Plasmids in antibiotic susceptible and antibiotic resistant commensal Escherichia coli from healthy Australian adults.

A collection of 111 commensal Escherichia coli isolated from 84 faecal samples from healthy Australian adults were screened using PCR-based replicon typing. Each isolate represented a distinct strain found in a particular faecal sample. Fifty-one isolates were resistant to one or more of 12 antibiotics tested. FII and FIB replicons were most common and usually found together. The FII replicon was detected in 63 isolates (35 susceptible, 28 resistant), the FIB replicon was present in 65 (32 susceptible, 33 resistant) and 54 (30 susceptible, 24 resistant) included both. Other replicon types were found infrequently (A/C, I1, K, L/M, P, R, Y, FIA and FIC) or not at all (HI1, HI2, N, T, U, W, X). Only the B/O amplicon, found in 21 resistant but only 4 susceptible isolates, was associated with antibiotic resistance. Detailed analysis of this group revealed that the B/O PCR also detected Z plasmids of several distinguishable types. PCR assays were developed to detect the two repA genes (repABKI and repAZ) found in members of the I-complex (I, B/O, K and Z plasmids). These assays distinguished the B/O and Z plasmids detected by the original "B/O" PCR. One isolate carried repABKI and the remainder carried repAZ. These genes were also detected in further isolates in the collection. Conjugative transfer of resistance genes was detected for the B/O plasmid and two Z groups. Evidence for transfer of repAZ plasmids in the human colon in the absence of antibiotic selection was also obtained.

Gene Electrotransfer via Conductivity-Clamped Electric Field Focusing Pivots Sensori-Motor DNA Therapeutics: "A Spoonful of Sugar Helps the Medicine Go Down".

Viral vectors and lipofection-based gene therapies have dispersion-dependent transduction/transfection profiles that thwart precise targeting. The study describes the development of focused close-field gene electrotransfer (GET) technology, refining spatial control of gene expression. Integration of fluidics for precise delivery of "naked" plasmid deoxyribonucleic acid (DNA) in sucrose carrier within the focused electric field enables negative biasing of near-field conductivity ("conductivity-clamping"-CC), increasing the efficiency of plasma membrane molecular translocation. This enables titratable gene delivery with unprecedently low charge transfer. The clinic-ready bionics-derived CC-GET device achieved neurotrophin-encoding miniplasmid DNA delivery to the cochlea to promote auditory nerve regeneration; validated in deafened guinea pig and cat models, leading to improved central auditory tuning with bionics-based hearing. The performance of CC-GET is evaluated in the brain, an organ problematic for pulsed electric field-based plasmid DNA delivery, due to high required currents causing Joule-heating and damaging electroporation. Here CC-GET enables safe precision targeting of gene expression. In the guinea pig, reporter expression is enabled in physiologically critical brainstem regions, and in the striatum (globus pallidus region) delivery of a red-shifted channelrhodopsin and a genetically-encoded Ca2+ sensor, achieved photoactivated neuromodulation relevant to the treatment of Parkinson's Disease and other focal brain disorders.

TRPC Channels Activated by G Protein-Coupled Receptors Drive Ca2+ Dysregulation Leading to Secondary Brain Injury in the Mouse Model.

Canonical transient receptor potential (TRPC) non-selective cation channels, particularly those assembled with TRPC3, TRPC6, and TRPC7 subunits, are coupled to Gαq-type G protein-coupled receptors for the major classes of excitatory neurotransmitters. Sustained activation of this TRPC channel-based pathophysiological signaling hub in neurons and glia likely contributes to prodigious excitotoxicity-driven secondary brain injury expansion. This was investigated in mouse models with selective Trpc gene knockout (KO). In adult cerebellar brain slices, application of glutamate and the class I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine to Purkinje neurons expressing the GCaMP5g Ca2+ reporter demonstrated that the majority of the Ca2+ loading in the molecular layer dendritic arbors was attributable to the TRPC3 effector channels (Trpc3KO compared with wildtype (WT)). This Ca2+ dysregulation was associated with glutamate excitotoxicity causing progressive disruption of the Purkinje cell dendrites (significantly abated in a GAD67-GFP-Trpc3KO reporter brain slice model). Contribution of the Gαq-coupled TRPC channels to secondary brain injury was evaluated in a dual photothrombotic focal ischemic injury model targeting cerebellar and cerebral cortex regions, comparing day 4 post-injury in WT mice, Trpc3KO, and Trpc1/3/6/7 quadruple knockout (TrpcQKO), with immediate 2-h (primary) brain injury. Neuroprotection to secondary brain injury was afforded in both brain regions by Trpc3KO and TrpcQKO models, with the TrpcQKO showing greatest neuroprotection. These findings demonstrate the contribution of the Gαq-coupled TRPC effector mechanism to excitotoxicity-based secondary brain injury expansion, which is a primary driver for mortality and morbidity in stroke, traumatic brain injury, and epilepsy.

Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice.

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the 'cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, "contralateral suppression" of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24-32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.

Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli.

OBJECTIVES: The context of antibiotic resistance genes can provide valuable information about the epidemiology of mobile genetic elements. This study examined the distribution of the closely related blaTEM transposons Tn1, Tn2 and Tn3, or blaTEM-containing fragments of them, in ampicillin-resistant human commensal Escherichia coli isolates. METHODS: A PCR mapping protocol was used to detect different segments of the transposons or to link partial copies to the insertion sequence IS26. Restriction digestion of one amplicon was used to assign transposons to Tn1, Tn2 or Tn3 groups and sequencing validated this approach. Restriction digestion and sequencing were used to determine how much of the transposon remained when blaTEM was linked to IS26. Sequences were compared with those in GenBank. RESULTS: Of 25 ampicillin-resistant E. coli strains recovered from the faecal flora of healthy humans that carried the blaTEM gene, 15 carried a complete copy of Tn2 or a Tn2 variant; one was interrupted by IS4. A further isolate carried Tn3. Tn2 was also most abundant in sequences available in GenBank. Two isolates carried Tn2 and an IS26-blaTEM fragment. The remaining 10 isolates carried only the blaTEM end of the transposon and 9 of these partial copies were flanked by IS26 at varying distances upstream of blaTEM. One configuration corresponded to that in Tn6029B and the complete transposon was shown to be present. CONCLUSIONS: Tn1, Tn2 and Tn3 can be simply and rapidly identified. Tn2 appears to be the most widely distributed. However, the blaTEM-containing end associated with an IS26 is also widely distributed.

Australian Scorpion Hormurus waigiensis Venom Fractions Show Broad Bioactivity Through Modulation of Bio-Impedance and Cytosolic Calcium.

Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da-13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.

Dual-Plasmid Bionic Array-Directed Gene Electrotransfer in HEK293 Cells and Cochlear Mesenchymal Cells Probes Transgene Expression and Cell Fate.

Naked plasmid DNA electrotransfer offers advantages over viral-based gene delivery, including being regulatory permissive, but factors influencing expression efficiency and cell fate impact on translational utility. This study compared co-expression of red and green fluorescence reporter plasmids with differing promoters in HEK293 cells and in vivo in guinea pig cochlear mesenchymal cells using Bionic array-Directed Gene Electrotransfer (BaDGE®). A functional plasmid copy number of ∼64 was established in HEK293 cells by co-transfecting with separate CMV-actin-globin (CAGp) promoter-driven mCherry and green fluorescent protein (GFP) reporters, where cell division diluted plasmids toward discrete red or green channels from 100% co-expression to 10% over 24 days (∼17 cell cycles). Cross-talk between promoters was identified by interchanging a cytomegalovirus promoter (CMVp)-driven GFP plasmid for the CAGp-GFP plasmid. Here, expression of the CMVp-GFP plasmid dominated, while a dual CAGp-based reporter plasmid cocktail showed persistent co-expression beyond 2 weeks. In contrast, in vivo, cochlear mesenchymal cells co-transduced with CAGp-mCherry and CMVp-GFP plasmids showed stable co-expression at ∼50%, while the total transfectant numbers diminished over 2 weeks. This is consistent with a lack of mitosis in the cochlear mesenchymal cells and shows that cell type is a factor in plasmid interaction.

Computational Simulation Expands Understanding of Electrotransfer-Based Gene Augmentation for Enhancement of Neural Interfaces.

The neural interface is a critical factor in governing efficient and safe charge transfer between a stimulating electrode and biological tissue. The interface plays a crucial role in the efficacy of electric stimulation in chronic implants and both electromechanical properties and biological properties shape this. In the case of cochlear implants, it has long been recognized that neurotrophins can stimulate growth of the target auditory nerve fibers into a favorable apposition with the electrode array, and recently such arrays have been re-purposed to enable electrotransfer (electroporation)-based neurotrophin gene augmentation to improve the "bionic ear." For both this acute bionic array-directed electroporation and for chronic conventional cochlear implant arrays, the electric fields generated in target tissue during pulse delivery are central to efficacy, but are challenging to map. We present a computational model for predicting electric fields generated by array-driven DNA electrotransfer in the cochlea. The anatomically realistic model geometry was reconstructed from magnetic resonance images of the guinea pig cochlea and an eight-channel electrode array embedded within this geometry. The model incorporates a description of both Faradaic and non-Faradaic mechanisms occurring at the electrode-electrolyte interface with frequency dependency optimized to match experimental impedance spectrometry measurements. Our simulations predict that a tandem electrode configuration with four ganged cathodes and four ganged anodes produces three to fourfold larger area in target tissue where the electric field is within the range for successful gene transfer compared to an alternate paired anode-cathode electrode configuration. These findings matched in vivo transfection efficacy of a green fluorescent protein (GFP) reporter following array-driven electrotransfer of the reporter-encoding plasmid DNA. This confirms utility of the developed model as a tool to optimize the safety and efficacy of electrotransfer protocols for delivery of neurotrophin growth factors, with the ultimate aim of using gene augmentation approaches to improve the characteristics of the electrode-neural interfaces in chronically implanted neurostimulation devices.

Neurotrophin gene augmentation by electrotransfer to improve cochlear implant hearing outcomes.

This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed 'close-field electroporation'. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4; plasmid Free of Antibiotic Resistance v.4) which could be used to package a 'humanized' neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies.

Computational Simulation of Array-based Electroporation in the Cochlea.

We present a computational model for predicting electric field distributions following array-based closed-loop electroporation in the cochlea. The model geometry was reconstructed from magnetic resonance images of the guinea pig cochlea and an eight-channel electrode array embedded within this geometry. The model's electrode voltage output waveform was obtained from electric potential mapping conducted in physiological solution following constant-current stimulation using the electrode array. Our simulations predict that a tandem electrode configuration with four ganged cathodes and four ganged anodes produces a larger area in target tissue where the electric field is within the range for successful gene transfer compared to an alternate paired anode-cathode electrode configuration. These findings corroborate published in vivo evidence comparing the two configurations and support the utility of the developed model as a tool to optimize the efficacy of electroporation electrodes.

Comparing perilymph proteomes across species.

OBJECTIVES/HYPOTHESIS: Biological components of perilymph affect the electrical performance of cochlear implants. Understanding the perilymph composition of common animal models will improve the understanding of this impact and improve the interpretation of results from animal studies and how it relates to humans. STUDY DESIGN: Analysis and comparison of the proteomes of human, guinea pig, and cat perilymph. METHODS: Multiple perilymph samples from both guinea pigs and cats were analysed via liquid chromatography with tandem mass spectrometry. Proteins were identified using the Mascot database. Human data were obtained from a published dataset. Proteins identified were refined to form a proteome for each species. RESULTS: Over 200 different proteins were found per species. There were 81, 39, and 64 proteins in the final human, guinea pig, and cat proteomes, respectively. Twenty-one proteins were common to all three species. Fifty-two percent of the cat proteome was found in the human proteome, and 31% of the guinea pig was common to human. The cat proteome had similar complexity to the human proteome in three protein classes, whereas the guinea pig had a similar complexity in two. The presence of albumin was significantly higher in human perilymph than in the other two species. Immunoglobulins were more abundant in the human than in the cat proteome. CONCLUSIONS: Perilymph proteomes were compared across three species. The degree of crossover of proteins of both guinea pig and cat with human indicate that these animals suitable models for the human cochlea, albeit the cat perilymph is a closer match. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E47-E52, 2018.

Understanding the cochlear implant environment by mapping perilymph proteomes from different species.

Cochlear implants operate within a bony channel of the cochlea, bathed in a fluid known as the perilymph. The perilymph is a complex fluid containing ions and proteins, which are known to actively interact with metallic electrodes. To improve our understanding of how cochlear implant performance varies in preclinical in vivo studies in comparison to human trials and patient outcomes, the protein composition (or perilymph proteome) is needed. Samples of perilymph were gathered from feline and guinea pig subjects and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to produce proteomes and compare against the recently published human proteome. Over 64% of the proteins in the guinea pig proteome were found to be common to the human proteome. The proportions of apolipoproteins, enzymes and immunoglobulins showed little variation between the two proteomes, with other classes showing similarity. This establishes a good basis for comparison of results. The results for the feline profile showed less similarity with the human proteome and would not provide a quality comparison. This work highlights the suitability of the guinea pig to model the biological environment of the human cochlear and the need to carefully select models of the biological environment of a cochlear implant to more adequately translate in vitro and in vivo studies to the clinic.

Close-field electroporation gene delivery using the cochlear implant electrode array enhances the bionic ear.

The cochlear implant is the most successful bionic prosthesis and has transformed the lives of people with profound hearing loss. However, the performance of the "bionic ear" is still largely constrained by the neural interface itself. Current spread inherent to broad monopolar stimulation of the spiral ganglion neuron somata obviates the intrinsic tonotopic mapping of the cochlear nerve. We show in the guinea pig that neurotrophin gene therapy integrated into the cochlear implant improves its performance by stimulating spiral ganglion neurite regeneration. We used the cochlear implant electrode array for novel "close-field" electroporation to transduce mesenchymal cells lining the cochlear perilymphatic canals with a naked complementary DNA gene construct driving expression of brain-derived neurotrophic factor (BDNF) and a green fluorescent protein (GFP) reporter. The focusing of electric fields by particular cochlear implant electrode configurations led to surprisingly efficient gene delivery to adjacent mesenchymal cells. The resulting BDNF expression stimulated regeneration of spiral ganglion neurites, which had atrophied 2 weeks after ototoxic treatment, in a bilateral sensorineural deafness model. In this model, delivery of a control GFP-only vector failed to restore neuron structure, with atrophied neurons indistinguishable from unimplanted cochleae. With BDNF therapy, the regenerated spiral ganglion neurites extended close to the cochlear implant electrodes, with localized ectopic branching. This neural remodeling enabled bipolar stimulation via the cochlear implant array, with low stimulus thresholds and expanded dynamic range of the cochlear nerve, determined via electrically evoked auditory brainstem responses. This development may broadly improve neural interfaces and extend molecular medicine applications.

Evolution of IncP-1α plasmids by acquisition of antibiotic and mercuric ion resistance transposons.

The aim of this study was to examine the relationship between the IncP-1α plasmid RP1 (representing RP4, RK2, R68, and R18) and two plasmids, R1033 and R934, that are known to be related to RP1. The region containing most of the antibiotic resistance genes in R1033 and R934 was mapped using polymerase chain reaction and sequenced. Both plasmids contained a copy of the class II transposon Tn1, but it was located at different sites between the position of Tn1 and the tet(A) determinant in RP1. Thus, Tn1 and the ampicillin resistance gene contained in it were acquired by the IncP1-α backbone on three separate occasions. In R1033, Tn1696 is located nearby, between Tn1 and the tet(A) determinant, and an IS186 insertion sequence was also found in this region. In R934, a defective class II transposon carrying a partial mercuric ion resistance (mer) module was found within the Tn1. These findings show that R1033 is not derived directly from RP1/RP4, refining the evolutionary pathways previously predicted for the IncP-1α plasmid family.

Commensal Escherichia coli of healthy humans: a reservoir for antibiotic-resistance determinants.

This study examined in detail the population structure of Escherichia coli from healthy adults with respect to the prevalence of antibiotic resistance and specific resistance determinants. E. coli isolated from the faeces of 20 healthy adults not recently exposed to antibiotics was tested for resistance to ten antibiotics and for carriage of integrons and resistance determinants using PCR. Strain diversity was assessed using biochemical and molecular criteria. E. coli was present in 19 subjects at levels ranging from 2.0×10(4) to 1.7×10(8) c.f.u. (g faeces)(-1). Strains resistant to one to six antibiotics were found at high levels (>30 %) in only ten individuals, but at significant levels (>0.5 %) in 14. Resistant isolates with the same phenotype from the same individual were indistinguishable, but more than one susceptible strain was sometimes found. Overall, individuals harboured one to four E. coli strains, although in 17 samples one strain was dominant (>70 % of isolates). Eighteen strains resistant to ampicillin, sulfamethoxazole, tetracycline and trimethoprim in 15 different combinations were observed. One resistant strain was carried by two unrelated individuals and a susceptible strain was shared by two cohabiting subjects. Two minority strains were derivatives of a more abundant resistant strain in the same sample, showing that continuous evolution is occurring in vivo. The trimethoprim-resistance genes dfrA1, dfrA5, dfrA7, dfrA12 or dfrA17 were in cassettes in a class 1 or class 2 integron. Ampicillin resistance was conferred by the bla(TEM) gene, sulfamethoxazole resistance by sul1, sul2 or sul3 and tetracycline resistance by tetA(A) or tetA(B). Chloramphenicol resistance (cmlA1 gene) was detected only once. Phylogenetic groups A and B2 were more common than B1 and D. Commensal E. coli of healthy humans represent an important reservoir for numerous antibiotic-resistance genes in many combinations. However, measuring the true extent of resistance carriage in commensal E. coli requires in-depth analysis.