Dual Drug Resistance Research Articles

S288T mutation altering MmpL3 periplasmic domain channel and H-bond network: a novel dual drug resistance mechanism.

Mycobacterial membrane proteins Large 3 (MmpL3) is responsible for the transport of mycobacterial acids out of cell membrane to form cell wall, which is essential for the survival of Mycobacterium tuberculosis (Mtb) and has become a potent anti-tuberculosis target. SQ109 is an ethambutol (EMB) analogue, as a novel anti-tuberculosis drug, can effectively inhibit MmpL3, and has completed phase 2b-3 clinical trials. Drug resistance has always been the bottleneck problem in clinical treatment of tuberculosis. The S288T mutant of MmpL3 shows significant resistance to the inhibitor SQ109, while the specific action mechanism remains unclear. The results show that MmpL3 S288T mutation causes local conformational change with little effect on the global structure. With MmpL3 bound by SQ109 inhibitor, the distance between D710 and R715 increases resulting in H-bond destruction, but their interactions and proton transfer function are still restored. In addition, the rotation of Y44 in the S288T mutant leads to an obvious bend in the periplasmic domain channel and an increased number of contact residues, reducing substrate transport efficiency. This work not only provides a possible dual drug resistance mechanism of MmpL3 S288T mutant but also aids the development of novel anti-tuberculosis inhibitors. In this work, molecular dynamics (MD) and quantum mechanics (QM) simulations both were performed to compare inhibitor (i.e., SQ109) recognition, motion characteristics, and H-bond energy change of MmpL3 after S288T mutation. In addition, the WT_SQ109 complex structure was obtained by molecular docking program (Autodock 4.2); Molecular Mechanics/ Poisson Boltzmann Surface Area (MM-PBSA) and Solvated Interaction Energy (SIE) methods were used to calculate the binding free energies (∆Gbind); Geometric criteria were used to analyze the changes of hydrogen bond networks.

Recombinant A(H6N1)-H274Y avian influenza virus with dual drug resistance does not require permissive mutations to retain the replicative fitness in vitro and in ovo

The possible emergence of drug-resistant avian flu raises concerns over the limited effectiveness of currently approved antivirals (neuraminidase inhibitors - NAIs) in the hypothetical event of a zoonotic spillover. Our study demonstrated that the recombinant avian A(H6N1) viruses showed reduced inhibition (RI) by multiple NAI drugs following the introduction of point mutations found predominantly in the neuraminidase gene (NA) of NAI-resistant human influenza strains (E119V, R292K and H274Y; N2 numbering). Moreover, A(H6N1)-H274Y showed increased replication efficiency in vitro, and a fitness advantage over wild-type (WT) when co-inoculated into embryonated hen’s eggs. The results presented in our study together with the zoonotic potential of the A(H6N1) virus as evidenced by the human infection from 2013, highlight the need for enhanced monitoring of NAI resistance-associated signatures in circulating LPAI (low pathogenic avian influenza) globally.

Inactivation of antibiotic resistant bacteria and elimination of transforming ability of plasmid carrying single and dual drug resistance genes by electro-oxidation using Ti/Sb-SnO2/PbO2 anode

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are one of the major components of environmental antimicrobial resistance (AMR). Wastewater treatment plant is an important source and a contributor of AMR components in environment. The existing tertiary treatment technologies viz., chlorination, ultraviolet treatment and ozonation are not effective in ARB disinfection or ARG removal. Therefore, it is important to evaluate the potential of advanced wastewater treatment technologies in removing ARB and ARGs. In this study, the potential of Ti/Sb-SnO2/PbO2 anode in disinfecting single ARB (pUC19-ampR), dual ARB (pBR322-ampR and tetR), regrowth inhibition, removal of intracellular and extracellular ARGs and loss of transformation efficiency were evaluated. Upto 7 log (99.99 %) removal of single ARB (0.212 min−1) and dual ARB (0.185 min−1) was achieved in 40 min and 35 min, respectively at an applied current density 30 mA cm−2. Scanning electron microscopy images confirmed extensive and irreparable cell damages. Therefore, no re-growth was observed upto 30 days. Agarose gel electrophoresis and qubit analysis confirmed degradation of intracellular and extracellular genetic material. Upto ∼7.5 log (53.14 ± 1.23 %) of intracellular and ∼5.3 log (41.43 ± 0.89 %) of extracellular ampR (pUC19) reduction was observed. Similarly, reduction of intracellular ampR and tetR (pBR322) were ∼6.5 log (55.19 ± 0.92 %) and ∼7.5 log (64.89 ± 2.39 %). While, extracellular ampR and tetR (pBR322) were ∼4.1 log (41.85 ± 1.62 %) and ∼4.9 log (52.49 ± 1.35 %), respectively. Despite low ARG removal efficiency, complete loss of transformation ability of intracellular and extracellular pUC19 and pBR322 was observed. From the results, it is evident that electro-oxidation is effective in disinfecting both the ARB, removal of intracellular and extracellular ARGs. Therefore, EOP is proposed as an effective and promising tertiary treatment technology.

Urine tenofovir-monitoring predicts HIV viremia in patients treated with high genetic-barrier regimens.

Access to viral load measurements is constrained in resource-limited settings. A lateral flow urine tenofovir (TFV) rapid assay (UTRA) for patients whose regimens include TFV offers an affordable approach to frequent adherence monitoring. We conducted a cross-sectional study of patients to assess the utility of UTRA to predict virologic failure, defined as a viral load greater than 400 copies/ml. We assessed urine TFV among 113 participants at increased risk of viral failure (who had previous viral failure on this regimen or had previously been ≥30 days out of care), comparing low genetic-barrier efavirenz (EFV) regimens (n = 60) to dolutegravir (DTG)-boosted or ritonavir-boosted protease inhibitor (PI/r)-based high genetic-barrier regimens (n = 53). Dried blood spots (DBS) for TFV-diphosphate and plasma for TFV concentrations were collected, with drug resistance assessed if viral failure present. Among 113 participants, 17 of 53 received DTG or PI/r had viral failure at the cross-sectional visit, with 11 (64.7%) demonstrating an undetectable urine TFV; the negative-predictive value (NPV) of undetectable UTRA for viral failure was 85% (34/40); none of the 16 sequenced had dual class drug resistance. In those treated with EFV regimens the sensitivity was lower, as only 1 (4.8%) of 21 with viral failure had an undetectable UTRA (P < 0.001). Urine tenofovir-testing had a high negative-predictive value for viral failure in patients treated with DTG or ritonavir-boosted protease inhibitor regimens, where viral failure was largely explained by poor drug adherence. Frequent monitoring with inexpensive lateral flow urine TFV testing should be investigated prospectively in between viral load visits to improve viral load suppression on DTG-based first-line therapy in resource-limited settings.

Assessment of Risk Factors for Drug Resistance of Dual Anti Platelet Therapy After PCI.

Since aspirin and clopidogrel are the widely and conventionally used drugs to treat acute myocardial infarction after percutaneous coronary intervention (PCI), it is important to explore potential risk factors of their resistance. The platelet aggregation rate with arachidonic acid (AA, PAg-AA%) and adenosine diphosphate (ADP, PAg-ADP%) of 219 PCI patients were measured after standard treatment for 24 h. The disease history and laboratory data (before PCI) were obtained. We found 101 (46.12%) patients to be aspirin-resistant, and PAg-ADP% was the most prominent risk factor of aspirin resistance. Clopidogrel resistance was present in 157 of 219 patients. Patients in the clopidogrel-resistant group carried more CYP2C19*3 or *2, which was associated with higher clopidogrel resistance in this group (69.11%, 47/68) than in the control group (64.29%, 36/56). Platelet count (109/L) and hemoglobin (g/L) were the prominent risk factors of clopidogrel resistance. Among the 219 patients, 98 showed dual antiplatelet drug resistance, for which platelet count (109/L) and monocyte count (g/L) were the risk factors. Aspirin resistance was found to usually accompany clopidogrel resistance.

ITGA5 Predicts Dual-Drug Resistance to Temozolomide and Bevacizumab in Glioma.

AimsAnti-angiotherapy (Bevacizumab) is currently regarded as a promising option for glioma patients who are resistant to temozolomide (TMZ) treatment. But ongoing clinical research failed to meet therapeutic expectations. This study aimed to explore the pivotal genetic feature responsible for TMZ and Bevacizumab resistance in glioma patients.MethodsWe downloaded the transcriptomic and methylation data of glioma patients from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and Gene Expression Omnibus (GEO) databases and grouped these patients into resistant and non-resistant groups based on their clinical profiles. Differentially expressed genes and pathways were identified and exhibited with software in R platform. A TMZ-resistant cell line was constructed for validating the expression change of the candidate gene, ITGA5. An ITGA5-overexpressing cell line was also constructed to investigate its biological function using the CCK8 assay, Western blot, periodic acid–Schiff (PAS) staining, and transcriptional sequencing.ResultsChange of the cell morphology and polarity was closely associated with TMZ mono-resistance and TMZ/Bevacizumab dual resistance in glioma patients. The expression level of ITGA5 was effective in determining drug resistance and the outcome of glioma patients, which is regulated by methylation on two distinct sites. ITGA5 was augmented in TMZ-resistant glioma cells, while overexpressing ITGA5 altered the cell-promoted TMZ resistance through enhancing vascular mimicry (VM) formation correspondingly.ConclusionsBoth the epigenetic and transcriptional levels of ITGA5 are effective in predicting TMZ and Bevacizumab resistance, indicating that ITGA5 may serve as a predictor of the treatment outcomes of glioma patients.

Frequency and Correlates of Mycoplasma genitalium Antimicrobial Resistance Mutations and Their Association With Treatment Outcomes: Findings From a National Sentinel Surveillance Pilot in England.

Mycoplasma genitalium infection is a public health concern due to extensive antimicrobial resistance. Using data from a pilot of M. genitalium antimicrobial resistance surveillance, we determined the prevalence and risk factors for resistance among specimens from sexual health clinic attendees and assessed treatment outcomes. Seventeen sexual health clinics in England sent consecutive M. genitalium-positive specimens to the national reference laboratory from January to March 2019. Regions of the 23S rRNA, parC, and gyrA genes associated with macrolide and fluoroquinolone resistance, respectively, were amplified and sequenced where appropriate. Fisher exact tests, and univariate and multivariable logistic regression models were used to determine associations between demographic, clinical, and behavioral factors and resistance-associated mutations. More than two-thirds (173 of 249 [69%]) of M. genitalium specimens had mutations associated with macrolide resistance, whereas predicted fluoroquinolone (21 of 251 [8%]) and dual-drug (12 of 237 [5%]) resistance were less prevalent. No specimens had both gyrA and parC resistance-associated mutations. Macrolide resistance was more common in specimens from men who have sex with men compared with heterosexual men (adjusted odds ratio, 2.64; 95% confidence interval, 1.09-6.38; P = 0.03). There was an association between both macrolide and fluoroquinolone resistance and having a previous sexually transmitted infection (P = 0.06).Only 19% of individuals returned for a test of cure. Of those infected with a macrolide-resistant genotype who were given azithromycin, 57 of 78 (73%) were known or assumed to be clinically cured; however, 43 of these 57 (75%) also received doxycycline. Of the 21 with a macrolide-resistant genotype who failed treatment, 18 of 21 (86%) also received doxycycline. Although macrolide resistance was widespread, particularly among specimens from men who have sex with men and those with a previous sexually transmitted infection diagnosis in the past year, resistance-associated mutations in M. genitalium did not seem to be unequivocally predictive of treatment failure.

Cyclodextrin-Based Hybrid Polymeric Complex to Overcome Dual Drug Resistance Mechanisms for Cancer Therapy.

Drug resistance always reduces the efficacy of chemotherapy, and the classical mechanisms of drug resistance include drug pump efflux and anti-apoptosis mediators-mediated non-pump resistance. In addition, the amphiphilic polymeric micelles with good biocompatibility and high stability have been proven to deliver the drug molecules inside the cavity into the cell membrane regardless of the efflux of the cell membrane pump. We designed a cyclodextrin (CD)-based polymeric complex to deliver chemotherapeutic doxorubicin (DOX) and Nur77ΔDBD gene for combating pumps and non-pump resistance simultaneously. The natural cavity structure of the polymeric complex, which was comprised with β-cyclodextrin-graft-(poly(ε-caprolactone)-adamantly (β-CD-PCL-AD) and β-cyclodextrin-graft-(poly(ε-caprolactone)-poly(2-(dimethylamino) ethyl methacrylate) (β-CD-PCL-PDMAEMA), can achieve the efficient drug loading and delivery to overcome pump drug resistance. The excellent Nur77ΔDBD gene delivery can reverse Bcl-2 from the tumor protector to killer for inhibiting non-pump resistance. The presence of terminal adamantyl (AD) could insert into the cavity of β-CD-PCL-PDMAEMA via host-guest interaction, and the releasing rate of polymeric inclusion complex was higher than that of the individual β-CD-PCL-PDMAEMA. The polymeric inclusion complex can efficiently deliver the Nur77ΔDBD gene than polyethylenimine (PEI-25k), which is a golden standard for nonviral vector gene delivery. The higher transfection efficacy, rapid DOX cellular uptake, and significant synergetic tumor cell viability inhibition were achieved in a pump and non-pump drug resistance cell model. The combined strategy with dual drug resistance mechanisms holds great potential to combat drug-resistant cancer.

Integrin-Src-YAP1 signaling mediates the melanoma acquired resistance to MAPK and PI3K/mTOR dual targeted therapy

Activation of PI3K/AKT pathway is one of the most recurrent resistant mechanisms for BRAF-targeted therapy, and the combination of MAPK and PI3K/AKT inhibitors becomes one of the most promising regimens for BRAF-targeted relapsed melanoma patients. Although the potent drug efficacy was observed in preclinical experiments and early clinical trials, the dual-drug resistance is inevitable observed. In this study, we systematically explored the mechanisms of dual-drug resistance to MAPKi and PI3K/mTORi in melanoma. With transcriptomic dissection of dual-drug resistant models, we identified that the drug tolerance was mediated by ECM-integrins α3β1 and α11β1 signaling. Upon binding ECM, the integrins activated downstream kinase Src rather than FAK, WNT, or TGFβ. Knockdown of integrins α3, α11, and β1 significantly inhibited the proliferation of dual-drug resistant sublines while with trivial effects on parental cells. Although Src inhibition suppressed the phosphorylation of AKT, c-JUN, and p38, none of inhibitors targeting these kinases reversed the dual-drug resistance in model cells. Notably, Src inhibitor promoted the phosphorylations of LATS1 and YAP1, subsequently, re-localized YAP1 from nucleus to cytosol facilitating further degradation. Both small molecule inhibitors and shRNAs targeting YAP1 or Src overcame the MAPKi and PI3K/mTORi dual-drug resistance. In conclusion, our data not only illuminated an integrin-Src-YAP1 pathway mediated MAPKi and PI3K/mTORi dual-drug resistant mechanism but also provided a potential combinatorial regimen for the drug-relapsed melanoma patients.

Synergistic Effects of Resveratrol and Temozolomide Against Glioblastoma Cells: Underlying Mechanism and Therapeutic Implications.

PurposeTemozolomide (TMZ) is a commonly used anti-glioblastoma (GBM) drug. However, glioblastoma cells frequently show primary and acquired resistance to TMZ. As a promising anti-GBM candidate, resveratrol (Res) faces the similar problem as TMZ. Although resveratrol combined with TMZ (Res/TMZ) has been reported to be used to treat GBMs, it remains unclear whether this combination is broad-spectrum for all glioma cells until now, especially for GBM cells/cases with dual drug resistance. The study aimed to evaluate the synergistic effects of resveratrol and TMZ against GBMs and identify the underlying mechanisms.Materials and MethodsDrug sensitivities of rat RG-2, human LN-18 and LN-428 cell lines and effectiveness of Res/TMZ combinations were investigated via multiple experimental methods. O6-methylguanine-DNA methyltransferase (MGMT) was observed by Western blotting and immunocytochemistry (ICC). Transducer and activator of transcription 3 (STAT3) signaling pathway and expression changes of STAT3-related gene were detected to explore the possible synergistic mechanism.ResultsOne hundred micromolar resveratrol and 500 μM TMZ inhibited the growth of RG-2 cells and the low-dose combination (25 μM/250 μM) showed similar suppressive effects. LN-18 and, especially, LN-428 cells were neither sensitive to 100 μM resveratrol nor to 500 μM TMZ, while their growth was suppressed by combination of 75 μM Res/750 μM TMZ with the suppressive rates of 62.5% and 28.6% and apoptosis rates of 11.9% and 7.4%, respectively. Resveratrol had regulatory effect on the expression of MGMT and it could significantly down-regulate MGMT overexpression caused by TMZ. In addition, STAT3/Bcl-2/survivin signaling pathway was also remarkably inhibited in Res/TMZ-treated GBM cells.ConclusionOur results demonstrated synergistic effects of Res/TMZ on RG-2 cells and their bilaterally sensitizing effects to LN-18 and LN-428 cells. Frequent upregulation of MGMT and activation of STAT3 are the unfavorable factors for the treatment of GBMs and they may be the potential targets of Res/TMZ therapy.

Genetic and Virulence Characteristics of Linezolid and Pretomanid Dual Drug-Resistant Strains Induced from Mycobacterium tuberculosis in vitro.

PurposeLinezolid (LZD) and pretomanid (PA-824) are promising candidates in regimens for the treatment of drug-resistant tuberculosis. However, research on LZD and PA-824 dual drug-resistant (LPDR) strains is rarely reported. This study aimed to investigate the genotypic and virulence characteristics of LPDR strains.MethodsTo obtain the LPDR strains (marked as LP or PL strains), we used a two-way induction method, namely, we first induced LZD- or PA-824-resistant mutants from the parental Mycobacterium tuberculosis (MTB) strain H37Rv in vitro, then we obtained the LPDR strains from induction of LZD- or PA-824-resistant mutants. Mutations in rplC, rrl, or ddn and fgd1 were identified in all mutants. To investigate the virulence of these strains, six strains were selected as representative strains, including LZD-resistant strains, PA-824-resistant strains and LPDR strains. We performed the animal survival study as virulence of MTB can be measured as survival time of an animal after being infected.ResultsWe induced 38 mutant strains of LZD and PA-824 mono or dual drug resistance from H37Rv in vitro. The mutation frequency of rplC (C154R) gene in LPDR strains was 100% and 86%, respectively. In the animal survival study, animals infected with different drug-resistant strains survived significantly longer than those infected with H37Rv; animals infected with LPDR strains and PA-824-resistant strains survived similarly and both of which survived significantly shorter than those infected with LZD-resistant strains.ConclusionOur study showed that rplC gene had a high mutation frequency in LPDR strains. The virulence of LPDR strains was similar to PA-824-resistant strains, and the virulence of the LZD-resistant strains was weaker than PA-824-resistant strains.

Isolation, characterization of Berberine from Berberis aristata DC for eradication of resistant Helicobacter pylori

H. pylori have gradually acquired resistance to the commonly used antibiotics because of their use in many parasitic and anaerobic infections, which leads to treatment failure of various gastric and duodenal diseases associated with H. pylori infection. The present research work aimed to isolate and characterize the bioactive compound from the methanol extract of stem of Berberis aristata DC. This is traditionally used for the treatment of dyspepsia, dysentery and diarrhea against antibiotic-resistant gastric pathogen H. pylori. The in vitro antimicrobial activity of Berberine an active isolated compound from methanol extract of stem of Berberis aristata DC against drug-resistant H. pylori strain isolated from North Indian GERD patients. The H. pylori strain was only collected from those, who were devoid of any kind of anti -H. Pylori therapy. The methodology was in determining the Minimum inhibition concentration (MIC) using the microdilution method and disk diffusion method. H. pylori isolate was included in this study. Berberine from methanol extract of stem of Berberis aristata DC showed its potency on H. pylori -infected isolated from GERD patients with a maximum inhibition at 0.000075 μg/ml. Prevalence of metronidazole resistance ranges between 50 and 90% in developing countries including India. The emergence of dual drug resistance was reported in various studies. This study suggests that Berberine an isolated compound from methanol extract of stem of Berberis aristata DC used commonly known as Daru Haldi potentially active for the treatment of drug-resistant H. pylori infection. Berberine from methanol extract of stem of Berberis aristata DC with a concentration of 0.000075 μg/ml shows a positive effect safely and effectively.

Case report: intravascular ultrasound sonography-guided re-entry technique in crushed stent.

BackgroundStent thrombosis (ST) is a rare, but potentially fatal complication. Procedural problems, such as stent under-dimension/under-expansion or dual antiplatelet drug resistance may result into ST. These conditions are more frequent during primary percutaneous coronary intervention for ST-elevation myocardial infarction (STEMI).Case summaryA 60-year-old male patient presented to our hospital with an inferior STEMI. In the emergency department, a dual antiplatelet therapy was administered with ticagrelor 180 mg and aspirin 250 mg IV. During the observation, the patient experienced a ventricular fibrillation. Urgent coronary angiography showed an occlusion of the proximal right coronary artery. Thrombus aspiration was performed followed by implantation of one drug-eluting stent. After 45 min early ST occurred and was treated by immediate thrombus aspiration and post-dilatation. Intravascular ultrasound sonography (IVUS) showed severe strut malapposition due to a partial crush after post-dilatation. Since it was not possible to directly insert the first guidewire in the stent lumen, the IVUS probe was placed between the vessel wall and the crushed stent to guide the manoeuvre.DiscussionCrushed stent is a rare complication, being caused by an incorrect passage of the guidewire between the stent’s struts and the vessel wall in case of severe underexpansion. In this case, an IVUS-guided re-entry could be an option to gain the stent true lumen and avoid a second stent implantation.

Deciphering extracellular antibiotic resistance genes (eARGs) in activated sludge by metagenome

Antibiotic resistance genes (ARGs) including extracellular ARGs (eARGs) and intracellular ARGs (iARGs), are recognized as emerging environmental contaminants. Despite extensive efforts to profile ARGs in their “hotspots” wastewater treatment plants (WWTPs), the contribution of eARGs to antibiotic resistance spread remains unclear. Here, we applied metagenomic sequencing to investigate the distribution, mobility and microbial hosts of eARGs in activated sludge from five WWTPs. The total relative abundance eARGs ranged from 9.5 × 10−6 to 1.3 × 10−4, mainly encompassing elfamycin, dual drug, and aminoglycoside resistance genes. Multiple eARGs (e.g., EF-Tu, ropB, and rpsL mutants) were shared among the five WWTPs, and some clustered in the same genetic element (e.g., EF-Tu-rpsJ). eARGs were found to frequently co-localize with the eMGEs (e.g., sul1-3′CS-TnAs3, sul2-intI1-ISVsa3, and tetX-p63039), which may facilitate the mobilization of eARGs. Most eARGs likely originated from the genera Mycobacterium (6.7%), Nitrosomonas (5.3%), Steroidobacter (5.3%), Nitrospira (5.2%) and Pseudomonas (5.1%). No significant difference in the diversity, abundance, and mobility was observed between eARGs and iARGs. The host composition of eARGs and iARGs from municipal WWTPs are consistently dominated by Nitrosomonas, Steroidobacter, Nitrospira and Pseudomonas, while some differentially enriched genera (especially Nitrospira) in the hosts of iARGs compared with those of eARGs from the swine WWTP were identified. Our findings corroborate the mobile eARGs reservoir in WWTPs, thereby laying foundation for mitigating widespread antibiotic resistance.

Antimicrobial susceptibility profiles of Helicobacter pylori isolated from patients in North India.

Helicobacter pylori-related gastroduodenal diseases are very common in India. Antibiotic resistance to commonly used antibiotics against H. pylori is increasing very rapidly. The aim of this study was to determine the antimicrobial susceptibility patterns of H. pylori strains from India against commonly used antibiotics in H. pylori treatment. Helicobacter pylori were cultured from 68 patients suffering from various gastroduodenal diseases in North India. Minimum inhibitory concentrations (MICs) to different antibiotics were determined by agar dilution. The clinical diagnosis of the 68 patients who were H. pylori culture-positive were gastro-oesophageal reflux disease (GERD) (n=23), non-erosive reflux disease (NERD) (n=22), non-ulcer dyspepsia (NUD) (n=13), antral gastritis (n=3), duodenal ulcer (n=2) and others (n=5). Of the 68 H. pylori isolates, 20 (29.4%) showed no resistance. The prevalence of drug resistance was 70.6%, including resistance to metronidazole (48.5%), furazolidone (22.1%), amoxicillin (17.6%), tetracycline (16.2%) and clarithromycin (11.8%). Dual and multiple drug resistance were found in 26.5% and 8.8% of cases, respectively. In conclusion, more than two-thirds of the isolated H. pylori strains showed resistance to at least one of the antibiotics for H. pylori treatment. Metronidazole resistance was most prevalent amongst the isolates tested. Emergence of dual and multidrug resistance is of great concern and there is an urgent need for regular antibiotic resistance surveillance studies. Amoxicillin- and clarithromycin-based anti-H. pylori regimens commonly prescribed for triple therapy in India show least resistance and hence are appropriate for anti-H. pylori management in India.

Tetrandrine reverses drug resistance in isoniazid and ethambutol dual drug-resistant Mycobacterium tuberculosis clinical isolates.

BackgroundTetrandrine is a natural chemical product purified from fourstamen stephania root which recently has been shown to act similarly as synthesized drug efflux pump inhibitor verapamil. The aim of the study is to examine whether tetrandrine could potentiate anti-tubercular drugs to which Mycobacterium tuberculosis (MTB) has turned resistant via efflux mechanisms.MethodsWe screened 200 MTB clinical isolates using drug sensitivity test to look for those who have turned resistant to the drugs most probably due to efflux mechanisms. We found 29 isoniazid (INH) and ethambutol (EMB) - dual resistant (IEDR) strains. Then we tested if treatment with tetrandrine or verapamil could reverse drug resistance to INH and/or EMB in IEDR isolates.ResultsThere is a parallel resistance among EMB- and INH-resistant strains in the tested clinical isolates. Among INH-resistant strains, 65% was also EMB-resistant. This suggests an involvement of efflux mechanisms which can lead to dual drug resistance in IEDR clinical isolates. Similar to a synthesized efflux pump inhibitor verapamil, tetrandrine treatment together with INH or EMB brought down the MICs from the clinical level of drug resistance to the sensitive range of both drugs. The effective rate reached 82% among IEDR clinical isolates.ConclusionsCombinational application of tetrandrine with INH or EMB increased drug efficacy. Drugs like tetrandrine may help to reduce drug dosage thus alleviate side effects.

Flumatinib, a selective inhibitor of BCR‐ABL/PDGFR/KIT, effectively overcomes drug resistance of certain KIT mutants

Activating mutations in KIT have been associated with gastrointestinal stromal tumors (GISTs). The tyrosine kinase inhibitor imatinib mesylate has revolutionized the treatment of GISTs. Unfortunately, primary or acquired resistance to imatinib does occur in GISTs and forms a major problem. Although sunitinib malate, a multi-kinase inhibitor, has shown effectiveness against imatinib-resistant GISTs, recent studies have indicated that some imatinib-resistant GISTs harboring secondary mutations in the KIT activation loop were also resistant to sunitinib. Therefore, new drugs capable of overcoming the dual drug resistance of GISTs probably have potential clinical utility. In this study, we investigated the efficacy of flumatinib, an inhibitor of BCR-ABL/PDGFR/KIT, against 32D cells transformed by various KIT mutants and evaluated its potency to overcome the drug resistance of certain mutants. Interestingly, our in vitro study revealed that flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P). Our in vivo study consistently suggested that flumatinib had superior efficacy compared with imatinib or sunitinib against 32D cells with the secondary mutation Y823D. Molecular modeling of flumatinib docked to the KIT kinase domain suggested a special mechanism underlying the capability of flumatinib to overcome the drug-resistance conferred by activation loop mutations. These findings suggest that flumatinib could be a promising therapeutic agent against GISTs resistant to both imatinib and sunitinib because of secondary mutations in the activation loop.

Low Prevalence of Antiretroviral Resistance Among HIV Type 1-Positive Prisoners in the Southeast United States

Drug-resistant HIV complicates management of HIV infection. Although an estimated 14% of all HIV-positive persons pass through a prison or jail in the United States each year, little is known about the overall prevalence of antiretroviral (ARV) resistance in incarcerated persons. All genotypic sequence data on HIV-positive prisoners in the North Carolina (NC) Department of Corrections (DOC) were obtained from LabCorp. Screening for major resistance mutations in protease (PI) and reverse transcriptase (NRTI and NNRTI) was done using Genosure and the Stanford HIV Database. For subjects with multiple genotype reports, each mutation was counted only once and considered present on all subsequent genotypes. Between October 2006 and February 2010, the NC DOC incarcerated 1,911 HIV(+) individuals of whom 19.2% (n=367) had at least one genotype performed. The overall prevalence of a major resistance mutation was 28.3% (95% CI 23.7, 33.0). Among prisoners ever exposed to an ARV during incarceration (n=329) prevalence of a major resistance mutation was 29.8% (95% CI 24.9, 34.7); resistance by class was 20.4% (95% CI 16.0, 24.7) for NRTIs, 19.8% (95% CI 15.5, 24.1) for NNRTIs, and 8.8% (95% CI 5.8,11.9) for PIs. Single class drug resistance was most prevalent at 14.2% (10.2,17.7) followed by dual 12.5% (I8.9,16.0) and triple class 3.3% (1.4,5.3) resistance. The three most prevalent mutations were K103N 15.8% (12.0, 20.2), M184V 14.3% (10.7,18.5), and M41L 4.9% (2.8,7.8). In the NC DOC ARV resistance prevalence, dual and triple class drug resistance was moderate over the study period. Resistance to PIs was lower than NNRTIs and NRTIs, likely reflecting higher usage of these two classes or a lower barrier to resistance.

Abstract 2559: Development of carboplatin, docetaxel and combined carboplatin/docetaxel resistant ovarian cancer cell lines

Abstract Carboplatin and docetaxel are two standard chemotherapeutics used to treat ovarian cancer. Patients often exhibit resistance to single agent therapy, and dual agent therapy was developed to overcome this resistance. However, resistance still commonly occurs in dual agent therapy. It is known that specific changes in gene expression occur in single agent resistance, but it is not known whether resistance to combined chemotherapy involves novel gene expression changes or if resistance is only the sum of changes seen in single agent resistant cells. Using the chemotherapy-naïve A2780 ovarian cancer cell line, we have generated three resistant cell lines including carboplatin and docetaxel single agent resistant lines (A2780CBN, and A2780DXL respectively), and a carboplatin/docetaxel dual resistant line (A2780CBNDXL) by exposing A2780 cells to increasing concentrations of drug until a dose is reached where resistance could no longer be developed. The A2780 parent line was co-cultured alongside selection to control for gene expression changes due to continuous culture and not drug resistance. The concentration of drug at which 50% of cells die (IC50) was measured for reach resistant cell line using a clonogenic assay. Cells were cultured in varying drug concentrations and plated in methylcellulose. Colonies were counted and normalized to the no drug control. The IC50 was calculated as a 50% survival fraction plotted against the log of drug (M). The IC50 of A2780CBN is 8×10−5M, 13-fold more resistant than the parent line. The IC50 of A2780DXL is 3×10−7M, 5000-fold more resistant than the parent line. The IC50 of A2780CBNDXL is 1×10−5M for carboplatin and 1×10−8M for docetaxel, 1000-fold more resistant than the parent line. This dual resistance may be caused by a synergistic effect of carboplatin with docetaxel leading to increased sensitivity of the co-cultured control to combination treatment. Total RNA was isolated from A2780DXL and its co-cultured counterpart and hybridized to Agilent 4×44k total human genome arrays to study gene expression. Agilent Feature Extraction software was used to scan the arrays and Partek Genomics suite was used to find statistical significance. A total of 1827 gene expression changes occurred, with 1061 genes up regulated and 766 genes down regulated in the resistant line. Signal intensity differences ≥ 2-fold and P ≤ 0.01 were considered significantly changed. Up regulated genes include ABCB4 (141) and ABCB1 (34) that encode two drug transporters implicated in taxane resistance. Down regulated genes include TUBB2B (−16), a beta-tubulin encoding gene, and growth factor receptors PDGFRB (−10), and PDGFRA (−8). Microarray analysis will also be performed on the A2780CBN and A2780CBNDXL lines. Gene expression changes associated with dual drug resistance may be used to develop clinically relevant biomarkers for carboplatin/docetaxel resistance in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2559.

Dual Antiplatelet Drug Resistance Is a Risk Factor for Cardiovascular Events after Percutaneous Coronary Intervention

Nonresponsiveness to clopidogrel and acetylsalicylic acid (ASA), a frequent result of platelet aggregometry studies, has unclear clinical and prognostic significance. We performed impedance aggregometry in 182 patients 12-24 h after percutaneous coronary intervention (PCI) and a 600-mg loading dose of clopidogrel, adding 5 micromol/L ADP and 1 mg/L collagen to diluted whole blood to determine platelet inhibition by clopidogrel and ASA, respectively. Samples from nonresponders were incubated in vitro with methyl-S-adenosine monophosphate or ASA to distinguish between pharmacodynamic and pharmacokinetic types of resistance. We assessed a combined primary endpoint of myocardial infarction, target vessel revascularization, late stent thrombosis, or cardiac death. Nineteen patients (10.4%) were dual nonresponders (nonresponsive to both ASA and clopidogrel), and 163 patients (89.6%) were designated responders. The latter group also included 15 and 14 single nonresponders (responsive to either clopidogrel or ASA, respectively), who exhibited endpoint frequencies comparable to those of full responders (n = 134). Pharmacokinetic resistance was most prevalent. Primary endpoints occurred more frequently in dual nonresponders (n = 6, 31.6%) than in responders (n = 20, 12.3%) (relative risk 2.57; 95% CI 1.18-5.61; log-rank P = 0.03). Multivariate analysis confirmed dual nonresponsiveness (hazard ratio 2.9; 95% CI 1.17-7.2; P = 0.02) as an independent risk factor. Dual nonresponders carry a high cardiovascular risk after PCI and should obtain intensified antiplatelet therapy and follow-up.