Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

Minimum Bactericidal Concentration of Ciprofloxacin to Pseudomonas aeruginosa Determined Rapidly Based on Pyocyanin Secretion.

Infections due to Pseudomonas aeruginosa (P. aeruginosa) often exhibit broad-spectrum resistance and persistence to common antibiotics. Persistence is especially problematic with immune-compromised subjects who are unable to eliminate the inhibited bacteria. Hence, antibiotics must be used at the appropriate minimum bactericidal concentration (MBC) rather than at minimum inhibitory concentration (MIC) levels. However, MBC determination by conventional methods requires a 24 h culture step in the antibiotic media to confirm inhibition, followed by a 24 h sub-culture step in antibiotic-free media to confirm the lack of bacterial growth. We show that electrochemical detection of pyocyanin (PYO), which is a redox-active bacterial metabolite secreted by P. aeruginosa, can be used to rapidly assess the critical ciprofloxacin level required for bactericidal deactivation of P. aeruginosa within just 2 hours in antibiotic-treated growth media. The detection sensitivity for PYO can be enhanced by using nanoporous gold that is modified with a self-assembled monolayer to lower interference from oxygen reduction, while maintaining a low charge transfer resistance level and preventing electrode fouling within biological sample matrices. In this manner, bactericidal efficacy of ciprofloxacin towards P. aeruginosa at the MBC level and bacterial persistence at the MIC level can be determined rapidly, as validated at later timepoints using bacterial subculture in antibiotic-free media.

Conductance-Based Biophysical Distinction and Microfluidic Enrichment of Nanovesicles Derived from Pancreatic Tumor Cells of Varying Invasiveness.

Diagnostics based on exosomes and other extracellular vesicles (EVs) are emerging as strategies for informing cancer progression and therapies, since the lipid content and macromolecular cargo of EVs can provide key phenotypic and genotypic information on the parent tumor cell and its microenvironment. We show that EVs derived from more invasive pancreatic tumor cells that express high levels of tumor-specific surface proteins and are composed of highly unsaturated lipids that increase membrane fluidity, exhibit significantly higher conductance versus those derived from less invasive tumor cells, based on dielectrophoresis measurements. Furthermore, through specific binding of the EVs to gold nanoparticle-conjugated antibodies, we show that these conductance differences can be modulated in proportion to the type as well as level of expressed tumor-specific antigens, thereby presenting methods for selective microfluidic enrichment and cytometry-based quantification of EVs based on invasiveness of their parent cell.

Innate Immune Response and Outcome of Clostridium difficile Infection Are Dependent on Fecal Bacterial Composition in the Aged Host.

Background: Clostridium difficile infection (CDI) is a serious threat for an aging population. Using an aged mouse model, we evaluated the effect of age and the roles of innate immunity and intestinal microbiota. Methods: Aged (18 months) and young (8 weeks) mice were infected with C difficile, and disease severity, immune response, and intestinal microbiome were compared. The same experiment was repeated with intestinal microbiota exchange between aged and young mice before infection. Results: Higher mortality was observed in aged mice with weaker neutrophilic mobilization in blood and intestinal tissue and depressed proinflammatory cytokines in early infection. Microbiota exchange improved survival and early immune response in aged mice. Microbiome analysis revealed that aged mice have significant deficiencies in Bacteroidetes phylum and, specifically, Bacteroides, Alistipes, and rc4-4 genera, which were replenished by cage switching. Conclusions: Microbiota-dependent alteration in innate immune response early on during infection may explain poor outcome in aged host with CDI.

Single-cell electro-phenotyping for rapid assessment of Clostridium difficile heterogeneity under vancomycin treatment at sub-MIC (minimum inhibitory concentration) levels.

Current methods for measurement of antibiotic susceptibility of pathogenic bacteria are highly reliant on microbial culture, which is time consuming (requires > 16 hours), especially at near minimum inhibitory concentration (MIC) levels of the antibiotic. We present the use of single-cell electrophysiology-based microbiological analysis for rapid phenotypic identification of antibiotic susceptibility at near-MIC levels, without the need for microbial culture. Clostridium difficile (C. difficile) is the single most common cause of antibiotic-induced enteric infection and disease recurrence is common after antibiotic treatments to suppress the pathogen. Herein, we show that de-activation of C. difficile after MIC-level vancomycin treatment, as validated by microbiological growth assays, can be ascertained rapidly by measuring alterations to the microbial cytoplasmic conductivity that is gauged by the level of positive dielectrophoresis (pDEP) and the frequency spectra for co-field electro-rotation (ROT). Furthermore, this single-cell electrophysiology technique can rapidly identify and quantify the live C. difficile subpopulation after vancomycin treatment at sub-MIC levels, whereas methods based on measurement of the secreted metabolite toxin or the microbiological growth rate can identify this persistent C. difficile subpopulation only after 24 hours of microbial culture, without any ability to quantify the subpopulation. The application of multiplexed versions of this technique is envisioned for antibiotic susceptibility screening.

Label-Free Quantification of Intracellular Mitochondrial Dynamics Using Dielectrophoresis.

Mitochondrial dynamics play an important role within several pathological conditions, including cancer and neurological diseases. For the purpose of identifying therapies that target aberrant regulation of the mitochondrial dynamics machinery and characterizing the regulating signaling pathways, there is a need for label-free means to detect the dynamic alterations in mitochondrial morphology. We present the use of dielectrophoresis for label-free quantification of intracellular mitochondrial modifications that alter cytoplasmic conductivity, and these changes are benchmarked against label-based image analysis of the mitochondrial network. This is validated by quantifying the mitochondrial alterations that are carried out by entirely independent means on two different cell lines: human embryonic kidney cells and mouse embryonic fibroblasts. In both cell lines, the inhibition of mitochondrial fission that leads to a mitochondrial structure of higher connectivity is shown to substantially enhance conductivity of the cell interior, as apparent from the significantly higher positive dielectrophoresis levels in the 0.5-15 MHz range. Using single-cell velocity tracking, we show ∼10-fold higher positive dielectrophoresis levels at 0.5 MHz for cells with a highly connected versus those with a highly fragmented mitochondrial structure, suggesting the feasibility for frequency-selective dielectrophoretic isolation of cells to aid the discovery process for development of therapeutics targeting the mitochondrial machinery.

Vancomycin Treatment Alters Humoral Immunity and Intestinal Microbiota in an Aged Mouse Model of Clostridium difficile Infection.

BACKGROUND: The elderly host is highly susceptible to severe disease and treatment failure in Clostridium difficile infection (CDI). We investigated how treatment with vancomycin in the aged host influences systemic and intestinal humoral responses and select intestinal microbiota. METHODS: Young (age, 2 months) and aged (age, 18 months) C57BL/6 mice were infected with VPI 10463 after exposure to broad-spectrum antibiotics. Vancomycin was given 24 hours after infection, and treatment was continued for 5 days. At select time points, specimens of serum and intestinal tissue and contents were collected for histopathologic analysis, to measure antibody levels and the pathogen burden, and to determine the presence and levels of select intestinal microbiota and C. difficile toxin. RESULTS: Levels of disease severity, relapse, and mortality were increased, and recovery from infection was slower in aged mice compared to young mice. Serum levels of immunoglobulin M, immunoglobulin A, and immunoglobulin G against C. difficile toxin A were depressed in aged mice, and vancomycin treatment reduced antibody responses in both age groups. While baseline levels of total bacterial load, Bacteroidetes, Firmicutes, and Enterobacteriaceae were mostly similar, aged mice had a significant change in the Firmicutes to Bacteroidetes ratio with vancomycin treatment. CONCLUSIONS: Vancomycin treatment decreases the systemic humoral response to CDI. Increased mortality from and recurrence of CDI in the aged host are associated with an impaired humoral response and a greater susceptibility to vancomycin-induced alteration of intestinal microbiota.

Treatment of Clostridium difficile infection using SQ641, a capuramycin analogue, increases post-treatment survival and improves clinical measures of disease in a murine model.

OBJECTIVES: Clostridium difficile infection (CDI) is a primary cause of antibiotic-associated diarrhoeal illness. Current therapies are insufficient as relapse rates following antibiotic treatment range from 25% for initial treatment to 60% for treatment of recurrence. In this study, we looked at the efficacy of SQ641 in a murine model of CDI. SQ641 is an analogue of capuramycin, a naturally occurring nucleoside-based compound produced by Streptomyces griseus. METHODS: In a series of experiments, C57BL/6 mice were treated with a cocktail of antibiotics and inoculated with C. difficile strain VPI10463. Animals were treated orally with SQ641 for 5 days at a dose range of 0.1-300 mg/kg/day, 20 mg/kg/day vancomycin or drug vehicle. Animals were monitored for disease severity, clostridial shedding and faecal toxin levels for 14 days post-infection. RESULTS: Five day treatment of CDI with SQ641 resulted in higher 14 day survival rates in mice compared with either vancomycin or vehicle alone. CDI survival rates were 100% (13 of 13) and 94% (32 of 34), respectively, in the 1 and 10 mg/kg/day SQ641 treatment groups, 37% (7 of 19) with vancomycin treatment at 20 mg/kg/day and 32% (14 of 44) in the vehicle-only control group. Secondary measures of efficacy, such as prevention of weight loss, decreased disease severity, decreased C. difficile shedding and decreased toxin in faeces, were observed with SQ641 and vancomycin treatment. CONCLUSIONS: SQ641 is effective for CDI treatment with prevention of relapse in the murine model of CDI.

Rapid detection of Clostridium difficile via magnetic bead aggregation in cost-effective polyester microdevices with cell phone image analysis.

Pathogen detection has traditionally been accomplished by utilizing methods such as cell culture, immunoassays, and nucleic acid amplification tests; however, these methods are not easily implemented in resource-limited settings because special equipment for detection and thermal cycling is often required. In this study, we present a magnetic bead aggregation assay coupled to an inexpensive microfluidic fabrication technique that allows for cell phone detection and analysis of a notable pathogen in less than one hour. Detection is achieved through the use of a custom-built system that allows for fluid flow control via centrifugal force, as well as manipulation of magnetic beads with an adjustable rotating magnetic field. Cell phone image capture and analysis is housed in a 3D-printed case with LED backlighting and a lid-mounted Android phone. A custom-written application (app.) is employed to interrogate images for the extent of aggregation present following loop-mediated isothermal amplification (LAMP) coupled to product-inhibited bead aggregation (PiBA) for detection of target sequences. Clostridium difficile is a pathogen of increasing interest due to its causative role in intestinal infections following antibiotic treatment, and was therefore chosen as the pathogen of interest in the present study to demonstrate the rapid, cost-effective, and sequence-specific detection capabilities of the microfluidic platform described herein.

Vancomycin treatment's association with delayed intestinal tissue injury, clostridial overgrowth, and recurrence of Clostridium difficile infection in mice.

Antibiotic treatment, including vancomycin, for Clostridium difficile infection (CDI) has been associated with recurrence of disease in up to 25% of infected persons. This study investigated the effects of vancomycin on the clinical outcomes, intestinal histopathology, and anaerobic community during and after treatment in a murine model of CDI. C57BL/6 mice were challenged with C. difficile strain VPI 10463 after pretreatment with an antibiotic cocktail. Twenty-four hours after infection, mice were treated daily with vancomycin, nitazoxanide, fidaxomicin, or metronidazaole for 5 days. Mice were monitored for either 6 or 12 days postinfection. Clinical, diarrhea, and histopathology scores were measured. Cecal contents or stool samples were assayed for clostridial or Bacteroides DNA and C. difficile toxins A and B. Vancomycin treatment of infected mice was associated with improved clinical, diarrhea, and histopathology scores and survival during treatment. However, after discontinuation of the drug, clinical scores and histopathology were worse in treated mice than in untreated infected controls. At the end of the study, 62% of the vancomycin-treated mice succumbed to recurrence, with an overall mortality rate equivalent to that of the untreated infected control group. Fidaxomicin-treated mice had outcomes similar to those of vancomycin-treated mice. C. difficile predominated over Bacteroides in cecal contents of vancomycin-treated mice, similar to findings for untreated infected mice. Decreasing the duration of vancomycin treatment from 5 days to 1 day decreased recurrence and deaths. In conclusion, vancomycin improved clinical scores and histopathology acutely but was associated with poor outcome posttreatment in C. difficile-infected mice. Decreasing vancomycin exposure may decrease relapse and improve survival in CDI.

Correlating Antibiotic-Induced Dysbiosis to Clostridioides difficile Spore Germination and Host Susceptibility to Infection Using an Ex Vivo Assay.

Antibiotic-induced microbiota disruption and its persistence create conditions for dysbiosis and colonization by opportunistic pathogens, such as those causing Clostridioides difficile (C. difficile) infection (CDI), which is the most severe hospital-acquired intestinal infection. Given the wide differences in microbiota across hosts and in their recovery after antibiotic treatments, there is a need for assays to assess the influence of dysbiosis and its recovery dynamics on the susceptibility of the host to CDI. Germination of C. difficile spores is a key virulence trait for the onset of CDI, which is influenced by the level of primary vs secondary bile acids in the intestinal milieu that is regulated by the microbiota composition. Herein, the germination of C. difficile spores in fecal supernatant from mice that are subject to varying degrees of antibiotic treatment is utilized as an ex vivo assay to predict intestinal dysbiosis in the host based on their susceptibility to CDI, as determined by in vivo CDI metrics in the same mouse model. Quantification of spore germination down to lower detection limits than the colony-forming assay is achieved by using impedance cytometry to count single vegetative bacteria that are identified based on their characteristic electrical physiology for distinction vs aggregated spores and cell debris in the media. As a result, germination can be quantified at earlier time points and with fewer spores for correlation to CDI outcomes. This sets the groundwork for a point-of-care tool to gauge the susceptibility of human microbiota to CDI after antibiotic treatments.

Current trends in vaginal labioplasty: a survey of plastic surgeons.

BACKGROUND: Labioplasty serves to resolve an anatomic variation that results in aesthetic and functional difficulties for many women. To date, little effort has been made to compare the efficacy or prevalence of various techniques, and furthermore, evidence-based practice guidelines have not yet been established for this procedure. The purpose of this study is to elucidate the current armamentarium and practice guidelines for labia minora reduction, as well as to examine self-reported outcomes for this procedure. METHODS: A 2009 web-based survey was sent to members of the American Society of Plastic Surgeons via electronic mail. The survey was used to assess surgeon demographics, practice guidelines for labioplasty, and self-reported outcomes measures. RESULTS: A total of 750 surgeons responded to the survey (19.7% response rate), and 51.0% of surgeons currently offered labioplasty. The total number of procedures over the past 24 months for all respondents was 2255. Per surgeon over 24 months, the mean number of procedures was 7.37 (range, 0-300). Surgeons that directly advertised that they performed labioplasty performed a mean of 14.2 procedures over 24 months versus a mean of 5.01 for those who did not (P = 0.001). The mean time suggested to refrain from intercourse was 31.3 days. Redundancy or inadequate resection and wound dehiscence were the 2 most common reasons that caused surgeons to reoperate. Surgeons using plain gut suture material had the highest reported rates of reoperation. The prevalence of each technique was as follows: simple amputation (52.7%), W-SHAPED resection (9.5%), S-shaped resection (8.8%), central v-wedge (36.1%), central wedge with z-plasty (13.9%), and deepithelialization (1.2%). The mean perceived patient satisfaction rate reported by surgeons was greater than 95% for all techniques, and there was no statistically significant difference when comparing perceived patient satisfaction among the techniques (P = 0.337). CONCLUSIONS: This study provides data suggesting that labioplasty has been safely and effectively used by many plastic surgeons. However, there is great variation with regard to both techniques and practice guidelines. In establishing these discrepancies among surgeons, we hope to provide the impetus for further academic dialogue and prospective trials.

Evaluating the role of postoperative prophylactic antibiotics in primary and secondary breast augmentation: a retrospective review.

BACKGROUND: The use of postoperative prophylactic antibiotics following augmentation mammaplasty remains a controversial topic, with many surgeons opting for extended prophylaxis. OBJECTIVES: The authors evaluate the role of postoperative prophylactic antibiotics in both primary and secondary cosmetic breast augmentation. METHODS: A five-year retrospective chart review was performed on all patients undergoing cosmetic breast augmentation at a single institution from January 2005 to December 2009. The four attending physicians in this study utilized similar perioperative protocol and implant materials. Patients were divided into two cohorts: those who had received three days of postoperative antibiotics (primarily cephalosporins) and those who had not. End points of particular interest included infection, capsular contracture (CC), and local wound complications. The mean follow-up time was 3.8 years. RESULTS: A total of 605 implants were included over the five-year study period. The overall infection rate was 0.66%. For primary augmentation, 493 implants were studied, with 52% of those patients having received postoperative antibiotics. There was no statistically significant reduction in infection, CC, or total complication rate for those receiving postoperative antibiotics. Similarly, 112 implants were studied for secondary augmentation, and again, postoperative antibiotics were not associated with a reduction in complications. CONCLUSIONS: The data suggest that there was no reduction in the overall rate of total complications, infection, or CC with postoperative prophylactic antibiotics for either primary or secondary cosmetic breast augmentation. This study provides Level 3 evidence in support of discontinuing prophylactic postoperative antibiotics following cosmetic breast augmentation.

Automated biophysical classification of apoptotic pancreatic cancer cell subpopulations by using machine learning approaches with impedance cytometry.

Unrestricted cell death can lead to an immunosuppressive tumor microenvironment, with dysregulated apoptotic signaling that causes resistance of pancreatic cancer cells to cytotoxic therapies. Hence, modulating cell death by distinguishing the progression of subpopulations under drug treatment from viable towards early apoptotic, late apoptotic, and necrotic states is of interest. While flow cytometry after fluorescent staining can monitor apoptosis with single-cell sensitivity, the background of non-viable cells within non-immortalized pancreatic tumors from xenografts can confound distinction of the intensity of each apoptotic state. Based on single-cell impedance cytometry of drug-treated pancreatic cancer cells that are obtained from tumor xenografts with differing levels of gemcitabine sensitivity, we identify the biophysical metrics that can distinguish and quantify cellular subpopulations at the early apoptotic versus late apoptotic and necrotic states, by using machine learning methods to train for the recognition of each phenotype. While supervised learning has previously been used for classification of datasets with known classes, our advancement is the utilization of optimal positive controls for each class, so that clustering by unsupervised learning and classification by supervised learning can occur on unknown datasets, without human interference or manual gating. In this manner, automated biophysical classification can be used to follow the progression of apoptotic states in each heterogeneous drug-treated sample, for developing drug treatments to modulate cancer cell death and advance longitudinal analysis to discern the emergence of drug resistant phenotypes.

Apoptotic Bodies in the Pancreatic Tumor Cell Culture Media Enable Label-Free Drug Sensitivity Assessment by Impedance Cytometry.

The ability to rapidly and sensitively predict drug response and toxicity using in vitro models of patient-derived tumors is essential for assessing chemotherapy efficacy. Currently, drug sensitivity assessment for solid tumors relies on imaging adherent cells or by flow cytometry of cells lifted from drug-treated cultures after fluorescent staining for apoptotic markers. Subcellular apoptotic bodies (ABs), including microvesicles that are secreted into the culture media under drug treatment can potentially serve as markers for drug sensitivity, without the need to lift cells under culture. However, their stratification to quantify cell disassembly is challenging due to their compositional diversity, with tailored labeling strategies currently needed for the recognition and cytometry of each AB type. It is shown that the high frequency impedance phase versus size distribution of ABs determined by high-throughput single-particle impedance cytometry of supernatants in the media of gemcitabine-treated pancreatic tumor cultures exhibits phenotypic resemblance to lifted apoptotic cells and enables shape-based stratification within distinct size ranges, which is not possible by flow cytometry. It is envisioned that this tool can be applied in conjunction with the appropriate pancreatic tumor microenvironment model to assess drug sensitivity and toxicity of patient-derived tumors, without the need to lift cells from cultures.

Label-Free Quantification of Cell Cycle Synchronicity of Human Neural Progenitor Cells Based on Electrophysiology Phenotypes.

The ability to coax human-induced pluripotent stem cells (hiPSCs) into human neural progenitor cells (hNPCs) can lead to novel drug discovery and transplant therapy platforms for neurological diseases. Since hNPCs can form organoids that mimic brain development, there is emerging interest in their label-free characterization for controlling cell composition to optimize organoid formation in three-dimensional (3D) cultures. However, this requires the ability to quantify hNPCs in heterogeneous samples with subpopulations of similar phenotype. Using high-throughput (>6000 cells per condition), single-cell impedance cytometry, we present the utilization of electrophysiology for quantification of hNPC subpopulations that are altered in cell cycle synchronicity by camptothecin (CPT) exposure. Electrophysiology phenotypes are determined from impedance magnitude and phase metrics for distinguishing each cell cycle phase, as validated by flow cytometry, for a wide range of subpopulation proportions. Using multishell dielectric models for each cell cycle phase, electrophysiology alterations with CPT dose could be predicted. This label-free detection strategy can prevent loss of cell viability to speed the optimization of cellular compositions for organoid development.

Paraspinous muscle flap reconstruction of complex midline back wounds: Risk factors and postreconstruction complications.

With increasingly complex spine surgeries now being performed on a more comorbid patient population, the reconstruction of midline back wounds from these procedures is becoming a frequent dilemma encountered by plastic surgery. The purpose of this study is to examine the effect of various preoperative risk factors on postoperative wound healing complications after paraspinous muscle flap reconstruction of midline back defects. An Institutional Review Board-approved, 11-year, retrospective, office and hospital chart review was conducted. All adult patients who underwent paraspinous muscle flap reconstruction during the study period were included. There were 92 patients in the study, representing the largest reported series to-date for the paraspinous muscle flap procedure. Mean follow-up was 120 days. Several wound-healing risk factors were present in this patient population: 72% were malnourished, 41% had hypertension, 37% were obese, 34% had a history of smoking, 32% had diabetes, 16% were on chronic steroids, 14% had a history of more than 2 previous spine surgeries, and 9% had a history of radiation to the wound area. Factors significantly (P < 0.05) associated with postreconstruction wound complications included history of traumatic spine injury, prereconstruction hardware removal, a history of more than 2 spine surgeries, hypertension, and lumbar wound location. This patient population possesses multiple comorbidities making complex wound healing difficult. Several specific risk factors are associated with an increased rate of postreconstruction wound complications after paraspinous muscle flaps. The paraspinous muscle flap remains an important tool for spinal wound reconstruction in the reconstructive surgeon's armamentarium.

Synthetic and biological mesh in component separation: a 10-year single institution review.

Definitive repair of recurrent ventral hernias using abdominal wall reconstruction techniques is an essential tool in the armentarium for general and plastic surgeons. Despite the great morbidity associated with incisional hernia, no consensus exists on the best means for treatment (Korenkov et al, Langenbecks Arch Surg. 2001;386:65-73). Ramirez et al (Plast Reconstr Surg. 1990;83:519-526) describes the "component separation" technique to mobilize the rectus-abdominus internal oblique and external oblique flap to correct the defect. This retrospective institutional study reviewed 10 years of myofascial flap reconstruction from 1996 to 2006 at Thomas Jefferson University Hospital and revealed an 18.3% recurrence rate in 545 component separations. We identified obesity (body mass index >30 kg/m2), age >65 years old, male gender, postoperative seroma, and preoperative infection as risk factors for hernia recurrence.

Rapid in Vitro Assessment of Clostridioides difficile Inhibition by Probiotics Using Dielectrophoresis to Quantify Cell Structure Alterations.

Clostridioides difficile (C. difficile) infection (CDI) is the primary cause of nosocomial antibiotic-associated diarrhea, with high recurrence rates following initial antibiotic treatment regimens. Restoration of the host gut microbiome through probiotic therapy is under investigation to reduce recurrence. Current in vitro methods to assess C. difficile deactivation by probiotic microorganisms are based on C. difficile growth inhibition, but the cumbersome and time-consuming nature of the assay limits the number of assessed permutations. Phenotypic alterations to the C. difficile cellular structure upon interaction with probiotics can potentially enable rapid assessment of the inhibition without the need for extended culture. Because supernatants from cultures of commensal microbiota reflect the complex metabolite milieu that deactivates C. difficile, we explore coculture of C. difficile with an optimal dose of supernatants from probiotic culture to speed growth inhibition assays and enable correlation with alterations to its prolate ellipsoidal structure. Based on sensitivity of electrical polarizability to C. difficile cell shape and subcellular structure, we show that the inhibitory effect of Lactobacillus spp. supernatants on C. difficile can be determined based on the positive dielectrophoresis level within just 1 h of culture using a highly toxigenic strain and a clinical isolate, whereas optical and growth inhibition measurements require far greater culture time. We envision application of this in vitro coculture model, in conjunction with dielectrophoresis, to rapidly screen for potential probiotic combinations for the treatment of recurrent CDI.

Quantifying bacterial spore germination by single-cell impedance cytometry for assessment of host microbiota susceptibility to Clostridioides difficile infection.

The germination of ingested spores is often a necessary first step required for enabling bacterial outgrowth and host colonization, as in the case of Clostridioides difficile (C. difficile) infection. Spore germination rate in the colon depends on microbiota composition and its level of disruption by antibiotic treatment since secretions by commensal bacteria modulate primary to secondary bile salt levels to control germination. Assessment of C. difficile spore germination typically requires measurement of colony-forming units, which is labor intensive and takes at least 24 h to perform but is regularly required due to the high recurrence rates of nosocomial antibiotic-associated diarrhea. We present a rapid method to assess spore germination by using high throughput single-cell impedance cytometry (>300 events/s) to quantify live bacterial cells, by gating for their characteristic electrophysiology versus spores, so that germination can be assessed after just 4 h of culture at a detection limit of ~100 live cells per 50 µL sample. To detect the phenotype of germinated C. difficile bacteria, we utilize its characteristically higher net conductivity versus that of spore aggregates and non-viable C. difficile forms, which causes a distinctive high-frequency (10 MHz) impedance phase dispersion within moderately conductive media (0.8 S/m). In this manner, we can detect significant differences in spore germination rates within just 4 h, with increasing primary bile salt levels in vitro and using ex vivo microbiota samples from an antibiotic-treated mouse model to assess susceptibility to C. difficile infection. We envision a rapid diagnostic tool for assessing host microbiota susceptibility to bacterial colonization after key antibiotic treatments.