Enzymatic Antimicrobial Susceptibility Testing with Bacteria Identification in 30 min.

Rapid antimicrobial susceptibility testing (AST) with the ability of bacterial identification is urgently needed for evidence-based antibiotic prescription. Herein, we propose an enzymatic AST (enzyAST) that employs ß-d-glucuronidase as a biomarker to identify pathogens and profile phenotypic susceptibilities simultaneously. EnzyAST enables to offer binary AST results within 30 min, much faster than standard methods (>16 h). The general applicability of enzyAST was verified by testing the susceptibility of two Escherichia coli strains to three antibiotics with different action mechanisms. The pilot study also shows that the minimal inhibitory concentrations can be determined by enzyAST with the statistical analysis of enzymatic activity of the bacteria population exposed to varying antibiotic concentrations. With further development of multiple bacteria and sample treatment, enzyAST could be able to evaluate the susceptibility of pathogens in clinical samples directly to facilitate the evidence-based therapy.

All-In-One Escherichia coli Viability Assay for Multi-dimensional Detection of Uncomplicated Urinary Tract Infections.

The urinary tract infections by antibiotic-resistant bacteria have been a serious public health problem and increase the healthcare costs. The conventional technologies of diagnosis and antimicrobial susceptibility testing (AST) relying on multiple culture-based assays are time-consuming and labor-intensive and thus compel the empirical antimicrobial therapies to be prescribed, fueling the prevalence of antimicrobial resistance. Herein, we propose an all-in-one Escherichia coli viability assay in an enclosed 3D microwell array chip, termed digital ß-d-glucuronidase (GUS)-AST assay. It employs GUS, a specific metabolism-related enzyme, to convert the presence of E. coli into bright fluorescence. The random distribution of single bacteria in microwell array enables to quantify the E. coli concentrations by counting the positive microwells. We incorporate the most probable number with digital quantification to lower the limit of detection and expand the dynamic range to 7 orders. The digital GUS-AST assay is able to indicate the potency of antibiotics and determine the minimum inhibitory concentrations. A streamlined procedure of urine removal, bacterial separation, and digital GUS-AST is established to perform the direct analysis of bacteria population in urine. The sample-to-result workflow can be finished in 4.5 h with a limit of detection of 39 CFU/mL. With further development for additional pathogens and multiple antibiotic conditions, the digital GUS-AST assay could help physicians to prescribe timely targeted therapies for better patient outcomes and the minimum emergence of resistant bacteria.

A large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking based on a thermosetting oil.

The rapid and accurate detection of viable bacteria is of great importance in food quality monitoring and clinical diagnosis. Escherichia coli (E. coli) is a major pathogenic bacterium, which causes potential threats to food safety and human health. Therefore, rapid and portable methods for preventing E. coli outbreaks are needed. Single cell analysis can be performed at the single-cell level, which has great advantages for analysis and diagnosis. Herein, we employed a thermosetting oil to generate a large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking. In this array, the droplets can be solidified without any inducers due to the cross-linking reaction of the hydrosilation of vinyl silicone oil and hydrosilicone oil. Single E. coli cells were encapsulated in solidified droplets to form a microcolony. Resazurin was used as a fluorescent indicator to achieve amplification of bacterial growth signals. This method can achieve digital counting of viable E. coli cells in 4 h. We achieved real-time monitoring of E. coli cell growth and division in droplets. It is rapid, simple, and does not require a pre-enrichment process when compared to the traditional plate counting method. We successfully applied the method for the enumeration of E. coli in milk. In conclusion, the thermosetting oil enables the immobilization of droplets to achieve real-time monitoring and digital counting of bacterial growth without impairing the flexibility of droplet microfluidics, and it has the potential to provide dynamic information at high resolution in this process.

Inoculum size-insensitive susceptibility determination of urine sample based on in-situ measurement of inducible enzyme activity after 20 min of antibiotic exposure.

BACKGROUND: The empirical antibiotic therapies for bacterial infections cause the emergence and propagation of multi-drug resistant bacteria, which not only impair the effectiveness of existing antibiotics but also raise healthcare costs. To reduce the empirical treatments, rapid antimicrobial susceptibility testing (AST) of causative microorganisms in clinical samples should be conducted for prescribing evidence-based antibiotics. However, most of culture-based ASTs suffer from inoculum effect and lack differentiation of target pathogen and commensals, hampering their adoption for evidence-based antibiotic prescription. Therefore, rapid ASTs which can specifically determine pathogens' susceptibilities, regardless of the bacterial load in clinical samples, are in urgent need. RESULTS: We present a pathogen-specific and inoculum size-insensitive AST to achieve the reliable susceptibility determination on Escherichia coli (E. coli) in urine samples. The developed AST is featured with an 1 h sample-to-result workflow in a filter, termed on-filter AST. The AST results can be obtained by using an inducible enzymatic assay to in-situ measure the cell response of E. coli collected from urine after 20 min of antibiotic exposure. The calculated detection limit of our AST (1.95 × 104 CFU/mL) is much lower than the diagnosis threshold of urinary tract infections. The specific expression of the inducible enzyme enables on-filter AST to correctly profile the susceptibilities of target pathogen to multi-type antibiotics without the interference from commensals. We performed the on-filter AST on 1 mL urine samples with bacterial loads varying from 105 CFU/mL to 107 CFU/mL and compared the results to that of standard method, demonstrating its insensitivity to inoculum size. SIGNIFICANCE: The developed AST is demonstrated to be of high sensitivity, specificity, and insensitive to inoculum size. With further developments for additional bacteria and clinical validation, on-filter AST is promising as a rapid and reliable surrogate of culture-based AST to promote the evidence-based prescription at the first visit and minimize the emergency of new multi-drug resistant microorganisms.

Direct single-cell antimicrobial susceptibility testing of Escherichia coli in urine using a ready-to-use 3D microwell array chip.

Empirical antibiotic therapies are prescribed for treating uncomplicated urinary tract infections (UTIs) due to the long turnaround time of conventional antimicrobial susceptibility testing (AST), leading to the prevalence of multi-drug resistant pathogens. We present a ready-to-use 3D microwell array chip to directly conduct comprehensive AST of pathogenic agents in urine at the single-cell level. The developed device features a highly integrated 3D microwell array, offering a dynamic range from 102 to 107 CFU mL-1, and a capillary valve-based flow distributor for flow equidistribution in dispensing channels and uniform sample distribution. The chip with pre-loaded reagents and negative pressure inside only requires the user to initiate AST by loading samples (∼3 s) and can work independently. We demonstrate an accessible sample-to-result workflow, including syringe filter-based bacteria separation and rapid single-cell AST on chip, which enables us to bypass the time-consuming bacteria isolation and pre-culture, speeding up the AST in ∼3 h from 2 days of conventional methods. Moreover, the bacterial concentration and AST with minimum inhibitory concentrations can be assessed simultaneously to provide comprehensive information on infections. With further development for multiple antibiotic conditions, the Dsc-AST assay could contribute to timely prescription of targeted drugs for better patient outcomes and mitigation of the threat of drug-resistant bacteria.

A Specific and Sensitive Aptamer-Based Digital PCR Chip for Salmonella typhimurium Detection.

Food poisoning and infectious diseases caused by Salmonella typhimurium (S. typhimurium) are serious public health concerns for human health and food safety. The diversity and complexity of food matrices pose great challenges for rapid and ultra-sensitive detection of S. typhimurium in food samples. A method capable of identification, detection, and quantification of S. typhimurium is essential for addressing these issues. In this study, aptamer-coated magnetic beads (Apt-MBs) are employed as capture bio-probes to specifically and selectively concentrate S. typhimurium in food samples. A self-priming chip-based digital PCR was then presented as another biosensor for on-site detection and quantification of S. typhimurium cells. The chip we developed was robust and did not require any external power for sample loading. The combination of Apt-MBs with an on-chip digital detection realized the integration into lab-on-a-chip-based biosensors for on-site monitoring of foodborne pathogens. It was possible to capture and detect S. typhimurium cells as low as 90 CFU/reaction with a capture efficiency of 94.5%. Additionally, the whole process only took about 2 h. This unique platform could also be used to monitor other target bacteria with high specificity and sensitivity by utilizing different aptamers. Furthermore, the platform has potential applications in point-of-care testing in the future.

Integrated microfluidic systems with sample preparation and nucleic acid amplification.

Rapid, efficient and accurate nucleic acid molecule detection is important in the screening of diseases and pathogens, yet remains a limiting factor at point of care (POC) treatment. Microfluidic systems are characterized by fast, integrated, miniaturized features which provide an effective platform for qualitative and quantitative detection of nucleic acid molecules. The nucleic acid detection process mainly includes sample preparation and target molecule amplification. Given the advancements in theoretical research and technological innovations to date, nucleic acid extraction and amplification integrated with microfluidic systems has advanced rapidly. The primary goal of this review is to outline current approaches used for nucleic acid detection in the context of microfluidic systems. The secondary goal is to identify new approaches that will help shape future trends at the intersection of nucleic acid detection and microfluidics, particularly with regard to increasing disease and pathogen detection for improved diagnosis and treatment.

Application of a Dielectric Barrier Discharge Atmospheric Cold Plasma (Dbd-Acp) for Eshcerichia Coli Inactivation in Apple Juice.

Atmospheric cold plasma (ACP) is a promising non-thermal technology in food industry. In this study, a dielectric barrier discharge (DBD)-ACP exhibited strong bactericidal effect on Escherichia coli in apple juice. Under a 30 to 50 W input power, less than 40 s treatment time was required for DBD-ACP to result in 3.98 to 4.34 log CFU/mL reduction of E. coli in apple juice. The inactivation behavior of ACP on E. coli was well described by the Weibull model. During the treatment, the cell membrane of E. coli was damaged severely by active species produced by plasma, such as hydrogen peroxide, ozone and nitrate. In addition, the ACP exposure had slight effect on the °Brix, pH, titratable acidity (TA), color values, total phenolic content, and antioxidant capacity of apple juice. However, higher level of DBD-ACP treatment, 50 W for more than 10 s in this case, resulted in significant change of the pH, TA, color and total phenolic content of apple juice. The results in this study have provided insight in potential use of DBD-ACP as an alternative to thermal processing for fruit juices in food industry. PRACTICAL APPLICATION: Escherichia coli O157:H7 in apple juice is a potential risk for public health. This study demonstrated that 30 s cold plasma treatment resulted in more than 4 log CFU/mL reduction under 50 W, while the quality attributes of apple juice were not significantly affected. Therefore, cold plasma technology is a promising alternative substitute of traditional thermal processing for juice pasteurization.

Effect of preliminary stresses on the resistance of Escherichia coli and Staphylococcus aureus toward non-thermal plasma (NTP) challenge.

As the development of hurdle technology, cross-protection of various stresses for pathogens posed the potential risk to food safety and public health. This study tried to explore various preliminary stresses including acidity, osmosis, oxidation, heat and cold on the resistance of microbial cells toward the non-thermal plasma (NTP) exposure. The results indicated that short-term (4h) exposure of Staphylococcus aureus and Escherichia coli to acidity, osmosis, oxidation, heat and cold stresses did not lead to the resistance to the subsequent NTP treatment. On the contrary, acidity, osmosis and heat preadaptation increased the vulnerability of E. coli cells to NTP treatment. After exposing S. aureus to osmosis, oxidation, heat and cold stress for longer period (24h), the reduction level showed significantly (P<0.05) higher. Interestingly, long-term (24h) preliminary exposure of acidic stress exhibited protective effect for S. aureus against the following NTP exposure with less damage in cell membrane integrity, membrane potential and intracellular enzyme activity. It might be due to the protein production for oxidative stress response during preliminary acidic adaptation. In general, the obtained result helped to grasp better understanding of the microbial stress response to NTP treatment and provided insight for the future research in order to accelerate the development of NTP technology in food industry.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment.

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

Significance of Viable but Nonculturable Escherichia coli: Induction, Detection, and Control.

Diseases caused by foodborne or waterborne pathogens are emerging. Many pathogens can enter into the viable but nonculturable (VBNC) state, which is a survival strategy when exposed to harsh environmental stresses. Pathogens in the VBNC state have the ability to evade conventional microbiological detection methods, posing a significant and potential health risk. Therefore, controlling VBNC bacteria in food processing and the environment is of great importance. As the typical one of the gram-negatives, Escherichia coli (E. coli) is a widespread foodborne and waterborne pathogenic bacterium and is able to enter into a VBNC state in extreme conditions (similar to the other gram-negative bacteria), including inducing factors and resuscitation stimulus. VBNC E. coli has the ability to recover both culturability and pathogenicity, which may bring potential health risk. This review describes the concrete factors (nonthermal treatment, chemical agents, and environmental factors) that induce E. coli into the VBNC state, the condition or stimulus required for resuscitation of VBNC E. coli, and the methods for detecting VBNC E. coli. Furthermore, the mechanism of genes and proteins involved in the VBNC E. coli is also discussed in this review.

Analysis of Staphylococcus aureus cell viability, sublethal injury and death induced by synergistic combination of ultrasound and mild heat.

This study was designed to investigate the combined effects of ultrasound and mild heat on the viability of S. aureus in association with the cell membrane integrity and intracellular enzyme activity. Cells were treated by ultrasound under 55°C for 3, 5, 7, 10, and 15min. The dynamic changes of S. aureus cell viability, sublethal injury and death were evaluated using flow cytometric assay. Microscopies were applied to identify the morphological appearance, ultrastructure and topography of S. aureus. The results showed the membrane damage was synchronous with esterase inhibition during the exposure to sonication, leading to the immediate lethal effect. On the other hand, bacteria under the mild heat at 55°C were inactivated via a sublethal injury process. The different lethal modes were observed between sonication and mild heat treatments, which could synergistically inactivate S. aureus. The antibacterial value of thermo-sonication was greater than the sum of the individual treatments. The thermo-sonication combination synergistically reduced the number of sublethal cells and also resulted in severe cell damage.