cells carefully balance how often they acquire new mutations. Cells have several mechanisms that limit the number of mutations by correcting errors in DNA. Occasionally these repair mechanisms may fail so that a small number of cells in a population accumulate mutations more quickly than other cells. This process is known as “hypermutation” and it enables some cells to rapidly adapt to changing conditions in order to avoid the entire population from becoming extinct. So far, studies on hypermutation have largely been carried out under conditions that are mildly stressful to the cells, which only cause low frequency of hypermutation. However, little is known about the role of this process in cells under near-lethal levels of stress, for example, when drugs target bacteria or cancer cells in the human body. Swings et al. studied hypermutation in populations of a bacterium called Escherichia coli exposed to levels of alcohol that cause the bacteria to experience extreme stress. The experiments show that hypermutation occurs rapidly in these conditions and is essential for bacteria to adapt to the level of alcohol and avoid extinction. Populations of bacteria in which hypermutation did not occur were unable to develop tolerance to the alcohol and perished. Further experiments show that an individual population of bacteria can alter the rate of mutation (that is, how often new mutations arise) several times as a result of changing stress levels. The findings of Swings et al. suggest that hypermutation can rapidly arise in populations of cells that are experiencing extreme stress. Therefore, this process may pose a serious risk in the development of drug resistant bacteria and cancer cells. In the future, developing new drugs that target hypermutation may help to fight bacterial infections and cancer. DOI: http://dx.doi.org/10.7554/eLife.22939.002

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 μg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Plant growth-promoting rhizobacteria (PGPR) have been isolated from legume nodules, displaying a potential to enhance nodulation, growth and yield of legume plants when co-inoculated with Rhizobium. This study genetically characterizes bacteria isolated from bean root nodules in Cuba and investigates the effect of Rhizobium–Pseudomonas co-inoculation on common bean (Phaseolus vulgaris L.) genotypes under two different Cuban soil conditions. Bacteria were identified by partial sequencing of the 16S rDNA gene. Growth-promoting effects of Rhizobium pisi–Pseudomonas monteilii co-inoculation were evaluated under field conditions in consecutive years. Two contrasting bean genotypes (BAT-477 and DOR-364) were co-inoculated and grown in two different soil types (Cambisol and Oxisol) in Cuban farm areas. Several growth parameters were evaluated at three time points during the crop cycle (21, 42 and 87 days after planting). In total, 20 strains were isolated belonging to six different genera, i.e. Arthrobacter, Chryseobacterium, Enterobacter, Stenotrophomonas, Pseudomonas and Rhizobium. It was observed that native Rhizobium–Pseudomonas co-inoculation as compared to single Rhizobium inoculation increased the nodulation, growth parameters and yield of the different genotypes. The response of BAT-477 was more pronounced than that of DOR-364. These findings contribute to the understanding of the interplay between Rhizobium, PGPR and the plant host under different soil conditions. Importantly, co-inoculation with R. pisi and P. monteilii could be an effective biofertilization strategy for common bean production.

Rhizobia are soil bacteria that can fix nitrogen in symbiosis with leguminous plants or exist free living in the rhizosphere. Crucial to their complex lifestyle is the ability to sense and respond to diverse environmental stimuli, requiring elaborate signaling pathways. In the majority of bacteria, the nucleotide-based second messenger cyclic diguanosine monophosphate (c-di-GMP) is involved in signal transduction. Surprisingly, little is known about the importance of c-di-GMP signaling in rhizobia. We have analyzed the genome sequences of six well-studied type species (Bradyrhizobium japonicum, Mesorhizobium loti, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium fredii, and Sinorhizobium meliloti) for proteins possibly involved in c-di-GMP signaling based on the presence of four domains: GGDEF (diguanylate cyclase), EAL and HD-GYP (phosphodiesterase), and PilZ (c-di-GMP sensor). We find that rhizobia possess a high number of these proteins. Conservation analysis suggests that c-di-GMP signaling proteins modulate species-specific pathways rather than ancient rhizobia-specific processes. Two hybrid GGDEF-EAL proteins were selected for functional analysis, R. etli RHE_PD00105 (CdgA) and RHE_PD00137 (CdgB). Expression of cdgA and cdgB is repressed by the alarmone (p)ppGpp. cdgB is significantly expressed on plant roots and free living. Mutation of cdgA, cdgB, or both does not affect plant root colonization, nitrogen fixation capacity, biofilm formation, motility, and exopolysaccharide production. However, heterologous expression of the individual GGDEF and EAL domains of each protein in Escherichia coli strongly suggests that CdgA and CdgB are bifunctional proteins, possessing both diguanylate cyclase and phosphodiesterase activities. Taken together, our results provide a platform for future studies of c-di-GMP signaling in rhizobia.

Bacterial populations are known to harbor a small fraction of so-called persister cells that have the remarkable ability to survive treatment with very high doses of antibiotics. Recent studies underscore the importance of persistence in chronic infections, yet the nature of persisters remains poorly understood. We recently showed that the universally conserved GTPase Obg modulates persistence via a (p)ppGpp-dependent mechanism that proceeds through expression of hokB. HokB is a membrane-bound toxin that causes the membrane potential to collapse. The resulting drop in cellular energy levels triggers a switch to the persistent state, yielding protection from antibiotic attack. Obgmediated persistence is conserved in the human pathogen Pseudomonas aeruginosa , making Obg a promising target for therapies directed against bacterial persistence. Over seven decades ago, Joseph Bigger observed that a small number of cells within a Staphylococcus pyogenes population was not killed by penicillin treatment. Curiously, these cells were not resistant to the antibiotic at hand, as they gave rise to a new population that again showed susceptibility towards penicillin. Bigger appropriately named the persevering cells persisters. Persister cells are now known to withstand antibiotic therapy and to resuscitate when the therapy is stopped, thereby contributing to the chronic nature of many infections. The development of novel techniques to study small fractions of cells that only transiently display multidrug tolerance, along with the recognition of their clinical importance, has substantially fueled persistence research. At present, it is clear that cells can switch to the persistent state as a result of stochastic expression of persister genes. In addition, persistence can develop as a result of environmental stimuli. The essential and conserved P-loop GTPase Obg has previously been implicated in the coordination of many cellular functions including ribosome biogenesis, DNA replication, and several stress responses. In addition, it has been dubbed a cell cycle sentinel, tuning its activities according to the cell’s energy status in order to enhance bacterial survival in hostile environments. These previously reported properties made Obg an attractive candidate to regulate entry into the persistent state. We assessed this by both overexpressing and depleting Obg in the model bacterium Escherichia coli . Our results show a direct and strong correlation between cellular Obg concentrations and persister levels of E. coli populations. Using fluorescence-activated cell sorting (FACS), we additionally demonstrated at the single-cell level that high Obg levels, originating from stochastic fluctuations in obg gene expression, significantly increase the likelihood of surviving lethal antibiotic treatment. Combined, our results indicate that naturally occurring cell-to-cell variations in expression of obg contribute to persistence. As well as noisy gene expression, environmental triggers have previously been shown to promote persistence development. Earlier research revealed an important role for the stringent response to nutrient limitation in the emergence of persistence. In E. coli , nutrient limitation is signaled by the alarmone (p)ppGpp that is produced by RelA and SpoT and broken down by SpoT. Interestingly, Obg is known to associate with both (p)ppGpp and SpoT. Using both mutants defective in the stringent response and mutants producing intermediate levels of (p)ppGpp, we

The Obg protein family belongs to the TRAFAC (translation factor) class of P-loop GTPases and is conserved from bacteria to eukaryotes. Essential roles in many different cellular processes have been suggested for the Obg protein from Escherichia coli (ObgE), and we recently showed that it is a central regulator of bacterial persistence. Here, we report the first crystal structure of ObgE at 1.85 Å resolution in the GDP-bound state, showing the characteristic N-terminal domain and a central G domain that are common to all Obg proteins. ObgE also contains an intrinsically disordered C-terminal domain, and we show here that this domain specifically contributed to GTP binding, while it did not influence GDP binding or GTP hydrolysis. Biophysical analysis, using small angle X-ray scattering and multi-angle light scattering experiments, revealed that ObgE is a monomer in solution, regardless of the bound nucleotide. In contrast to recent suggestions, our biochemical analyses further indicate that ObgE is neither activated by K+ ions nor by homodimerization. However, the ObgE GTPase activity was stimulated upon binding to the ribosome, confirming the ribosome-dependent GTPase activity of the Obg family. Combined, our data represent an important step toward further unraveling the detailed molecular mechanism of ObgE, which might pave the way to further studies into how this GTPase regulates bacterial physiology, including persistence. ispartof: Journal of Biological Chemistry vol:292 issue:14 pages:5871-5883 ispartof: location:United States status: published

The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans . We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 μM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 μM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans . Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.

Oral infections are among the most common diseases worldwide. Many protocols for the prevention and treatment of oral infections have been described, yet no golden standard has been developed so far. The antiseptic chlorhexidine and antibiotics are often used in these treatment procedures. However, long-term use of chlorhexidine can lead to side effects and extensive use of antibiotics can promote the development of antibiotic-resistant bacteria, which in turn can compromise the effectiveness of the treatment. Consequently, it remains important to search for new antibacterial agents for the treatment of oral infections. In this study, we report on the antibacterial activity of the anti-asthma drug zafirlukast against oral pathogens Porphyromonas gingivalis and Streptococcus mutans. Furthermore, its activity against oral biofilms grown on titanium surfaces was confirmed. In addition, we demonstrated that zafirlukast displays no cytotoxicity against human osteoblasts. Combined, this study paves the way for further research to determine the potential of zafirlukast to be used as a new antibiotic against oral pathogens. ispartof: FEMS Microbiology Letters vol:364 issue:2 ispartof: location:England status: published

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. ispartof: Journal of Orthopaedic Research vol:34 issue:12 pages:2191-2198 ispartof: location:United States status: published