Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro.

Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5α, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor-recipient pairings (10-2 to 10-8). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10-2 to 10-7. A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

Microfluidic dual picoinjection based encapsulation of hemoglobin in alginate microcapsules reinforced by a poly(L-lysine)-g-poly(ethylene glycol).

Hemoglobin (Hb) encapsulation inside polysaccharide hydrogels has been considered a possible red blood cell (RBC) surrogate in transfusiology. Here we report on the microfluidic dual picoinjection assisted synthesis of Hb encapsulated alginate-poly(L-lysine)-g-poly(ethylene glycol) beads. This process is realized by the on-chip injections of blended Hb alginate solutions in emulsified aqueous calcium chloride (CaCl2) droplets followed by a subsequent injection of an aqueous PLL-g-PEG into each emulsified aqueous droplet. The proposed fabrication approach was realized using a flow-focusing and two picoinjection sites in a single PDMS device. Aqueous CaCl2 solution was emulsified and infused with Hb-alginate solution as the squeezed droplet passed through the first picoinjection site. The injection of PLL-g-PEG to reinforce the microgel and minimize the protein leaching was realized in the second picoinjection site located downstream from the first in the same microfluidic channel. In this process, monodisperse Hb-alginate-PLL-g-PEG particles with a diameter around the size of RBCs (9 µm) were obtained with around 80% of the 7.5 mg ml-1 Hb included in the injected aqueous alginate retaining in the obtained microparticles. Microparticles with Hb loading (32.8 pg per bead) and retention (28.8 pg per bead) over a week of storage at 4 °C are in accordance with the average amount of Hb per RBC. The Hb-alginate-PLL-g-PEG microbeads fabricated in the size range of RBCs are significant for further exploration.

Zoonotic Transmission of Campylobacter jejuni to Caretakers From Sick Pen Calves Carrying a Mixed Population of Strains With and Without Guillain Barré Syndrome-Associated Lipooligosaccharide Loci.

Campylobacter jejuni causes foodborne gastroenteritis and may trigger acute autoimmune sequelae including Guillain Barré Syndrome. Onset of neuromuscular paralysis is associated with exposure to C. jejuni lipooligosaccharide (LOS) classes A, B, C, D, and E that mimic and evoke antibodies against gangliosides on myelin and axons of peripheral nerves. Family members managing a Michigan dairy operation reported recurring C. jejuni gastroenteritis. Because dairy cattle are known to shed C. jejuni, we hypothesized that calves in the sick pen were the source of human infections. Fecal samples obtained from twenty-five calves, one dog, and one asymptomatic family member were cultured for Campylobacter. C. jejuni isolates were obtained from thirteen calves and the family member: C. coli from two calves, and C. hyointestinalis from two calves. Some calves had diarrhea; most were clinically normal. Typing of lipooligosaccharide biosynthetic loci showed that eight calf C. jejuni isolates fell into classes A, B, and C. Two calf isolates and the human isolate possessed LOS class E, associated mainly with enteric disease and rarely with Guillain Barré Syndrome. Multi-locus sequence typing, porA and flaA typing, and whole genome comparisons of the thirteen C. jejuni isolates indicated that the three LOS class E strains that included the human isolate were closely related, indicating zoonotic transmission. Whole-genome comparisons revealed that isolates differed in virulence gene content, particularly in loci encoding biosynthesis of surface structures. Family members experienced diarrheal illness repeatedly over 2 years, yet none experienced GBS despite exposure to calves carrying invasive C. jejuni with LOS known to elicit antiganglioside autoantibodies.

Cadmium Sub-Lethal Concentration Effect on Growth, Haematological and Biochemical Parameters of Mystus seenghala (Sykes, 1839).

The current study aims to assess the cadmium sub-lethal concentration influence on growth and haematological and biochemical parameters of Mystus seenghala. A total of 60 fish of three different length groups (20 each) were collected from Head Qadirabad, Pakistan. The fish were treated to the sub-lethal concentration viz. one-third of LC50, for 16 weeks except for the control groups. Water quality parameters were kept constant during the entire course of the research, and the major parameters were measured as temperature (28.03 ± 0.03 °C), DO (5.82 ± 0.14 mg L-1), pH (8.00 ± 0.01) and total hardness (249.98 ± 0.01 mg L-1). Findings revealed that the growth of three treated variant length groups was affected negatively by cadmium exposure and showed significantly (P < 0.05) lower average wet weight, body length and condition factor as compared to control groups, while the feed conversion ratio (FCR) increases by increasing the exposure duration. The haematological parameters including values of Hct, Hb and MCHC were significantly (P < 0.05) reduced in all Cd-treated groups than control groups, whereas the level of MCH and MCV were significantly higher, but no significant difference was found in the value of RBCs in all treated groups. Biochemical parameters such as ALT, AST, total lipid and glucose level in Cd exposure groups were significantly higher, while the total protein level was significantly (P < 0.05) reduced in all treated groups as compared to control groups. From the current study, it has been concluded that the growth, haematology and biochemical parameters are important indicators of ecotoxicology particularly contamination of the cadmium and health of the fish.

Effect of Fortified Feed with Phyto-Extract on the First Physical Barrier (Mucus) of Labeo rohita.

The aim of the current study was to assess the effect of two different fortified feeds with different concentrations of two important medicinal plants (Withania coagulans and Zingiber officinale) on the mucosal immunity of Labeo rohita. After a dietary intervention, mucus was tested against five pathogenic bacteria (in-vitro), while experimental fish were tested against the ectoparasite (Lernaea) (in-vivo). Our results revealed that all fish groups fed with different concentrations (1, 1.5, and 2%) of Z. officinale had low molecular weight proteins and did not develop any significant signs of parasitic infection, with low mortality rate; whereas the groups that were fed with W. coagulans (particularly with 1% and 2%), including a control group, developed rapid signs of infection with high mortality rate. The highest hemagglutination titer value was recorded for the fish fed with 1% and 1.5% of Z. officinale. The lowest value was found for the fish fed with 2% of W. coagulans. The mucus of all fish of fortified groups was active and inhibited the growth of tested bacterial pathogens as compared to the control group. Further, Z. officinale groups showed greater efficacy against bacteria as compared to the W. coagulans groups. In conclusion, Z. officinale can be considered as a potential and functional ingredient in aquaculture feed. Furthermore, future studies should be conducted to investigate more details on the subject.

On the Determination of Mechanical Properties of Aqueous Microgels-Towards High-Throughput Characterization.

Aqueous microgels are distinct entities of soft matter with mechanical signatures that can be different from their macroscopic counterparts due to confinement effects in the preparation, inherently made to consist of more than one domain (Janus particles) or further processing by coating and change in the extent of crosslinking of the core. Motivated by the importance of the mechanical properties of such microgels from a fundamental point, but also related to numerous applications, we provide a perspective on the experimental strategies currently available and emerging tools being explored. Albeit all techniques in principle exploit enforcing stress and observing strain, the realization differs from directly, as, e.g., by atomic force microscope, to less evident in a fluid field combined with imaging by a high-speed camera in high-throughput strategies. Moreover, the accompanying analysis strategies also reflect such differences, and the level of detail that would be preferred for a comprehensive understanding of the microgel mechanical properties are not always implemented. Overall, the perspective is that current technologies have the capacity to provide detailed, nanoscopic mechanical characterization of microgels over an extended size range, to the high-throughput approaches providing distributions over the mechanical signatures, a feature not readily accessible by atomic force microscopy and micropipette aspiration.

Fabrication of monodisperse alginate microgel beads by microfluidic picoinjection: a chelate free approach.

Micron-sized alginate hydrogel beads are extensively employed as an encapsulation medium for biochemical and biomedical applications. Here we report on the microfluidic assisted fabrication of calcium alginate (Ca-alginate) beads by on-chip picoinjection of aqueous calcium chloride (CaCl2) in emulsified aqueous sodium alginate (Na-alginate) droplets or by picoinjection of Na-alginate solution in emulsified aqueous CaCl2 droplets. There is no added chelator to reduce the Ca activity in either of the two strategies. The two fabrication strategies are implemented using a flow-focusing and picoinjection modules in a single PDMS chip. Aqueous alginate solution was emulsified and infused with CaCl2 solution as the squeezed droplet pass the picoinjection channel; consequently, monodisperse, spherical, and structurally homogeneous Ca-alginate beads were obtained. Monodisperse alginate microgel populations with a mean diameter in the range of 8 to 28 µm and standard deviation less than 1 µm were successfully generated using microfluidic channels with various dimensions and controlling the flow parameters. Monodisperse but also non-spherical particles with diameters 6 to 15 µm were also fabricated when picoinjecting Na-alginate solution in emulsified aqueous CaCl2 droplets. The Ca-alginate microbeads fabricated with tailormade size in the range from sub-cellular and upwards were in both strategies realized without any use of chelators or change in pH conditions, which is considered a significant advantage for further exploitation as encapsulation process for improved enzymatic activity and cell viability.

Microparticle self-assembly induced by travelling surface acoustic waves.

We present an acoustofluidic method based on travelling surface acoustic waves (TSAWs) to induce self-assembly of microparticles inside a microfluidic channel. The particles are trapped above an interdigitated transducer, placed directly beneath the microchannel, by the TSAW-based direct acoustic radiation force (ARF). This approach was applied to trap 10 µm polystyrene particles, which were pushed towards the ceiling of the microchannel by 72 MHz TSAWs to form single- and multiple-layer colloidal structures. The repair of cracks and defects within the crystal lattice occurs as part of the self-assembly process. The sample flow through the first inlet can be switched with a buffer flow through the second inlet to control the number of particles assembled in the crystalline structure. The constant flow-induced Stokes drag force on the particles is balanced by the opposing TSAW-based ARF. This force balance is essential for the acoustics-based self-assembly of microparticles inside the microchannel. Moreover, we studied the effects of varying input voltage and fluid flow rate on the position and shape of the colloidal structure. The active self-assembly of microparticles into crystals with multiple layers can be used in the bottom-up fabrication of colloidal structures with dimensions greater than 500 µm × 500 µm, which is expected to have important applications in various fields.

Sheathless Focusing and Separation of Microparticles Using Tilted-Angle Traveling Surface Acoustic Waves.

Sheathless focusing and separation of microparticles is an important preprocessing step in various biochemical assays in which enriched sample isolation is critical. Most of the previous microfluidic particle separation techniques have used sheath flows to achieve efficient sample focusing. The sheath flow dilutes the analyte and requires additional microchannels and accurate flow control. We demonstrated a tilted-angle traveling surface acoustic wave (taTSAW)-based sheathless focusing and separation of particles in a continuous flow. The proposed device consists of a piezoelectric substrate with a pair of interdigitated transducers (IDTs) deposited at two different angles relative to the flow direction. A Y-shaped polydimethylsiloxane (PDMS) microchannel having one inlet and two outlet ports was positioned on top of the IDTs such that the acoustic energy coupling into the fluid was maximized and wave attenuation by the PDMS walls was minimized. The two IDTs independently produced high-frequency taTSAWs, which propagated at ±30° with respect to the flow direction and imparted a direct acoustic radiation force onto the target particles. A sample mixture of 4.8 and 3.2 µm particles was focused and then separated by the actuation of the IDTs at 194 and 136 MHz frequencies, respectively, without using an additional sheath flow. The proposed taTSAW-based particle separation device offered a high purity >99% at the both outlets over a wide range of flow speeds (up to 83.3 mm/s).

Vertical Hydrodynamic Focusing and Continuous Acoustofluidic Separation of Particles via Upward Migration.

A particle suspended in a fluid within a microfluidic channel experiences a direct acoustic radiation force (ARF) when traveling surface acoustic waves (TSAWs) couple with the fluid at the Rayleigh angle, thus producing two components of the ARF. Most SAW-based microfluidic devices rely on the horizontal component of the ARF to migrate prefocused particles laterally across a microchannel width. Although the magnitude of the vertical component of the ARF is more than twice the magnitude of the horizontal component, it is long ignored due to polydimethylsiloxane (PDMS) microchannel fabrication limitations and difficulties in particle focusing along the vertical direction. In the present work, a single-layered PDMS microfluidic chip is devised for hydrodynamically focusing particles in the vertical plane while explicitly taking advantage of the horizontal ARF component to slow down the selected particles and the stronger vertical ARF component to push the particles in the upward direction to realize continuous particle separation. The proposed particle separation device offers high-throughput operation with purity >97% and recovery rate >99%. It is simple in its fabrication and versatile due to the single-layered microchannel design, combined with vertical hydrodynamic focusing and the use of both the horizontal and vertical components of the ARF.

Development of reliable techniques for the differential diagnosis of avian tumour viruses by immunohistochemistry and polymerase chain reaction from formalin-fixed paraffin-embedded tissue sections.

A variety of techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek's disease virus from those induced by avian leukosis virus and reticuloendotheliosis virus. However, most current techniques are unreliable when used in formalin-fixed paraffin-embedded (FFPE) tissues, which often is the only sample type available for definitive diagnosis. A collection of tumours was generated by the inoculation of different strains of Marek's disease virus, reticuloendotheliosis virus or avian leukosis virus singularly or in combination. FFPE tissue sections from tumour and non-tumour tissues were analysed by optimized immunohistochemistry (IHC) techniques and traditional as well as quantitative polymerase chain reaction (PCR) with newly designed primers ideal for DNA fragmented by fixation. IHC and PCR results were highly sensitive and specific in tissues from single-infected birds. Virus quantity was higher in tumours compared to non-tumour spleens from Marek's disease (MD) virus-infected birds. Thus, using FFPE sections alone may be sufficient for the diagnosis of MD by demonstration of high quantities of viral antigens or genome in tumour cells, along with the absence of other tumour viruses by traditional PCR, and if standard criteria are met based on clinical history and histology. IHC furthermore allowed detection of the specific cells that were infected with different viruses in tumours from birds that had been inoculated simultaneously with multiple viruses. Following validation with field samples, these new protocols can be applied for both diagnostic and research purposes to help accurately identify avian tumour viruses in routine FFPE tissue sections.

Microfluidic flow switching via localized acoustic streaming controlled by surface acoustic waves.

We propose an acoustic flow switching device that utilizes high-frequency surface acoustic waves (SAWs) produced by a slanted-finger interdigitated transducer. As the acoustic field induced by the SAWs was attenuated in the fluid, it produced an acoustic streaming flow in the form of a pair of symmetrical microvortices, which induced flow switching between two fluid streams in a controlled manner. The microfluidic device was composed of a piezoelectric substrate attached to a polydimethylsiloxane (PDMS) microchannel having an H-shaped junction that connected two fluid streams in the middle. The two immiscible fluids, separated by the PDMS wall, flowed in parallel, briefly came in contact at the junction, and separated again into the downstream microchannels. The acoustic streaming flow induced by the SAWs rotated the fluid streams within the microchannel cross-section, thereby altering the respective positions of the two fluids and directing them into the opposite flow paths. The characteristics of the flow switching mechanism were investigated by tuning the input voltage and the flowrates. On-demand acoustic flow switching was successfully achieved without additional moving parts inside the microchannel. This technique may be useful for fundamental studies that integrate complex experimental platforms into a single chip.

On-demand acoustic droplet splitting and steering in a disposable microfluidic chip.

On-chip droplet splitting is one of the fundamental droplet-based microfluidic unit operations to control droplet volume after production and increase operational capability, flexibility, and throughput. Various droplet splitting methods have been proposed, and among them the acoustic droplet splitting method is promising because of its label-free operation without any physical or thermal damage to droplets. Previous acoustic droplet splitting methods faced several limitations: first, they employed a cross-type acoustofluidic device that precluded multichannel droplet splitting; second, they required irreversible bonding between a piezoelectric substrate and a microfluidic chip, such that the fluidic chip was not replaceable. Here, we present a parallel-type acoustofluidic device with a disposable microfluidic chip to address the limitations of previous acoustic droplet splitting devices. In the proposed device, an acoustic field is applied in the direction opposite to the flow direction to achieve multichannel droplet splitting and steering. A disposable polydimethylsiloxane microfluidic chip is employed in the developed device, thereby removing the need for permanent bonding and improving the flexibility of the droplet microfluidic device. We experimentally demonstrated on-demand acoustic droplet bi-splitting and steering with precise control over the droplet splitting ratio, and we investigated the underlying physical mechanisms of droplet splitting and steering based on Laplace pressure and ray acoustics analyses, respectively. We also demonstrated droplet tri-splitting to prove the feasibility of multichannel droplet splitting. The proposed on-demand acoustic droplet splitting device enables on-chip droplet volume control in various droplet-based microfluidic applications.

A Pumpless Acoustofluidic Platform for Size-Selective Concentration and Separation of Microparticles.

We have designed a pumpless acoustofluidic device for the concentration and separation of different sized particles inside a single-layered straight polydimethylsiloxane (PDMS) microfluidic channel. The proposed device comprises two parallel interdigitated transducers (IDTs) positioned underneath the PDMS microchannel. The IDTs produce high-frequency surface acoustic waves that generate semipermeable virtual acoustic radiation force field walls that selectively trap and concentrate larger particles at different locations inside the microchannel and allow the smaller particles to pass through the acoustic filter. The performance of the acoustofluidic device was first characterized by injecting into the microchannel a uniform flow of suspended 9.9 µm diameter particles with various initial concentrations (as low as 10 particles/mL) using a syringe pump. The particles were trapped with ∼100% efficiency by a single IDT actuated at 73 MHz. The acoustofluidic platform was used to demonstrate the pumpless separation of 12.0, 4.8, and 2.1 µm microparticles by trapping the 12 and 4.8 µm particles using the two IDTs actuated at 73 and 140 MHz, respectively. However, most of the 2.1 µm particles flowed over the IDTs unaffected. The acoustofluidic device was capable of rapidly processing a large volume of sample fluid pumped through the microchannel using an external syringe pump. A small volume of the sample fluid was processed through the device using a capillary flow and a hydrodynamic pressure difference that did not require an external pumping device.

Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation.

We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin-biotin linkage to form the PS-apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS-apt15-th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS-apt15-th complex was formed, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS-apt15-th separation from the non-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.

Acoustothermal tweezer for droplet sorting in a disposable microfluidic chip.

Precise control over droplet position within a microchannel is fundamental to droplet microfluidic applications. This article proposes acoustothermal tweezer for the control of droplet position, which is based on thermocapillary droplet migration actuated by acoustothermal heating. The proposed system comprises an acoustothermal heater, which is composed of a slanted finger interdigital transducer patterned on a piezoelectric substrate and a thin PDMS membrane, and a PDMS microchannel. In the proposed system, droplets moving in a droplet microfluidic chip experience spatiotemporally varying thermal stimuli produced by acoustothermal heating and thus migrate laterally. In comparison to previous methods for droplet sorting, the acoustothermal tweezer offers significant advantages: first, the droplet position can be manipulated in two opposite directions, which enables bidirectional droplet sorting to one of three outlets downstream; second, precise control over the droplet position as well as improved droplet lateral displacement on the order of hundreds of micrometers can be achieved in a deterministic manner, thereby enabling multichannel droplet sorting; third, the PDMS microfluidic chip is disposable and thus can be easily replaced since it is attached to the substrate by reversible bonding, which allows the acoustothermal heater to be reused. Given these advantages, the proposed droplet sorting system is a promising droplet microfluidic lab-on-a-chip platform for tunable, on-demand droplet position control.

On-Demand Droplet Capture and Release Using Microwell-Assisted Surface Acoustic Waves.

We demonstrate an acoustofluidic platform that uses surface acoustic waves (SAWs) for the facile capture of droplets inside microwells and their on-demand release. When the ac signal applied to the device is tuned to modulate the location of the SAW, the SAW-based acoustic radiation force retracts or pushes the droplets into or out of one of three microwells fabricated inside a microchannel to selectively capture or release the droplet.

In-droplet microparticle separation using travelling surface acoustic wave.

Droplets in microfluidic systems can contain microscale objects such as cells and microparticles. The control of the positions of microscale objects within a microchannel is crucial for practical applications in not only continuous-flow-based but also droplet-based systems. This paper proposes an active method for the separation of microparticles inside moving droplets which uses travelling surface acoustic waves (TSAWs). We demonstrate the preconcentration and separation of 5 and 10 µm polystyrene microparticles in moving water-in-oil droplets through the application of TSAWs with two different frequencies. The microparticles inside the droplets are affected by the acoustic radiation force induced by the TSAWs to move laterally in the direction of the TSAW propagation and are thereby separated according to their size. In-droplet separation is then demonstrated through droplet splitting at a Y-junction. Compared to our previous studies, this acoustic approach offers the label-free and on-demand separation of different-sized micro-objects in moving droplets. The present method has potential uses such as in-droplet sample purification and enrichment.

Particle Separation inside a Sessile Droplet with Variable Contact Angle Using Surface Acoustic Waves.

A sessile droplet of water carrying polystyrene microparticles of different diameters was uniformly exposed to high frequency surface acoustic waves (SAWs) produced by an interdigitated transducer (IDT). We investigated the concentration behavior of the microparticles as the SAWs generated a strong acoustic streaming flow (ASF) inside the water droplet and exerted a direct acoustic radiation force (ARF) on the suspended particles, the magnitude of which depended upon the particle diameter. As a result of the ARF, the microparticles were concentrated according to their diameters at different positions inside the sessile droplet placed in the path of the SAW, right in front of the IDT. The microparticle concentration behavior changed as the sessile droplet contact angle with the substrate was varied by adding surfactant to the water or by gradually evaporating the water. The positions at which the smaller and larger microparticles were concentrated remained distinguishable, even at very different experimental conditions. The long-term exposure of the droplets to the SAWs was accompanied by the gradual evaporation of the carrier fluid, which dynamically changed the droplet contact angle as well as the concentration of particles. Complete evaporation of the fluid left behind several concentrated yet separated clusters of particles on the substrate surface. The effect of the droplet contact angle on particles' concentration behavior and consequent separation of particles has been uniquely studied in this SAW-based report.