Stinging ant anaphylaxis: Advances in diagnosis and treatment.

Stinging ants represent a wide range of over 200 different species across the world, of which Solenopsis, Myrmecia, Pogonomyrmex, and Brachyponera genera, account for a substantial economic and healthcare burden. S. invicta (red imported fire ant; IFA) and M. pilosula (jack jumper ant; JJA) are 2 species of high clinical importance, known to cause anaphylaxis in humans, with numerous reported fatalities. Diagnostic testing should be performed in patients with a history of a systemic reaction with skin testing and/or in vitro specific-IgE testing. In vitro testing is commercially available for IFA through whole-body extract (WBE) specific-IgE and JJA venom specific-IgE, but not widely available for other stinging ant species. Commercial venom component testing for IFA and JJA is currently not available. Patients with a clinical history and positive specific-IgE testing, should undergo treatment with specific immunotherapy, which is currently available for IFA and JJA. Build-up may be performed using conventional, semi-rush, rush, or ultra-rush schedules with similar risk profiles for IFA. Optimal duration for WBE immunotherapy for IFA and specific JJA venom immunotherapy is not well-studied, but generally recommended for at least 3-5 years. Sting challenges are used in research settings, primarily to assess treatment efficacy of immunotherapy.

Toward a more realistic estimate of exposure to chromium and nickel in soils of geogenic and/or anthropogenic origin: importance of oral bioaccessibility.

To enhance risk assessment for contaminated sites, incorporating bioavailability through bioaccessibility as a corrective factor to total concentration is essential to provide a more realistic estimate of exposure. While the main in vitro tests have been validated for As, Cd, and/or Pb, their potential for assessing the bioaccessibility of additional elements remains underexplored. In this study, the physicochemical parameters, pseudototal Cr and Ni concentrations, soil phase distribution, and oral bioaccessibility of twenty-seven soil samples were analysed using both the ISO 17924 standard and a simplified test based on hydrochloric acid. The results showed wide variability in terms of the concentrations (from 31 to 21,079 mg kg-1 for Cr, and from 26 to 11,663 mg kg-1 for Ni) and generally low bioaccessibility for Cr and Ni, with levels below 20% and 30%, respectively. Bioaccessibility variability was greater for anthropogenic soils, while geogenic enriched soils exhibited low bioaccessibility. The soil parameters had an influence on bioaccessibility, but the effects depended on the soils of interest. Sequential extractions provided the most comprehensive explanation for bioaccessibility. Cr and Ni were mostly associated with the residual fraction, indicating limited bioaccessibility. Ni was distributed in all phases, whereas Cr was absent from the most mobile phase, which may explain the lower bioaccessibility of Cr compared to that of Ni. The study showed promising results for the use of the simplified test to predict Cr and Ni bioaccessibility, and its importance for more accurate human exposure evaluation and effective soil management practices.

Guidelines for Manufacturing and Application of Organoids: Liver.

Recent amendments to regulatory frameworks have placed a greater emphasis on the utilization of in vitro testing platforms for preclinical drug evaluations and toxicity assessments. This requires advanced tissue models capable of accurately replicating liver functions for drug efficacy and toxicity predictions. Liver organoids, derived from human cell sources, offer promise as a reliable platform for drug evaluation. However, there is a lack of standardized quality evaluation methods, which hinders their regulatory acceptance. This paper proposes comprehensive quality standards tailored for liver organoids, addressing cell source validation, organoid generation, and functional assessment. These guidelines aim to enhance reproducibility and accuracy in toxicity testing, thereby accelerating the adoption of organoids as a reliable alternative or complementary tool to animal testing in drug development. The quality standards include criteria for size, cellular composition, gene expression, and functional assays, thus ensuring a robust hepatotoxicity testing platform.

Oral Drug Product Administration via Enteral Feeding Tubes: In Vitro Testing.

Medication administration via enteral feeding tubes (EFT) is a necessary practice for patients unable to swallow oral dosage forms due to a medical condition or treatment that affects the ability to swallow or the function of the gastrointestinal tract. Off-label administration of oral drug products via EFT raises concerns for pharmaceutical sponsors, regulators, and healthcare practitioners (HCPs) because of the potential risks this practice introduces to both the patient and the caregiver. These risks can be mitigated by generating data-supported instructions that patients and HCPs can use to ensure safe and accurate administration of oral drug products via EFT. This commentary presents an industry perspective on the testing that should be conducted to enable development of product-specific instructions in the labeling to support or advise against administration of oral drug products via enteral feeding tube. The proposal outlined in this commentary takes a risk-based approach, addressing recommendations from both regulatory agencies as well as considerations for expanding this testing to address needs specific to neonatal and pediatric populations.

Dispersion and Dosimetric Challenges of Hydrophobic Carbon-Based Nanoparticles in In Vitro Cellular Studies.

Methodologies across the dispersion preparation, characterization, and cellular dosimetry of hydrophilic nanoparticles (NPs) have been developed and used extensively in the field of nanotoxicology. However, hydrophobic NPs pose a challenge for dispersion in aqueous culture media using conventional methods that include sonication followed by mixing in the culture medium of interest and cellular dosimetry. In this study, a robust methodology for the preparation of stable dispersions of hydrophobic NPs for cellular studies is developed by introducing continuous energy over time via stirring in the culture medium followed by dispersion characterization and cellular dosimetry. The stirring energy and the presence of proteins in the culture medium result in the formation of a protein corona around the NPs, stabilizing their dispersion, which can be used for in vitro cellular studies. The identification of the optimal stirring time is crucial for achieving dispersion and stability. This is assessed through a comprehensive stability testing protocol employing dynamic light scattering to evaluate the particle size distribution stability and polydispersity. Additionally, the effective density of the NPs is obtained for the stable NP dispersions using the volumetric centrifugation method, while cellular dosimetry calculations are done using available cellular computational modeling, mirroring approaches used for hydrophilic NPs. The robustness of the proposed dispersion approach is showcased using a highly hydrophobic NP model (black carbon NPs) and two culture media, RPMI medium and SABM, that are widely used in cellular studies. The proposed approach for the dispersion of hydrophobic NPs results in stable dispersions in both culture media used here. The NP effective density of 1.03-1.07 g/cm3 measured here for black carbon NPs is close to the culture media density, resulting in slow deposition on the cells over time. So, the present methodology for dispersion and dosimetry of hydrophobic NPs is essential for the design of dose-response studies and overcoming the challenges imposed by slow particle deposition.

Electrochemical polishing, characterisation and in vitro evaluation of additively manufactured CoCr stents with personalised designs.

Additive manufacturing (AM) technology has paved the way for manufacturing personalised stents. However, there is a notable gap in comprehensive microstructure analyses and in vitro evaluations of the AM CoCr stents using advanced methodologies. To address this gap, this study focuses on investigating the microstructure and in vitro performance of personalised CoCr stents manufactured through micro-laser powder bed fusion (µ-LPBF). The evaluation process begins with the measurements of dimensions and surface roughness, followed by in-depth microstructural analyses. To improve surface roughness and reduce excessive strut size, the µ-LPBF stents undergo electrochemical polishing. Importantly, in vitro stent deployments are carried out in artificial arteries manufactured based on actual patients' data. Compared to the commercial MULTI-LINK VISION CoCr stent, the µ-LPBF personalised stents have rough surface finish (average roughness: 1.55 µm for µ-LPBF vs. 1.09 µm for commercial) and compromised grain microstructures (elongated for µ-LPBF vs. equiaxed for commercial). However, the personalised stents demonstrate better performances in in vitro tests. Notably, compared to the commercial stent in the two studied cases, they deliver larger lumen gains (up to 11.24 %) and reduced recoils (up to 4 times). This study validates the merit of the lesion-specific designs and the feasibility of using AM technology for stent fabrication.

Identification of two novel chemical classes of Autotaxin (ATX) inhibitors using Enalos Asclepios KNIME nodes.

Autotaxin is a secreted lysophospholipase D which is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family converting extracellular lysophosphatidylcholine and other non-choline lysophospholipids, such as lysophosphatidylethanolamine and lysophosphatidylserine, to the lipid mediator lysophosphatidic acid. Autotaxin is implicated in various fibroproliferative diseases including interstitial lung diseases, such as idiopathic pulmonary fibrosis and hepatic fibrosis, as well as in cancer. In this study, we present an effort of identifying ATX inhibitors that bind to allosteric ATX binding sites using the Enalos Asclepios KNIME Node. All the available PDB crystal structures of ATX were collected, prepared, and aligned. Visual examination of these structures led to the identification of four crystal structures of human ATX co-crystallized with four known inhibitors. These inhibitors bind to five binding sites with five different binding modes. These five binding sites were thereafter used to virtually screen a compound library of 14,000 compounds to identify molecules that bind to allosteric sites. Based on the binding mode and interactions, the docking score, and the frequency that a compound comes up as a top-ranked among the five binding sites, 24 compounds were selected for in vitro testing. Finally, two compounds emerged with inhibitory activity against ATX in the low micromolar range, while their mode of inhibition and binding pattern were also studied. The two derivatives identified herein can serve as "hits" towards developing novel classes of ATX allosteric inhibitors.

The study of loading mode with in-vitro fatigue testing for mitral annuloplasty ring.

To maintain the physiological dynamics of the mitral annulus, mitral annuloplasty rings (MAR) must be flexible. Enhanced flexibility implies decreased resistance to fatigue and potential for fatigue fracture. This study established new methods to test the flexible fatigue life of MAR in-vitro using numerical analysis; the purpose is that the fatigue test could reflect the real stress distribution in-vivo. Based on the conventional test methods (C1, D1), this paper presents a novel test method (C2, D2). Four testing methods for open-end annuloplasty rings (C1, C2) and closed-end annuloplasty rings (D1, D2) were modelled and their stress distribution calculated by finite element analysis. The mean absolute error (Χ) and the Pearson correlation coefficient (Φ) were used to quantify the difference in stress distribution between the loading modes in-vivo and in-vitro. For closed-end annuloplasty rings, the novel test method (D2) is not obvious better than conventional test methods(D1) in duplicating the stress distribution (ΦD1 = 0.88 vs ΦD2 = 0.92). However, the maximum values of stress in the novel test method are closer to the maximum value of stress under in-vivo loading (ΧD1 = 5.2Mpa vs ΧD2 = 4.4Mpa). For open-end annuloplasty rings, the novel test method(C2) is obviously superior to the conventional test method(C1) in duplicating both the stress distribution and the stress peak values of the in-vivo loading (ΦC1 = 0.22 vs ΦC2 = 0.98; ΧC1 = 59.1Mpa vs ΧC2 = 11.0Mpa). The in-vitro loading methods described in this article more closely approximated in-vivo conditions compared to traditional methods. They are simpler to operate, more efficient and can help manufacturers expedite new product development, assist regulatory agencies with product quality oversight.

Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Recombinantly Expressed Tagged SUrface Protein (RETSUP) assay: a new diagnostic system for the detection of antibodies to platelets.

BACKGROUND: Current assays for the detection of (allo)antibodies to platelet antigens are often laborious and widely based on the presence of well-characterized donor platelets. OBJECTIVES: To develop an easy-to-perform, sensitive, and specific test for the detection of antibodies against platelet antigens, in particular, glycoprotein (GP) antigens, called "Recombinantly Expressed Tagged SUrface Protein" (RETSUP) assay, which does not require donor platelets. METHODS: Twin-Strep-tagged GP complexes were recombinantly expressed in human embryonic kidney 293 cells after stable transfection. These cell lines were used as antigen sources in the RETSUP assay, combining cell-based and enzyme-linked immunosorbent assay-based assay procedures. The assay performance was tested with recombinant antibodies, anti-human platelet antigen (HPA) reference plasmas, and anti-HPA patient sera. RESULTS: Human embryonic kidney 293 cell lines stably expressing either Twin-Strep-labeled GPIa/IIa, GPIIb/IIIa, GPIb/IX, or GPIb/IX/V complexes or GPV as well as the distinct HPA-1, HPA-3, and HPA-5 epitopes were successfully generated. Applying the generated GP-expressing cell lines, the developed RETSUP assay proved very sensitive and specific with recombinant antibodies targeting different GPs and human plasma/serum samples. The results of the test were not affected by the GP carrying the Twin-Strep-tag or by using freshly harvested or cryopreserved cells. CONCLUSION: The RETSUP assay is an easy-to-perform, sensitive, and specific assay for the detection of plasma/serum antibodies to platelet GP, with performance comparable to or better than those of current state-of-the-art assays in antiplatelet antibody diagnostics. Owing to the recombinant nature of the target antigens, it can be easily adapted to detect antibodies in other antibody-mediated diseases.

Effect of Receptor Solution in Studies of In Vitro Permeation Test (IVPT).

In Vitro Permeation Test (IVPT) is commonly used to evaluate skin penetration of chemicals and performance of dermatological products. For a permeant with low aqueous solubility, an additive that is expected not to alter the skin barrier can be used in the receptor solution to improve permeant solubility. The objective of this study was to (a) evaluate the effects of these additives in IVPT receptor solution on skin permeability of model permeants and skin electrical resistance and (b) determine the solubility of the permeants in these receptor solutions. Bovine serum albumin (BSA), 2-hydroxypropyl-beta-cyclodextrin (HPCD), ethanol, nonionic surfactant Brij-98, and propylene glycol were the additives, and phosphate buffered saline (PBS) was the control. Steady-state skin permeability coefficients and resistances were determined. The receptor solutions examined in this study did not cause a significant increase in skin permeability or decrease in resistance (less than 40 % changes) except 25 % ethanol. The receptor solution containing 25 % ethanol induced an approximately twofold average increase in skin permeability and reduced skin electrical resistance by approximately threefold. The receptor solution of 2.5 % HPCD provided the highest levels of solubility for the model lipophilic permeants, while 0.2 % Brij-98 and 5 % ethanol showed the lowest solubility enhancement from those in PBS.

Development of Novel Sutureless Balloon Expandable Fetal Heart Valve Device Using Absorbable Polycaprolactone Leaflets.

Congenital heart disease (CHD) accounts for nearly one-third of all congenital defects, and patients often require repeated heart valve replacements throughout their lives, due to failed surgical repairs and lack of durability of bioprosthetic valve implants. This objective of this study is to develop and in vitro test a fetal transcatheter pulmonary valve replacement (FTPVR) using sutureless techniques to attach leaflets, as an option to correct congenital defects such as pulmonary atresia with intact ventricular septum (PA/IVS), in utero. A balloon expandable design was analyzed using computational simulations to identify areas of failure. Five manufactured valves were assembled using the unique sutureless approach and tested in the fetal right heart simulator (FRHS) to evaluate hemodynamic characteristics. Computational simulations showed that the commissural loads on the leaflet material were significantly reduced by changing the attachment techniques. Hemodynamic analysis showed an effective orifice area of 0.08 cm2, a mean transvalvular pressure gradient of 7.52 mmHg, and a regurgitation fraction of 8.42%, calculated over 100 consecutive cardiac cycles. In conclusion, the FTPVR exhibited good hemodynamic characteristics, and studies with biodegradable stent materials are underway.

A Dynamic Elbow Testing Apparatus for Simulating Elbow Joint Motion in Varying Shoulder Positions.

Purpose: To develop and evaluate the capabilities of a dynamic elbow testing apparatus that simulates unconstrained elbow motion throughout the range of humerothoracic (HTA) abduction. Methods: Elbow flexion was generated by six computer-controlled electromechanical actuators that simulated muscle action, while six degree-of-freedom joint motion was measured using an optical tracking device. Repeatability of joint kinematics was assessed at four HTA angles (0°, 45°, 90°, 135°) and with two muscle force combinations (A1-biceps brachialis, brachioradialis and A2-biceps, brachioradialis). Repeatability was determined by comparing kinematics at every 10° of flexion over five flexion-extension cycles (0° to 100°). Results: Multiple muscle force combinations can be used at each HTA angle to generate elbow flexion. Trials showed that the testing apparatus produced highly repeatable joint motion at each HTA angle and with varying muscle force combinations. The intraclass correlation coefficient was greater than 0.95 for all conditions. Conclusions: Repeatable smooth cadaveric elbow motion was created that mimicked the in vivo situation. Clinical relevance: These results suggest that the dynamic elbow testing apparatus can be used to characterize elbow biomechanics in cadaver upper extremities.

Non-animal approaches for photoallergenicity safety assessment: Needs and perspectives for the toxicology for the 21st century.

Certain chemicals and/or their byproducts are photoactivated by UV/VIS and trigger a dermal allergenic response, clinically recognized as photoallergic contact dermatitis (PACD). It is important to identify the chemicals which are potentially photoallergenic, not only for establishing the correct differential diagnosis between PACD and other photodermatoses, but also as causative agents which should be avoided as a preventative measure. Moreover, materials with photoallergenic properties need to be correctly identified to allow thorough safety assessments for their use in finished products (e.g. cosmetics). Development of methods for predicting photoallergenicity potential of chemicals has advanced at slow pace in recent years. To date, there are no validated methods for photosensitisation potential of chemicals for regulatory purposes, although it remains a required endpoint in some regions. The purpose of this review is to explore the mechanisms potentially involved in the photosensitisation process and discuss the methods available in the literature for identification of photosensitisers. The review also explores the possibilities of further research investment required to develop human-relevant new approach methodologies (NAMs) and next generation risk assessment (NGRA) approaches, considering the current perspectives and needs of the Toxicology for the 21st Century.

Mining the ZINC database of natural products for specific, testosterone-like, OXER1 antagonists.

OXER1, the receptor for the oxidized arachidonic acid metabolite 5-oxo-ETE has been reported to play a significant role in inflammatory responses, being responsible for leucocyte chemotactic responses. Recently, we have identified OXER1 (GPR170) as a membrane receptor for androgens in prostate and breast cancer cells. Testosterone action via OXER1 induces specific Ca2+ release from intracellular organelles, modifies polymerized actin distribution induces apoptosis and decreases cancer cell migration. These actions are antagonized by 5-oxo-ETE. In addition, 5-oxo-ETE through a Gαi protein decreases cAMP, an action antagonized by testosterone. In this work, we mined the ZINC15 database, using QSAR, for natural compounds able to signal through Gαi and Gßγ simultaneously, mimicking testosterone actions, as well as for specific Gßγ interactors, inhibiting 5-oxo-ETE tumor promoting actions. We were able to identify four druggable Gαßγ and seven Gßγ specific OXER1 interactors. We further confirmed by bio-informatic methods their binding to the 5-oxo-ETE/testosterone binding groove of the receptor, their ADME properties and their possible interaction with other receptor and/or enzyme targets. Two compounds, ZINC04017374 (Naphthofluorescein) and ZINC08589130 (Puertogaline A) were purchased, tested in vitro and confirmed their OXER1 Gßγ and Gαßγ activity, respectively. The methodology followed is useful for a better understanding of the mechanism by which OXER1 mediates its actions, it has the potential to provide structural insights, in order to design small molecular specific interactors and ultimately design new anti-inflammatory and anti-cancer agents. Finally, the methodology may also be useful for identifying specific agonists/antagonists of other GPCRs.

Developing a Prolamin-Based Gel for Food Packaging: In-Vitro Assessment of Cytocompatibility.

Growing environmental concerns drive efforts to reduce packaging waste by adopting biodegradable polymers, coatings, and films. However, biodegradable materials used in packaging face challenges related to barrier properties, mechanical strength, and processing compatibility. A composite gel was developed using biodegradable compounds (prolamin, d-mannose, citric acid), as a coating to increase the oxygen barrier of food packaging materials. To improve gel stability and mechanical properties, the gels were physically cross-linked with particles synthesized from tetraethyl orthosilicate and tetramethyl orthosilicate precursors. Additionally, biocompatibility assessments were performed on human keratinocytes and fibroblasts, demonstrating the safety of the gels for consumer contact. The gel properties were characterized, including molecular structure, morphology, and topography. Biocompatibility of the gels was assessed using bioluminescent ATP assay to detect cell viability, lactate dehydrogenase assay to determine cell cytotoxicity, and a leukocyte stimulation test to detect inflammatory potential. A composite gel with strong oxygen barrier properties in low-humidity environments was prepared. Increasing the silane precursor to 50 wt% during gel preparation slowed degradation in water. The addition of citric acid decreased gel solubility. However, higher precursor amounts increased surface roughness, making the gel more brittle yet mechanically resistant. The increase of precursor in the gel also increased gel viscosity. Importantly, the gels showed no cytotoxicity on human keratinocytes or fibroblasts and had no inflammatory effects on leukocytes. This composite gel holds promise for oxygen barrier food packaging and is safe for consumer contact. Further research should focus on optimizing the stability of the oxygen barrier in humid environments and investigate the potential sensitizing effects of biodegradable materials on consumers.

Advanced in vitro hemocompatibility assessment of biomaterials using a new flow incubation system.

Physiologically relevant in vitro hemocompatibility assessment of biomaterials remains challenging. We present a new setup that enables standardized whole blood incubation of biomedical materials under flow. A blood volume of 2 mL is recirculated over test surfaces in a custom-made parallel plate incubation system to determine the activation of hemostasis and inflammation. Controlled physiological shear rates between 125 s-1 and 1250 s-1 and minimized contact to air are combined with a natural-like pumping process. A unique feature of this setup allows tracing adhesion of blood cells to test surfaces microscopically in situ. Validation testing was performed in comparison to previously applied whole blood incubation methodologies. Experiments with the newly developed setup showed that even small obstacles to blood flow activate blood (independent of materials-induced blood activation levels); that adhesion of blood cells to biomaterials equilibrates within 5 to 10 min; that high shear rates (1250 compared to 375 s-1) induce platelet activation; and that hemolysis, platelet factor 4 (PF4) release and platelet loss - but not thrombin formation - depend on shear rate (within the range investigated, 125 to 1250 s-1).

Significance of cell culture phases for utilizing 3D cultures of A549 cells for in vitro studies.

Three-dimensional (3D) culture systems of human cancer cell lines have become popular experimental models for a variety of applications including drug screening. It is understood that the 2D and 3D cultures of the same cell line behave differently in several aspects. One such difference is in the duration of cell culture phases (the lag, log, plateau and the decline stages). We obtained 3D cultures of A549 cells on agarose hydrogels. We observed and compared the morphological differences in the progression of 2D and 3D cultures of A549 cells in a time-dependent manner. The morphological features along with the cell counts and viabilities obtained for the 2D and 3D cultures at different time intervals clearly indicate that the cell culture phases occurred as more extended one for the 3D cultures compared to that of the 2D counterparts. The plateau stage for the 2D and 3D cultures occurred at 48 and 69 h, respectively. Such cell culture phase durations can be different for different cell lines as a function of their doubling times. We propose that the cell culture phase durations for any cell line should be first established before using them for drug testing or for studies involving toxicity to obtain useful results from 3D cell cultures. Also, we propose that the late-exponential (lag) phase of 3D cultures of cancer cell lines is the most ideal one for drug testing owing to the various optimal features of the aggregates in this cell culture phase.

Insight into Antibiotic Synergy Combinations for Eliminating Colistin Heteroresistant Klebsiella pneumoniae.

Colistin heteroresistance has been identified in several bacterial species, including Escherichia coli and Klebsiella pneumoniae, and may underlie antibiotic therapy failures since it most often goes undetected by conventional antimicrobial susceptibility tests. This study utilizes population analysis profiling (PAP) and time-kill assay for the detection of heteroresistance in K. pneumoniae and for evaluating the association between in vitro regrowth and heteroresistance. The mechanisms of colistin resistance and the ability of combination therapies to suppress resistance selection were also analysed. In total, 3 (18%) of the 16 colistin-susceptible strains (MIC ≤ 2 mg/L) were confirmed to be heteroresistant to colistin by PAP assay. In contrast to the colistin-susceptible control strains, all three heteroresistant strains showed regrowth when exposed to colistin after 24 h following a rapid bactericidal action. Colistin resistance in all the resistant subpopulations was due to the disruption of the mgrB gene by various insertion elements such as ISKpn14 of the IS1 family and IS903B of the IS5 family. Colistin combined with carbapenems (imipenem, meropenem), aminoglycosides (amikacin, gentamicin) or tigecycline was found to elicit in vitro synergistic effects against these colistin heteroresistant strains. Our experimental results showcase the potential of combination therapies for treatment of K. pneumoniae infections associated with colistin heteroresistance.