Yong-Gyun Jung

A rapid antimicrobial susceptibility test based on single-cell morphological analysis

An antimicrobial susceptibility test rapidly identifies bacterial response to drugs based on imaged morphology of single cells. Microbes Get in Shape for Antibiotic Testing Conventional tests that measure bacteria susceptibility to antibiotics rely on a change in occupied area. Thus, a positive change in area indicates that the bacteria is growing and is resistant to the drug, right? Not always: Bacteria can also take on different shapes, such as filaments, or swell; these changes increase the area, but the bug is still susceptible to the antibiotic. Choi et al. therefore devised an imaging-based antibiotic susceptibility test (AST) that factors in changes in morphology, to rapidly determine whether single bacteria, confined to microwells, respond to various drugs. The authors looked at four standard strains, including antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), as well as clinical samples, in response to dozens of different antibiotics used in the clinic. The single-cell morphology AST performed as well as the gold standard, culture-based broth microdilution test, with read-out in only 4 hours. Such a rapid and accurate screen could improve time-to-answer in the clinic by avoiding conventional culture methods, thus allowing for faster decision making in administering proper antibiotics to patients. A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum β-lactamase–positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration.

Capillary Based Patterning of Cellular Communities in Laterally Open Channels

In order to offer an easier way to study interactions between multiple cellular populations, we have developed a novel method to precisely place cells in a variety of nonoverlapping patterns using surface tension in laterally open microchannels. Our design is fundamentally different from previous strategies such as compartmentalization, stamping, stenciling, or mechanical approaches. It relies on capillary action or the propensity for liquid to move more readily through narrow spaces as a result of surface tension. Until now, capillary based patterning has been limited to coating chemically isolated areas. Here, we demonstrate, through use of surface tension and controlled flooding, that it is possible to pattern multiple cells and proteins using laterally open channels in a variety of designs. We demonstrate the relevance of the concept by coculturing different mammalian cell types and evaluating the behavior of engineered quorum sensing circuits in E. coli. In the future, we believe the laterally open channel designs shown here can be useful for rapidly creating and studying cellular ecologies using simple pipetting.

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K33FEHH37FEEC41SPC44LEK47 that encompass the conserved HX3CX2C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.

Bacteriocins as food preservatives: Challenges and emerging horizons

ABSTRACT The increasing demand for fresh-like food products and the potential health hazards of chemically preserved and processed food products have led to the advent of alternative technologies for the preservation and maintenance of the freshness of the food products. One such preservation strategy is the usage of bacteriocins or bacteriocins producing starter cultures for the preservation of the intended food matrixes. Bacteriocins are ribosomally synthesized smaller polypeptide molecules that exert antagonistic activity against closely related and unrelated group of bacteria. This review is aimed at bringing to lime light the various class of bacteriocins mainly from gram positive bacteria. The desirable characteristics of the bacteriocins which earn them a place in food preservation technology, the success story of the same in various food systems, the various challenges and the strategies employed to put them to work efficiently in various food systems has been discussed in this review. From the industrial point of view various aspects like the improvement of the producer strains, downstream processing and purification of the bacteriocins and recent trends in engineered bacteriocins has also been briefly discussed in this review.

Embedded biofilm, a new biofilm model based on the embedded growth of bacteria.

ABSTRACT A variety of systems have been developed to study biofilm formation. However, most systems are based on the surface-attached growth of microbes under shear stress. In this study, we designed a microfluidic channel device, called a microfluidic agarose channel (MAC), and found that microbial cells in the MAC system formed an embedded cell aggregative structure (ECAS). ECASs were generated from the embedded growth of bacterial cells in an agarose matrix and better mimicked the clinical environment of biofilms formed within mucus or host tissue under shear-free conditions. ECASs were developed with the production of extracellular polymeric substances (EPS), the most important feature of biofilms, and eventually burst to release planktonic cells, which resembles the full developmental cycle of biofilms. Chemical and genetic effects have also confirmed that ECASs are a type of biofilm. Unlike the conventional biofilms formed in the flow cell model system, this embedded-type biofilm completes the developmental cycle in only 9 to 12 h and can easily be observed with ordinary microscopes. We suggest that ECASs are a type of biofilm and that the MAC is a system for observing biofilm formation.

Bedaquiline susceptibility test for totally drug-resistant tuberculosis Mycobacterium tuberculosis

This study aimed to provide information that bedaquilline is significantly effective for treatment of totally drug resistant (TDR) Mycobacterium tuberculosis that shows resistant to all first- and second-line drugs-using an innovative disc agarose channel (DAC) system. Time-lapse images of single bacterial cells under culture conditions with different concentrations of bedaquiline were analysed by image processing software to determine minimum inhibitory concentrations (MICs). Bedaquiline inhibited the growth of TDR M. tuberculosis strains, with MIC values ranging from 0.125 to 0.5 mg/L. The results of the present study demonstrate that bedaquiline, newly approved by the United States Food and Drug Administration (FDA), may offer therapeutic solutions for TDR-TB.

A rapid culture system uninfluenced by an inoculum effect increases reliability and convenience for drug susceptibility testing of Mycobacterium tuberculosis

The Disc Agarose Channel (DAC) system utilizes microfluidics and imaging technologies and is fully automated and capable of tracking single cell growth to produce Mycobacterium tuberculosis (MTB) drug susceptibility testing (DST) results within 3~7 days. In particular, this system can be easily used to perform DSTs without the fastidious preparation of the inoculum of MTB cells. Inoculum effect is one of the major problems that causes DST errors. The DAC system was not influenced by the inoculum effect and produced reliable DST results. In this system, the minimum inhibitory concentration (MIC) values of the first-line drugs were consistent regardless of inoculum sizes ranging from ~103 to ~108 CFU/mL. The consistent MIC results enabled us to determine the critical concentrations for 12 anti-tuberculosis drugs. Based on the determined critical concentrations, further DSTs were performed with 254 MTB clinical isolates without measuring an inoculum size. There were high agreement rates (96.3%) between the DAC system and the absolute concentration method using Löwenstein-Jensen medium. According to these results, the DAC system is the first DST system that is not affected by the inoculum effect. It can thus increase reliability and convenience for DST of MTB. We expect that this system will be a potential substitute for conventional DST systems.

Marker Pen Device with Concentration Gradient Nib for Antibiotic Susceptibility Testing

Background Pen-based devices have emerged as useful tools for measuring pH and glucose, and for fabricating microchannels and microarrays. Pen-based devices take advantage of flexible patterning, inexpensive costs, and small volumes, thereby saving time and increasing efficiency. We have developed a gradient nib marker pen device that generated simultaneously different antibiotic concentrations in bacteria antibiotic susceptibility testing (AST). Methods The device can deposit on the target surface with the antibiotic gradient. The designed polyester fiber nibs are a highly uniform porosity with unidirectional orientation and produce a visible gradient pattern. Results We have demonstrated and quantitatively analyzed bacterial growth after antibiotic marking. The antibiotic marking produces an inhibition zone of bacterial growth. The inhibition zones of bacterial growth are captured and converted to 8-bit grayscale images, and then quantified by gray values using the Image J program. A profile of the inhibition zone showed different gray values in response to bacterial viability. Conclusion The gradient nib marker pen device can be used to determine the quantitative antibiotic concentration based on the relationship between gray values and bacterial density conveniently without requiring a series of dilution tubes, including nutrient medium, and diversely diluted antibiotics.