Phase I Safety and Feasibility Pilot of Hepatic Artery Infusion Chemotherapy in a Rural Catchment Area Using The Codman Vascular Catheter with The Medtronic SynchroMed II Pump for Intrahepatic Cancers.

BACKGROUND: Discontinuation of the Codman 3000 pump in 2018 left no Food and Drug Administration (FDA)-approved hepatic artery infusion (HAI) device for unresectable colorectal liver metastases (uCLM) and intrahepatic cholangiocarcinoma (uIHC). Historically, HAI has been performed at academic medical centers in large metropolitan areas, which are often inaccessible to rural patients. Consequently, feasibility of dissemination of HAI to rural populations is unknown. PATIENTS AND METHODS: Under an FDA investigational device exemption, we opened the only HAI program in Kentucky and enrolled patients with uCLM and uIHC in a phase I clinical trial. The trial examined the safety of the hybrid Codman catheter/Medtronic SynchroMed II pump (hCMP) combination, defined as successful completion of one cycle of HAI chemotherapy. Rural feasibility was assessed by number of missed pump fills appointments. RESULTS: A total of 21 patients (n = 17 uCLM, n = 4 uIHC) underwent hCMP implantation before accrual was stopped early owing to FDA approval of the Intera 3000 pump. 20/21 (95%) patients met the primary safety endpoint. Serious adverse events (AEs) included a grade 5 coronavirus disease 2019 (COVID-19) infection (n = 1) and a grade 3 catheter erosion into the bowel (n = 1). Biliary sclerosis developed in two patients (9.5%). Median distance to infusion center was 47.6 miles (2-138 miles), and 62% were from Appalachia, yet there were no missed pump fill appointments. The 2-year overall survival was 82.4% (uCLM) and 50% (uIHC). CONCLUSIONS: The hCMP device had an acceptable safety profile. Despite the complexity of starting a new HAI program, early results showed feasibility for HAI delivery in a rural catchment area and comparable outcomes to larger urban-based HAI centers.

Elevated Levels of Three Reactive Oxygen Species and Fe(II) in the Antibiotic-Surviving Population of Mycobacteria Facilitate De Novo Emergence of Genetic Resisters to Antibiotics.

We had earlier reported the de novo emergence of genetic resisters of Mycobacterium tuberculosis and Mycobacterium smegmatis to rifampicin and moxifloxacin from the antibiotic-surviving population containing elevated levels of the non-DNA-specific mutagenic reactive oxygen species (ROS) hydroxyl radical. Since hydroxyl radical is generated by Fenton reaction between Fe(II) and H2O2, which is produced by superoxide dismutation, we here report significantly elevated levels of these three ROS and Fe(II) in the M. smegmatis rifampicin-surviving population. Elevated levels of superoxide and the consequential formation of high levels of H2O2 and Fe(II) led to the generation of hydroxyl radical, facilitating de novo high frequency emergence of antibiotic resisters. The M. smegmatis cultures, exposed to nontoxic concentrations of the ROS scavenger, thiourea (TU), and the NADH oxidase (one of the superoxide producers) inhibitor, diphenyleneiodonium chloride (DPI), showed a reduction in the levels of the three ROS, Fe(II), and antibiotic resister generation frequency. The non-antibiotic-exposed cultures grown in the absence/presence of TU/DPI did not show increased ROS, Fe(II) levels, or antibiotic resister generation frequency. The antibiotic-surviving population showed significantly increased expression and activity of superoxide-producing genes and decreased expression of antioxidant and DNA repair genes, revealing an environment conducive for the acquisition and retention of mutations. Since we recently reported significant comparability between the antibiotic-survival gene expression profiles of the saprophyte-cum-opportunistic pathogens M. smegmatis and the M. tuberculosis in tuberculosis patients undergoing treatment, we discuss the clinical relevance of the findings on the mechanism of emergence of antibiotic-resistant mycobacterial strains.

Mycobacterium smegmatis moxifloxacin persister cells produce high levels of hydroxyl radical, generating genetic resisters selectable not only with moxifloxacin, but also with ethambutol and isoniazid.

Bacterial antibiotic persister cells tolerate lethal concentrations of antibiotics but emerge as the antibiotic-sensitive population upon antibiotics withdrawal. However, the possibility of antibiotic-resistant genetic mutants emerging from the antibiotic persister population in the continued exposure to microbicidal concentrations of antibiotics needed investigation. We explored this possibility using the fast-growing Mycobacterium smegmatis as a model organism for Mycobacterium tuberculosis biology, as it is known to incur antibiotic-resistant mutations identical to and at identical target positions as found in the clinical isolates of M. tuberculosis. Here we report that the moxifloxacin (MXF) persister population generate significantly elevated levels of hydroxyl radical. Hydroxyl radical being a sequence-non-specific mutagen, resulted in the emergence of moxifloxacin-resistant genetic mutants at 8-log10 higher frequency from the persister population. Luria-Delbruck experiment (in modified format) confirmed that MXF-resistant mutants emerged de novo from the persister population and were not pre-existent. The nature of the mutations in the quinolone resistance determining region indicated that they were generated due to oxidative stress. These mutations were identical to and at identical positions as found in the clinical isolates of MXF-resistant M. tuberculosis. Interestingly, from the MXF persister population, resisters to microbicidal concentrations of ethambutol and isoniazid could also be selected. These observations implied that the significantly high levels of hydroxyl radical might have generated genome-wide mutations, creating a pool of mutants in the MXF persister population, facilitating selection of resisters to other antibiotics also. These findings may be of clinical relevance to the emergence of drug-resistant strains during prolonged tuberculosis treatment regimen with high doses of multiple antibiotics.

Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility.

Phenotypically heterogeneous but genetically identical mycobacterial subpopulations exist in in vitro cultures, in vitro-infected macrophages, infected animal models and tuberculosis patients. In this regard, we recently reported the presence of two subpopulations of cells, which are phenotypically different in length and buoyant density, in mycobacterial cultures. These are the low-buoyant-density short-sized cells (SCs), which constitute ~10-20 % of the population, and the high-buoyant-density normal/long-sized cells (NCs), which form ~80-90 % of the population. The SCs were found to be significantly more susceptible to rifampicin (RIF), isoniazid (INH), H2O2 and acidified nitrite than the NCs. Here we report that the RIF-/INH-/H2O2-exposed SCs showed significantly higher levels of oxidative stress and therefore higher susceptibility than the equivalent number of exposed NCs. Significantly higher levels of hydroxyl radical and superoxide were found in the antibiotic-exposed SCs than in the equivalently exposed NCs. Different proportions of the subpopulation of SCs were found to have different levels of reactive oxygen species (ROS). The hydroxyl radical quencher, thiourea, and the superoxide dismutase mimic, TEMPOL, significantly reduced hydroxyl radical and superoxide levels, respectively, in the antibiotic-exposed SCs and NCs and thereby decreased their differential susceptibility to antibiotics. Thus, the present study shows that the heterogeneity of the reactive oxygen species (ROS) levels in these mycobacterial subpopulations confers differential susceptibility to antibiotics. We have discussed the possible mechanisms that can generate differential ROS levels in the antibiotic-exposed SCs and NCs. The present study advances our current understanding of the molecular mechanisms underlying antibiotic tolerance in mycobacteria.

In Situ Salification in Polar Solvents: a Paradigm for Enabling Drug Delivery of Weakly Ionic Drugs as Amorphous Solid Dispersion.

Solubility challenge for a poorly water-soluble drug gets further intensified when it is weakly ionic because the most common solubility enhancement technique, salt formation, becomes less feasible. Salt screening for such drugs often concludes with either a difficult to crystalize salt or an unstable salt, leading the scientists to explore other solubility enhancement technique like amorphous solid dispersions which is comparatively costlier, time-consuming and may require use of hazardous organic solvents. Present study evaluated in situ salification in polar protic solvents for dissolving poorly water-soluble drug Itraconazole which is weakly ionic and not very amenable to formation of stable inorganic salts. Through systematic selection of solvents, counterions and polymers, an amorphous solid dispersion of drug salt was obtained. In vitro characterizations with polarized light microscopy (PLM), modulated differential scanning calorimetry (mDSC), Fourier transform infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD) confirmed the physical and chemical stability of the amorphous solid dispersion. In vivo pharmacokinetic study showed that the drug salt amorphous solid dispersion achieved 45 times higher plasma exposure compared to crystalline drug. This study provides one of the first data sets for the hypothesis that in situ drug salts can be utilized for manufacturing amorphous solid dispersions of weakly ionic drugs and leverages the solubility advantage of salts and amorphous state.

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro.

Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

Nonalcoholic steatohepatitis is strongly associated with sarcopenic obesity in patients with cirrhosis undergoing liver transplant evaluation.

BACKGROUND: Sarcopenia is the most common complication of cirrhosis and adversely affects quality of life and outcomes before, during, and after liver transplantation. We studied predictors of sarcopenia and sarcopenic obesity in patients with cirrhosis undergoing liver transplant (LT) evaluation. METHODS: A retrospective analysis of 207 adult cirrhotic patients that underwent LT from January 2008 to December 2013 was performed at our institution. RESULTS: Two hundred seven patients were evaluated, 68% were male with a mean age of 54 ± 8 years. The most common etiology of cirrhosis was alcoholic liver disease (38.6%), followed by chronic hepatitis C (38.2%), nonalcoholic steatohepatitis (NASH) (21.7%), and hepatocellular carcinoma (HCC) (24.6%). The mean body mass index of the cohort was of 30.1 ± 5.7 kg/m(2) . Forty-eight percent of these patients were obese. Of the 207 patients, 88% had computed tomographic (CT) scans within 90 days before transplant; of these, 59% had sarcopenia found during LT evaluation. Of the patients with pretransplant sarcopenia, 59 had CT scan at 6 months posttransplant and 56 (95%) remained sarcopenic. Of the 56 patients who had sarcopenia at 6 months, 31 had available CT scans at 1 year, and 100% persisted with sarcopenia. These 31 subjects had a mean skeletal muscle index of 35 at 6 months and 36 at 1 year. SO was found in 41.7% of our patients. On multivariable regression analysis, obesity and age were found to be independently associated with pretransplant sarcopenia after controlling for gender and alcohol liver disease diagnosis (P = 0.00001, odds ratio [OR] 0.22, and P = 0.008, OR 2.0, respectively). A multivariable logistic regression analysis found that NASH as cause of cirrhosis and model of end-stage liver disease score are independent predictors of sarcopenic obesity after controlling for age, gender, alcoholic liver disease diagnosis, and HCC (P = 0.014 and 0.038, respectively; 95% confidence interval, 1.44-25.26 and 1.00-1.15, respectively; OR 6.03, 1.08, respectively). CONCLUSIONS: Sarcopenia and sarcopenic obesity is seen in a significant number of patients with cirrhosis undergoing LT evaluation. Sarcopenia progresses after LT initially and does not recover at least within the first year after surgery. Obesity is an independent predictor of pretransplant sarcopenia and NASH was associated with 6-fold increased risk of having sarcopenic obesity in cirrhotic patients in our cohort.

Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis.

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis.

Changes in metabolic pathways of Desulfovibrio alaskensis G20 cells induced by molybdate excess.

The activity of sulfate-reducing bacteria (SRB) intensifies the problems associated to corrosion of metals and the solution entails significant economic costs. Although molybdate can be used to control the negative effects of these organisms, the mechanisms triggered in the cells exposed to Mo-excess are poorly understood. In this work, the effects of molybdate ions on the growth and morphology of the SRB Desulfovibrio alaskensis G20 (DaG20) were investigated. In addition, the cellular localization, ion uptake and regulation of protein expression were studied. We found that molybdate concentrations ranging between 50 and 150 µM produce a twofold increase in the doubling time with this effect being more significant at 200 µM molybdate (five times increase in the doubling time). It was also observed that 500 µM molybdate completely inhibits the cellular growth. On the context of protein regulation, we found that several enzymes involved in energy metabolism, cellular division and metal uptake processes were particularly influenced under the conditions tested. An overall description of some of the mechanisms involved in the DaG20 adaptation to molybdate-stress conditions is discussed.

TupA: a tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20.

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, ß = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.

The C-terminus is essential for the stability of the mycobacterial channel protein MspA.

Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.

Comparison of the remaining dentin thickness in the root after hand and four rotary instrumentation techniques: an in vitro study.

AIM: The aim of the present study was to compare the remaining dental thickness (RDT) in the mesiobuccal root of mandibular first molars at 3 and 7 mm from the anatomic apex after instrumentation with ProTaper, light speed LSX, K3 and M2 and to compare with that of K-files. MATERIALS AND METHODS: In this study, 60 extracted, untreated human mandibular first molars with fully formed apices, with curvature less than 35° and no root resorption were used. Prepared specimens were cut horizontally at 3 and 7 mm short of anatomic apex. The least dentin thickness from canal to external root surface was observed under 3× magnification and recorded using Clemax measuring tool and the sections were reassembled. Group I-instrumentation with ProTaper, group II-instrumentation with K3, group III-instrumentation with Light Speed LSX, group IV-instrumentation with M2 and group V- instrumentation with K-files and RDT was measured. RESULTS: Results showed that group V removed lesser amount of dentin compared to all other groups while all the three instrumentation techniques removed almost equal amount of dentin apically. CLINICAL SIGNIFICANCE: Cleaning and shaping of the root canal space involves the elimination of pathogenic contents as well as attaining a uniform specific shape. However, the RDT following the use of various intraradicular procedures is an important factor to be considered as an iatrogenic cause that may result in root fracture. To avoid this, newer rotary instruments are being introduced.

Influence of post fit and post length on fracture resistance: an in vitro study.

BACKGROUND AND OBJECTIVES: As root filled teeth often have insufficient coronal tooth structure, placement of a post is occasionally necessary to provide adequate retention for the core and final restoration. The aim of the present study was to investigate (i) the impact of post fit (form-congruence) and (ii) the influence of post length on the fracture resistance of severely damaged root filled extracted teeth. MATERIALS AND METHODS: Forty single-rooted human teeth were root filled and divided into four groups (n = 10 per group). Post spaces were prepared with a depth of 6 mm (groups 1, 3) and 3 mm (groups 2, 4). Form-congruence with a maximal fit of the post within the root canal space was obtained in groups 1 and 2, whereas there was no form-congruence in groups 3 and 4. In all groups, glass fiber reinforced composite (FRC) posts were adhesively cemented and direct composite crown buildups were fabricated without a ferrule. Specimens were subjected to thermocycling and cyclic loading followed by application of static load until failure. Loads-to-failure [in N] were compared among the groups. RESULTS: Post fit did not have a significant influence on fracture resistance, irrespective of the post length. Both groups with post insertion depths of 6 mm resulted in significantly higher mean failure loads (group 1: 274.27 N; group 3: 277.16 N) than the groups with post space preparation of 3 mm (group 2: 250.40 N; group 4: 255.48 N). INTERPRETATION AND CONCLUSION: Within the limitations of this study, the fracture resistance of teeth restored with FRC posts and direct resin composite crowns without ferrules was not influenced by post fit within the root canal. These results imply that excessive post space preparation aimed at producing an optimal circumferential post fit is not required to improve fracture resistance of roots.

'You don't compare horrors, you just don't do that': Examining assumptions and extending the scope of comparative victim beliefs.

Social psychological research on collective victimhood has often focused on comparisons between the ingroup's and outgroups' collective victimization (i.e. comparative victim beliefs such as competitive victimhood or inclusive victim beliefs). This qualitative study examines how people in different contexts of collective victimization and its aftermath make sense of items commonly used to assess comparative victim beliefs, and how they extend or challenge these constructs and their underlying assumptions. We used thematic analysis to analyse eight focus group discussions among four minority groups in the United States with historical or more recent experiences of collective victimization (Armenian Americans, Burundian refugees, Jewish Americans and Nepali-speaking Bhutanese refugees). Findings extend commonly assessed comparative victim beliefs and reveal participants' critical perspectives on these constructs. The findings also highlight the dialectical structure of collective victim beliefs: Participants not only endorsed but also rejected comparative victim beliefs, and relatedly described both ingroup power and outgroup power in the context of their group's victimization. These findings extend existing social psychological literature on comparative victim beliefs and intergroup relations.

MRI defecography revisited. At-rest pelvic floor measurements with and without rectal gel. Is there a difference?

PURPOSE: The authors sought to test if there was a difference in key pelvic floor measurements obtained during MR defecography at-rest, i.e., H-line, M-line and anorectal angle (ARA), before and after rectal gel administration. The authors also sought to determine if any observed differences would affect the interpretation of the defecography studies. METHODS: Institutional Review Board approval was obtained. An abdominal fellow retrospectively reviewed the images of all patients who underwent MRI defecography at our institution from January 2018 through June 2021. The H-line, M-line and ARA values were remeasured on T2-weighted sagittal images, with and without rectal gel for each patient. RESULTS: One hundred and eleven (111) studies were included in the analysis. 18% (N = 20) of patients satisfied the criterion for pelvic floor widening before gel administration based on H-line measurement. This increased to 27% (N = 30) after rectal gel (p = 0.08). 14.4% (N = 16) met the M-line measurement criterion for pelvic floor descent before gel administration. This increased to 38.7% after rectal gel (N = 43) (p < 0.001). 67.6% (N = 75) demonstrated an abnormal ARA prior to administration of rectal gel. This decreased to 58.6% (N = 65) after rectal gel administration (p = 0.07). The overall reporting discrepancies incurred by the presence or absence of rectal gel were 16.2%, 29.7% and 23.4% for H-line, M-line and ARA, respectively. CONCLUSION: The instillation of gel during MR defecography can cause significant changes to the observed pelvic floor measurements at-rest. This in turn can influence the interpretation of defecography studies.

I saw the "kissing ovaries" sign: Too close for comfort.

The Kissing ovaries sign is a radiological sign seen in women with deep pelvic endometriosis. It refers to abutment of the ovaries within the cul-de-sac. The term kissing ovaries was first described by Ghezzi et al. (2005) and has been since used widely. When seen on imaging it indicates moderate to severe endometriosis with the ovaries tethered within abnormal pelvic soft tissue, which may warrant surgical management.

Percoll discontinuous density gradient centrifugation method for the fractionation of the subpopulations of Mycobacterium smegmatis and Mycobacterium tuberculosis from in vitro cultures.

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis.•The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density.•The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis.•Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity.