Measurement Of Bacterial Growth Research Articles

Comparison of Five Escherichia coli Strains to Achieve the Maximum Yield of a Recombinant Immunotoxin Consisting of an Antibody against VEGF Conjugated with MAP30 Toxin in a Benchtop Bioreactor.

Cancer is among the leading causes of death worldwide, imposing high costs on the health systems of all societies. Extensive biological studies are required to discover appropriate therapies. Escherichia coli has long been regarded as one of the main biotechnological bio-factories to produce recombinant protein-based therapeutics. In the present study, five strains of E. coli were compared to achieve the maximum production of a previously designed recombinant immunotoxin-carrying MAP30 toxin against VEGF-overexpressed cancer cells in a benchtop bioreactor. The recombinant immunotoxin coding gene sequence was extracted from the NCBI database. The host used to produce the recombinant immunotoxin were five E. coli strains of BL21 (DE3), DH5α, SHuffle®T7, XL1-Blue, and Rosetta-gamiTM (DE3). CaCl2 method was used for bacterial transformation. Bacterial growth measurements were performed using optical density measurements at 600 nm. The immunotoxin production was measured using SDS-PAGE analysis. The best-producing strain was cultivated in a 10-L benchtop stirred tank bioreactor. Recent patents on this field were also studied. The results demonstrated that the BL21 (DE3) strain had the highest expression of recombinant protein in comparison to other strains. Moreover, the cell growth of E. coli BL21 (DE3) and SHuffle®T7 strains before transformation in the LB medium, were significantly higher in comparison to other strains. Additionally, the transformation of Rosettagami was associated with decreased cell proliferation. The transformation of the XL1-Blue strain did not effect cell growth. Analysis of the growth kinetics demonstrated appropriate proliferation of the transformed BL21 (DE3) cells in the laboratory benchtop bioreactor. Based on the results of this study, the BL21 (DE3) strain could be used as a suitable host for the production of the recombinant immunotoxin against VEGF in stirred tank bioreactor, which can be employed for the treatment of tumors. Yet, its precise mechanism must be explored in extensive studies.

A novel rapid bioluminescence-based antimicrobial susceptibility testing method based on adenosine triphosphate consumption.

Standard, phenotypic antimicrobial susceptibility testing (AST) methods require 16-20 h of incubation and are considered as the bottleneck in providing timely input for appropriate antimicrobial treatment. In this study, a novel adenosine triphosphate (ATP)-bioluminescence-based method which allows rapid AST within 3 h was described. Standard AST was performed for 56 Enterobacterales isolates using EUCAST disk diffusion (DD) methodology. For the bioluminescence-based rapid AST, suspensions of bacteria were prepared using Mueller-Hinton broth to obtain a turbidity of 0.5 McFarland. The suspensions were distributed into 96-well microtiter plates. ATP (20 mM) and fixed concentrations of different antibiotics were added. Following incubation at 37°C for 1 h, a luminescent reaction mixture, including the substrate luciferin and luciferase enzyme solutions, was added. The chemiluminescence was monitored using an imaging system. Light production demonstrated the presence of ATP, indicating that the isolate was susceptible to the antibiotic in the well. Absence or decrease of light intensity, compared with the growth control well, indicated the use of ATP as an indirect measure of bacterial growth, and therefore resistance to the antibiotic in the well. The novel AST method was tested using a total of 348 test wells. Concordance was achieved for 290 (83.3%) of the tests, whereas 52 (14.9%) and 6 (1.7%) tests caused minor and major errors, respectively. In this study, a bioluminescence-based rapid AST was developed based on the consumption of ATP by bacteria. Our method's uniqueness relies on determining ATP consumption by microorganisms in the presence or absence of an antibiotic. The novel AST method described in this study lays the groundwork for obtaining rapid results, which should be considered as a proof of concept. With further optimization studies, this novel method can provide higher accuracy and be introduced into clinical practice as a routine AST method.

Thermal adaptation of soil microbial growth traits in response to chronic warming.

Soils are the largest terrestrial carbon sink and the foundation of our food, fiber, and fuel systems. Healthy soils are carbon sinks, storing more carbon than they release. This reduces the amount of carbon dioxide released into the atmosphere and buffers against climate change. Soil microbes drive biogeochemical cycling and contribute to soil health through organic matter breakdown, plant growth promotion, and nutrient distribution. In this study, we determined how soil microbial growth traits respond to long-term soil warming. We found that bacterial isolates from warmed plots showed evidence of adaptation of optimum growth temperature. This suggests that increased microbial biomass and growth in a warming world could result in greater carbon storage. As temperatures increase, greater microbial activity may help reduce the soil carbon feedback loop. Our results provide insight on how atmospheric carbon cycling and soil health may respond in a warming world.

Utilization of molasses wastewater for medium production of Bacillus cereus

This study aims to analyze the potential of molasses wastewater as a production medium for the Bacillus cereus SN7 and B. cereus consortium. The research was conducted experimentally using several B. cereus strains such as B. cereus SN7, B. cereus SP4, B. cereus S5, B. cereus Xmb051, and B. cereus BF2. The Analysis of the potential of molasses wastewater as a production medium was carried out by growing the B. cereus SN7 and B. cereus consortium on molasses liquid waste medium with added liquid skim milk. In the comparison, the nutrient broth medium was used as a positive control, and sterile distilled water as a negative control. Bacterial growth measurements were carried out for 24 hours with different observations, sequentially, 0, 6, 12,18, and 24 hours and observed every six hours using the total plate count method. It can be shown that the highest cell biomass is 1,029 g/ml and the optimum growth time of each treatment is at 18th hours. Thus, the use of molasses as a bacterium medium can be used for the production of B. cereus SN7, and B. cereus consortium.

Multimodal CMOS Biosensor for Microbial Growth Monitoring

This paper describes a new biosensor prototype that uses microscale electrodes to measure impedance, pH, and temperature changes caused by bacterial growth <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in-vitro</i> . The prototype uses multiple sensor geometries to optimize sensitivity, and employs custom circuits including an integrated CMOS lock-in amplifier (LIA) for multi-frequency impedance measurement. The gold-plated interdigitated electrodes (AuIDEs), iridium oxide (IrO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> )-based pH electrodes, and snake-shaped gold resistance-temperature detectors (RTDs) electrodes are implemented on a flexible PCB. The custom integrated LIA is designed and fabricated in a 0.18-μm CMOS technology with 1.8 V supply voltage. The overall sensitivity of the LIA is 240 mV/nA with a current detection sensitivity down to 1 pA. A pH measuring circuit is designed using an operational amplifier with a very low input-bias-current. A Wheatstone bridge circuit with a network of resistors and RTD electrode is used to measure RTD resistance changes caused by temperature fluctuations. The whole system is enclosed into a self-contained platform developed to monitor bacterial growth by measuring the impedance as well as the environmental growth parameters such as pH and temperature. The fabricated multi-frequency LIA was employed to test the impedance of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> cells with different concentrations at multiple frequencies. The results showed that the IDE can measure the impedance variation caused by the bacterial activity with sensitivities of 36.62%, 34.37%, 33.29% and 20.23% at 1, 2, 4, and 10 kHz frequency, respectively. The fabricated pH electrodes showed linearity with a linear regression (R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> ) value of 0.99 and can measure pH changes ranging from 4 to 10, with a sensitivity of 36, 52, and 68 mV/pH for sensing areas of 4 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , 9 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , and 16 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , respectively. The temperature sensor measurements showed that the 4 mm × 4 mm RTD gold-printed temperature electrodes had a linear relationship between resistance and temperature with a R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> value of 0.996, and the higher sensitivity of 36 (Ω/°C) compared to other tested RTD geometries. The prototype was successfully used to perform bacterial growth measurements <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in-vitro</i> .

Enabling direct microcalorimetric measurement of metabolic activity and exothermic reactions onto microfluidic platforms via heat flux sensor integration

All biological processes use or produce heat. Traditional microcalorimeters have been utilized to study the metabolic heat output of living organisms and heat production of exothermic chemical processes. Current advances in microfabrication have made possible the miniaturization of commercial microcalorimeters, resulting in a few studies on the metabolic activity of cells at the microscale in microfluidic chips. Here we present a new, versatile, and robust microcalorimetric differential design based on the integration of heat flux sensors on top of microfluidic channels. We show the design, modeling, calibration, and experimental verification of this system by utilizing Escherichia coli growth and the exothermic base catalyzed hydrolysis of methyl paraben as use cases. The system consists of a Polydimethylsiloxane based flow-through microfluidic chip with two 46 µl chambers and two integrated heat flux sensors. The differential compensation of thermal power measurements allows for the measurement of bacterial growth with a limit of detection of 1707 W/m3, corresponding to 0.021OD (2 ∙ 107 bacteria). We also extracted the thermal power of a single Escherichia coli of between 1.3 and 4.5 pW, comparable to values measured by industrial microcalorimeters. Our system opens the possibility for expanding already existing microfluidic systems, such as drug testing lab-on-chip platforms, with measurements of metabolic changes of cell populations in form of heat output, without modifying the analyte and minimal interference with the microfluidic channel itself.

Seasonal variation in temperature sensitivity of bacterial growth in a temperate soil and lake.

Faster bacterial biomass turnover is expected in water compared to soil, which would result in more rapid community adaption to changing environmental conditions, including temperature. Bacterial community adaptation for growth is therefore predicted to have larger seasonal amplitudes in lakes than in soil. To test this prediction, we compared the seasonal variation in temperature adaptation of bacterial community growth in a soil and lake in Southern Sweden (Tin situ 0–20°C, mean 10°C) during 1.5 years, based on monthly samplings including two winters and summers. An indicator of community adaptation, minimum temperature for growth (Tmin), was calculated from bacterial growth measurements (Leu incorporation) using the Ratkowsky model. The seasonal variation in Tmin (sinusoidal function, R2 = 0.71) was most pronounced for the lake bacterial community, with an amplitude for Tmin of 3.0°C (−4.5 to −10.5°C) compared to 0.6°C (−7 to −8°C) for the soil. Thus, Tmin in water increased by 0.32°C/degree change of Tin situ. Similar differences were also found when comparing four lakes and soils in the winter and summer (amplitudes 2.9°C and 0.9°C for lakes and soils, respectively). Thus, seasonal variation in temperature adaptation has to be taken into account in lakes, while for soils a constant Tmin can be used.

Antimicrobial effect of Eysenhardtia polystachya homemade extracts on bacteria causing urinary tract infections

Objective: To evaluate the in vitro antimicrobial effect of homemade preparations of Eysenhardtia polystachya, obtained by maceration or by infusion, on bacteria that cause urinary tract infections. Methodology: Microdilution assays were performed in 96-well plates where bacterial suspensions were co-incubated with different dilutions of homemade extracts, as well as a control for comparison using a commercial extract of the plant. Subsequently, the absorbance at 600 nm was measured to be considered as a direct measure of bacterial growth. For each culture, the values were normalized to growth percentage from the absorbance value obtained in the wells without extract. Contribution: The results show that the homemade extracts have a partial antimicrobial effect on the growth of the bacteria used in this work. The effect was the same between the extract by infusion and the extract by maceration against Gram negative bacteria. On the other hand, the extract by infusion showed a better effect than the extract by maceration on Gram positive bacteria. None of the homemade extracts achieved the antimicrobial effect of the commercial extract. This work corroborates the empirical knowledge of the use of homemade preparations of Eysenhardtia polystachya to treat urinary tract infections.

Biodegradability of Proteolytic Bacteria in Mangrove Ecosystems

The purpose of this study was to awareness the optimal time for the growth of proteolytic bacteria in producing the protease enzyme and determine the effectiveness of the protease enzyme from proteolytic bacteria in degrading mangrove litter. The method used was the experimental method by using two proteolytic bacteria isolates, namely Bacillus manliponensis (K6) and Bacillus toyonensis (K20). This method used one treatment for each proteolytic bacterial isolate, namely the addition of different extract containing protease enzyme (0%, 25%, and 50%) with three replications. Measurement of bacterial growth was carried out every 6 hours for 24 hours using Total Plate Count (TPC) and spectrophotometric method. B. manliponensis and B. toyonensis have enzyme activity as indicate by the presence of a clear zone on Zobell Marine Agar 2216 and skim milk 1%. The growth bacteria based on the TPC measurement was directly proportional to the spectrophotometric measurement results. The highest bacterial count was (1.39 x 108 CFU's/ml) K6 bacteria and (1.52 x 108 CFU's/ml) K20 bacteria. The results of the measurement of cell growth in the spectrophotometric method occurred at the 6th hour, namely 10.36 x 108 CFU's/ml (K6 bacteria) and 10.97 x 108 CFU's/ml (K20 bacteria). The optimum time of the protease enzyme occurred at 6 hours, which was 0.0258 mg/ml (K6 bacteria) and 0.0262 mg/ml (K20 bacteria). The highest dissolved protein content was obtained at the 50% dose of 0.054 mg/ml (K6 bacteria) and 0.055 mg/ml (K20 bacteria), while the lowest was at 0%, namely 0.050 mg/ml (K6 bacteria) and 0.051 mg/ml (K20 bacteria)

The Impact of Norepinephrine on Mono-Species and Dual-Species Staphylococcal Biofilms.

The effect of norepinephrine (“NE”) on Gram-negative bacteria is well characterized; however, little is known about the impact of NE on cutaneous Gram-positive skin residents, especially staphylococci. In this study, the impact of NE on monospecies and dual-species biofilms of Staphylococcus epidermidis and S. aureus model strains was investigated for the first time. Biofilms were grown in two different models (on polytetrafluoroethylene (“PTFE”) cubes and glass microfiber filters (“GMFFs”)) and additionally kinetic measurements of bacterial growth was performed. We have shown that NE can affect the biofilm formation of both species with a strong dependence on aerobic or anaerobic culture conditions in different models. It was shown that S. epidermidis suppresses S. aureus growth in dual-species biofilms and that NE can accelerate this process, contributing to the competitive behavior of staphylococci.

Antibacterial Effect of Amino Acid-Silver Complex Loaded Montmorillonite Incorporated in Dental Acrylic Resin.

Several dental materials contain silver for antibacterial effect, however the effect is relatively low. The reason for the lower antibacterial efficacy of silver is considered to be the fact that silver ions bind to chloride ions in saliva. To develop new effective silver antibacterial agents that can be useful in the mouth, we synthesized two novel amino acid (methionine or histidine)–silver complexes (Met or His–Ag) loaded with montmorillonite (Mont) and analyzed their antibacterial efficacy. At first the complexes were characterized using nuclear magnetic resonance (NMR), and amino acid–Ag complex-loaded Mont (amino acid–Ag–Mont) were characterized using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The antibacterial efficacy of these materials in dental acrylic resin was then investigated by bacterial growth measurement using a spectrophotometer. As controls, commercially available silver-loaded zeolite and silver-zirconium phosphate were also tested. Dental acrylic resin incorporating His–Ag–Mont strongly inhibited Streptococcus mutans growth. This was explained by the fact that His-Ag complex revealed the highest amounts of silver ions in the presence of chloride. The structure of the amino acid–Ag complexes affected the silver ion presence in chloride and the antibacterial efficacy. His–Ag–Mont might be used as antibacterial agents for dental materials.

Bacterial Growth Control Mechanisms Inferred from Multivariate Statistical Analysis of Single-Cell Measurements

Analysis of single-cell measurements of bacterial growth and division often relied on testing preconceived models of cell size control mechanisms. Such an approach could limit the scope of data analysis and prevent us from uncovering new information. Here, we take an "agnostic" approach by applying regression methods to multiple simultaneously measured cellular variables, which allow us to infer dependencies among those variables from their apparent correlations. Besides previously observed correlations attributed to particular cell size control mechanisms, we identify dependencies that point to potentially new mechanisms. In particular, cells born smaller than their sisters tend to grow faster and make up for the size difference acquired during division. We also find that sister cells are correlated beyond what single-cell, size-control models predict. These trends are consistently found in repeat experiments, although the dependencies vary quantitatively. Such variation highlights the sensitivity of cell growth to environmental variations and the limitation of currently used experimental setups.

Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth.

Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

Using sliding mode observers to estimate BtuB concentration from measured vitamin B12 concentration.

A simple model for the B12-riboswitch regulatory network in Escherichia coli is first described and the same analysis is applied when changing the strain to Salmonella enterica. Model validation is undertaken by linking the dynamics of the riboswitch model to bacterial growth and comparing the results obtained with in vivo experimental measurements. Measurements of bacterial growth are relatively straightforward to obtain experimentally, but experimental measurements relating to the operation of the riboswitch are more difficult. Using the validated model, sliding mode observer design methods are used to estimate BtuB given measurements of the concentration of vitamin B12. The sliding mode approach is selected because of its inherent robustness properties as well as for the ease of implementation. Validation of the estimates of BtuB produced by the observer is undertaken by comparing the BtuB and vitamin B12 concentrations estimated from the observer with green fluorescent protein production and the concentration of vitamin B12 obtained experimentally. These experimental results also provide further validation of the underpinning mathematical model. The results establish that using a sliding mode observer as a soft sensor is a useful approach to explore the operation of a vitamin B12 riboswitch given measurements of the concentration of vitamin B12.

Antimicrobial activity of functionalised carbon nanotubes against pathogenic microorganisms.

Carbon nanotubes represent one of the best examples of novel nanostructures, exhibit a range of extraordinary physical properties, strong antimicrobial activity and can pierce bacterial cell walls. This investigation handles the antimicrobial activity of functionalised multiwall carbon nanotubes (F-MWNTs) as an alternative antimicrobial material compared to the commercial antibiotics. Antibacterial activities of F-MWNTs are investigated through two different kinds of bacteria, E. coli and S. aureus. The results demonstrate that the best concentration of F-MWNTs for the maximum inhibition and antibacterial functionality is 80 and 60 μg/ml for E. coli and S. aureus, respectively. The transmission electron microscope reveals the morphological changes damage mechanism for the cellular reliability on these microorganisms. F-MWNTs are capable of biologically isolating the cell from their microenvironment, contributing to the development of toxic substances and placing the cell under oxidative stress leading to cellular death. The efficiency of F-MWNTs is compared with the common antibiotics and shows an enhancement in the inhibitory effect with percentages reaches 85%. To account for the bactericidal performance of F-MWNTs towards these pathogens, the dielectric conductivity and the bacterial growth measurements are conducted. The present study endeavour that F-MWNTs could be exploited in biomedical devices and altering systems for hospital and industrial cleaning applications.

The EcoChip 2: An Autonomous Sensor Platform for Multimodal Bio-environmental Monitoring of the Northern Habitat.

This paper presents the EcoChip 2, an autonomous multimodal bio-environmental sensor platform for the monitoring of microorganisms in the northern habitat. The EcoChip 2 prototype includes an array of 96-wells for the continuous monitoring of microbiological growth through a multichannel electrochemical impedance analyzer circuit. In addition, the platform includes luminosity, humidity, temperature sensors and monitoring. The developed electronic board uses an ultra-low-power microcontroller unit, a custom power management unit, a low-power wireless ISM-2.45 GHz transceiver, and a flash memory to accumulate and store the sensor data over extended monitoring periods. When a wireless base station is placed within the transmission range of the EcoChip 2, an embedded low-power wireless transceiver transmits the 96-wells impedance data and the other sensor data stored in the flash memory to the user interface. We present the measured performance of the prototype, along with laboratory test results of bacterial growth measurements inside the 96 wells in parallel. We show that the EcoChip 2 can successfully measure the impedances associated with bacterial growth over several hours using an excitation frequency of 2 kHz with power consumption of 114.6 mW under operating mode.

Multichannel QCM-Based System for Continuous Monitoring of Bacterial Biofilm Growth

Quartz crystal microbalance (QCM) sensors are becoming a good alternative to analytical methods for the measurement of bacterial growth in liquid media culture. For this purpose, two essential resonance parameters allow the monitoring of biofilm formation: the series resonance frequency shift and the change of the resistance at this frequency. Nevertheless, several problems arise in determining these parameters, as their relative variation is very small. This means that an accurate procedure must be implemented for the measurement of the QCM resonance parameters, including the automatic calibration of the frequency response effects of the measurement circuits and the automatic compensation of the static electrical capacitance of the QCM. In this article, a novel multichannel system for on-line monitoring of biofilm formation based on QCM sensors is proposed. QCM resonance parameters are determined from the electrical impedance analysis by means of an auto-balanced impedance bridge. This configuration has allowed the implementation of an affordable multichannel measurement instrument. Obtained results, based on binary mixtures of water–glycerol measurements and real microorganism experiments, are in good agreement with the theoretical behavior. These results show the great potential of this instrument to be used for monitoring microbial growth and biofilm formation.

Isolation and characterization of indole-3-acetic acid producing bacteria from red onion rhizosphere

There are many bacteria classified as growth enhancer in plants which known as Plant Growth Promoting Rhizobacteria (PGPR). PGPR can directly induce plant growth by producing of phytohormone Indole-3-Acetic Acid (IAA). The objective of this study was to obtain bacterial isolates of IAA producing rhizobacteria from red onion rhizosphere and to characterize the potential isolates. The methods used in the study were a sampling of soil, isolation of IAA producing rhizobacteria, measurement of IAA by using colorimetric assay, morphological identification of bacterial isolates, measurement of bacterial growth and IAA production, and test of hypersensitivity on tobacco leaves. There were fourteen IAA producing bacterial from red onion rhizosphere. The isolates could produce IAA by colorimetric assay detection. Three isolates produced IAA was higher than other isolates i.e. BIT 2,4 (61.72 ppm), BIS 3,4 (60.92 ppm) and BIT 2,1 (49.3 ppm). Meanwhile, isolate BIT 4,1 produced the lowest IAA as much as 3 ppm. Four isolates were gram-positive bacteria. Isolates BIS 3,4 and BIT 2,1 produced exogenous (extracellular) IAA on stationary phase. Two isolates potential (BIS 3,4 and BIT 2,1) did not cause necrotic symptoms or negative results for the hypersensitivity test on tobacco leaves.

Recent Developments of Chip-based Phenotypic Antibiotic Susceptibility Testing

Antibiotic susceptibility testing (AST), the screening of effective types and dosages of antibiotics, has become significantly important in the antimicrobial- resistance era over the last few decades. In order to overcome the limitations of conventional AST methods, several recent studies have developed AST platforms which exhibit the advantages of microfluidics. They demonstrated the performance of the platforms by determining effective antimicrobials for bacterial strains and their minimum inhibitory concentrations within hours. In this review, we cover recent developments of on-chip approaches for measurements of bacterial growth as well as for dilutions of antibiotic concentrations. We also discuss Point-of-Care AST devices that employ inexpensive materials and simple working principles to operate screenings near the site of care, which can potentially bring current laboratory- limited assays to clinical standards. All thing considered, emerging microfluidic AST devices have the potential to be decent alternatives to commonplace macro-scale AST methods.

A micro luminescence-based assay to measure serum susceptibility in Escherichia coli

Neonatal meningitis Escherichia coli (NMEC) is the leading cause of gram-negative meningitis in neonates, and it contributes to neonatal morbidity and mortality globally. The prototypic strain of NMEC, E. coli RS218 possesses the K1 capsule and has been widely employed in the study of NMEC pathogenesis. Previously, our laboratory has utilised relatively large volumes of culture to assay serum bactericidal activities, to garner valuable insights into bacterial immune evasion strategies. However, these methods can be labour intensive, time consuming and are not easily adaptable for high throughput applications. To overcome these limitations, a smaller volume real-time assay was developed. Bacteria were cultured in 100 µl volumes and were sub-inoculated for logarithmic growth. A slow kinetic absorbance assay was established on the Optima Fluorostar microplate reader, which enabled the accurate and reliable measurement of bacterial growth. Subsequently, bioluminescence was incorporated into the assay to facilitate the measurement of bacterial viability in real-time. Bacteria were rendered bioluminescent via electroporation of the pIlux plasmid or alternatively by the addition of exogenous beetle luciferin and recombinant firefly luciferase. The utility of these approaches in the determination of bacterial complement evasion is reported below.