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PubMed1987

DNA-mediated transfer and cloning of a human multidrug-resistant gene of adriamycin-resistant myelogenous leukemia K562.

We have successfully transferred and cloned a fragment of a human multidrug-resistant gene by using DNA-mediated gene transfer. Macromolecular DNA of human multidrug-resistant K562 cells was transfected to drug-sensitive mouse Ltk- cells to obtain a drug-resistant transfectant with a human resistant gene. Both primary and secondary transfectants showed similar patterns of cross-resistance to Adriamycin and vincristine. The mechanism of drug resistance of the transfectants was attributed to decreased retention of the drug. Three secondary transfectants obtained independently contained common Alu-containing EcoRI fragments 15, 6.5, 3.7, 2.6, and 1.9 kilobases long. The 2.6-kilobase EcoRI fragment was cloned from a lambda phage genomic library made from DNA of a secondary transfectant. The 2.6-kilobase fragment was detected in the primary and secondary transfectants but not in the parental Ltk-, Adriamycin-resistant Ltk-, and Adriamycin-resistant P388 cells. This sequence was found to be amplified in several multidrug-resistant cell lines such as Adriamycin-resistant ovarian carcinoma A2780 and colchicine-resistant KB carcinoma cells. The 2.6-kilobase fragment hybridized with a 4.5-kilobase mRNA which is overexpressed in the Adriamycin-resistant K562 cells and the Adriamycin-resistant A2780 cells but not detected in the parental K562 cells. The gene transferred and cloned in this study seems to be related to the P-glycoprotein gene as judged from the size of mRNA and its overexpression in some of the multidrug-resistant cell lines where P-glycoprotein was found to be highly expressed.

Animals↗
PubMed2026

Investigation of pmrCAB and mcr associated resistance in colistin-resistant A. baumannii isolates.

BACKGROUND & OBJECTIVES: Colistin is one of the last-resort antibiotics for multidrug-resistant Acinetobacter baumannii. Increasing resistance to colistin limits treatment options, particularly in intensive care units (ICUs). The aim of this study was to compare the expression levels of pmrC, pmrA, and pmrB, among colistin-resistant and colistin-susceptible clinical A. baumannii isolates, to investigate the presence of plasmid-mediated mcr-1-5 genes, and to determine clonal relationships among colistin-resistant isolates. METHODS: A total of 110 A. baumannii isolates recovered from ICU patients in 2020 were included. Colistin minimum inhibitory concentrations were determined using the broth microdilution method. Expression levels of pmrC, pmrA, and pmrB were analyzed by RT-qPCR and compared with the reference strain A. baumannii ATCC 19606. Colistin-resistant isolates (Group 1) were compared with 10 randomly selected colistin-susceptible isolates (Group 2). Detection of mcr-1-5 genes was performed by in-house multiplex PCR. Clonal relationships among resistant isolates were assessed by PFGE. RESULTS: Colistin resistance was detected in 15.45% (17/110) of isolates. The median relative expression levels of pmrC, pmrB, and pmrA in colistin-resistant isolates were 47.84-fold (IQR: 19.29-67.18), 14.72-fold (IQR: 10.13-16.68), and 8.57-fold (IQR: 5.17-12.82), respectively. In colistin-susceptible isolates, the corresponding median expression levels were 5.32-fold (IQR: 3.60-7.97), 3.29-fold (IQR: 0.85-5.95), and 3.31-fold (IQR: 2.58-6.55). Expression levels were significantly higher in colistin-resistant isolates for pmrC (p < 0.001), pmrB (p = 0.002), and pmrA (p = 0.024). None of the resistant isolates carried mcr-1-5 genes. PFGE analysis revealed 12 distinct genotypes among 17 resistant isolates. INTERPRETATION & CONCLUSIONS: Colistin-resistant A. baumannii isolates exhibited significantly higher expression levels of the pmrC, pmrA, and pmrB genes compared to colistin-susceptible isolates. Among the genes evaluated, pmrC showed the largest effect size and the strongest association with the colistin-resistant phenotype. No changes were found in the mcr-1-5 genes among the isolates studied. Further studies, including genomic and functional analyses, are needed to elucidate the underlying mechanisms of these expression changes and their contribution to colistin resistance.

Journal Article↗
PubMed2003

[Antibiotic resistance in 622 Streptococcus pneumoniae isolated from blood and cerebrospinal fluid in 33 Spanish hospitals participating in the European Antimicrobial Resistance Surveillance System (2000)].

INTRODUCTION: In 1998 the European Union founded the European Antimicrobial Resistance Surveillance System (EARSS) for the control of antimicrobial resistance in invasive pathogens. This report present the results obtained for S. pneumoniae in the year 2000 in Spain. METHODS: Participants included 33 hospitals covering approximately 25% of the Spanish population. The laboratories carried out isolation, identification and sensitivity studies with their usual methods. Quality control was performed by the UK National External Quality Assurance Scheme (NEQAS). A form was filled out with clinical information on the patient and data on the hospital, department and specimen isolated. This information was entered in a central database for analysis and validation by the Whonet.5 program. RESULTS: Invasive S. pneumoniae was isolated in 622 patients. Overall incidence was 6.4 cases/100,000 inhabitants. Among the strains isolated, 33.1% (CI 95%: 29.6-37) showed resistance to penicillin (22.2% intermediate; 10.9% resistant); 11% showed resistance to cefotaxime (10.1% intermediate; 0.9% resistant); and 21.1% were resistant to erythromycin. Erythromycin resistance was 7% in penicillin-sensitive strains and 46.4% in penicillin-resistant strains (p < 0.001). A total of 51.7% of strains were resistant to penicillin in children < or = 4 years old, as compared to 29% in patients > 4 years old (p < 0.001). A ciprofloxacin MIC > 2 mg/L was found for 2.4% of isolates. CONCLUSIONS: Penicillin resistance in invasive S. pneumoniae in Spain is among the highest in the European Union. The decrease in sensitivity to penicillin was significantly associated with cefotaxime and erythromycin resistance. Children < or = 4 years old have a significantly higher risk than adults of acquiring infection with strains resistant to penicillin, cefotaxime and erythromycin.

Adolescent↗
PubMed1997

Metronidazole resistance reduces efficacy of triple therapy and leads to secondary clarithromycin resistance.

There has been a significant increase in the prevalence of H. pylori resistance to metronidazole in recent years, while clarithromycin resistance is still relatively rare. In this study we assessed: (1) the effect of primary H. pylori resistance to metronidazole and clarithromycin on the clinical efficacy of a one-week regimen consisting of omeprazole, metronidazole, and clarithromycin; and (2) the rate of acquisition of secondary antimicrobial resistance after treatment failure. Eighty-seven patients with duodenal ulceration or nonulcer dyspepsia were included in the study. The primary metronidazole and clarithromycin resistance rates were 35.6% and 3.4%, respectively (all three pretreatment clarithromycin resistant strains had concurrent metronidazole resistance). H. pylori was eradicated in 81.6% of patients. The eradication rate for fully sensitive isolates was 98.2% (55/56) but was significantly reduced to 57.1% (16/28) for isolates that were resistant to metronidazole alone and 0% (0/3) in cases of dual resistance (P < 0.001). Secondary resistance to clarithromycin was acquired in 58.3% of cases of treatment failure. In areas of high prevalence of primary metronidazole resistance, this is a significant cause of treatment failure with this triple therapy regimen. This leads to the selection of strains with dual resistance that are difficult to eradicate and may contribute to an increase in the prevalence of clarithromycin resistance. In such areas an alternative first-line treatment should be prescribed.

Adult↗
PubMed1998

Global surveillance for antituberculosis-drug resistance, 1994-1997. World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance.

BACKGROUND: Drug-resistant tuberculosis threatens efforts to control the disease. This report describes the prevalence of resistance to four first-line drugs in 35 countries participating in the World Health Organization-International Union against Tuberculosis and Lung Disease Global Project on Anti-Tuberculosis Drug Resistance Surveillance between 1994 and 1997. METHODS: The data are from cross-sectional surveys and surveillance reports. Participating countries followed guidelines to ensure the use of representative samples, accurate histories of treatment, standardized laboratory methods, and common definitions. A network of reference laboratories provided quality assurance. The median number of patients studied in each country or region was 555 (range, 59 to 14,344). RESULTS: Among patients with no prior treatment, a median of 9.9 percent of Mycobacterium tuberculosis strains were resistant to at least one drug (range, 2 to 41 percent); resistance to isoniazid (7.3 percent) or streptomycin (6.5 percent) was more common than resistance to rifampin (1.8 percent) or ethambutol (1.0 percent). The prevalence of primary multidrug resistance was 1.4 percent (range, 0 to 14.4 percent). Among patients with histories of treatment for one month or more [corrected], the prevalence of resistance to any of the four drugs was 36.0 percent (range, 5.3 to 100 percent), and the prevalence of multidrug resistance was 13 percent (range, 0 to 54 percent). The overall prevalences were 12.6 percent for resistance to any of the four drugs [corrected] (range, 2.3 to 42.4 percent) and 2.2 percent for multidrug resistance (range, 0 to 22.1 percent). Particularly high prevalences of multidrug resistance were found in the former Soviet Union, Asia, the Dominican Republic, and Argentina. CONCLUSIONS: Resistance to antituberculosis drugs was found in all 35 countries and regions surveyed, suggesting that it is a global problem.

Antitubercular Agents↗
PubMed2006

The importance of addressing multidrug resistance and not assuming single-drug resistance in case-control studies.

BACKGROUND: Case-control studies analyzing antibiotic exposure as a risk factor for antimicrobial resistance usually assume single-drug resistance in the bacteria under study, even though resistance to multiple antimicrobials may be present. Since antibiotic selection pressures differ depending on the susceptibility profile of the antimicrobial-resistant bacteria, an accurate assessment of whether exposure to an individual antimicrobial is a risk factor for the emergence of resistance should distinguish between single-drug-resistant and multidrug-resistant bacteria. OBJECTIVE: To determine whether the exposures to individual antibiotics that were identified as independent risk factors in case-control studies differed depending on whether single-drug-resistant or multidrug-resistant bacteria were evaluated. DESIGN: Two retrospective case-control studies were performed with data on patients harboring Pseudomonas aeruginosa strains resistant only to ciprofloxacin (CRPA) and patients harboring P. aeruginosa strains resistant to ciprofloxacin and other antibiotics (multidrug-resistant P. aeruginosa [MDR-PA]). These 2 groups were compared with patients not harboring P. aeruginosa. SETTING: Two tertiary care hospitals. RESULTS: A total of 41 patients harboring CRPA and 151 patients harboring MDR-PA were identified and matched to 192 control subjects. By conditional logistic regression, independent risk factors associated with presence of CRPA were nonambulatory status (OR, 5.6 [95% confidence interval {CI}, 1.4-23]; P=.02) and prior ciprofloxacin exposure (OR, 5.0 [95% CI, 1.2-21]; P=.03). Independent risk factors for presence of MDR-PA were a Charlson score greater than 2 (OR, 3.3 [95% CI 1.8-6.0]; P<.001) and exposure to quinolones (OR, 2.8 [95% CI, 1.2-5.0]; P=.001), third- and fourth-generation cephalosporins (OR, 3.5 [95% CI, 1.7-7.1]; P<.001), imipenem (OR, 3.8 [95% CI, 1.2-12.1]; P=.02), and/or aminoglycosides (OR, 2.3 [95% CI, 1.04-5.1]; P=.04). CONCLUSION: There were substantial differences in exposure to individual antimicrobials between patients harboring CRPA and patients harboring MDR-PA. Future case-control studies addressing risk factors for single-drug-resistant bacteria should consider the complete susceptibility profile of the bacteria under investigation.

Case-Control Studies↗
PubMed2002

Prevalence and antibiotic resistance profile of mercury-resistant oral bacteria from children with and without mercury amalgam fillings.

Genes encoding resistance to mercury and to antibiotics are often carried on the same mobile genetic element and so it is possible that mercury-containing dental materials may select for bacteria resistant to mercury and to antibiotics. The main aim of this study was to determine whether the prevalence of Hg-resistant oral bacteria was greater in children with mercury amalgam fillings than in those without. A secondary aim was to determine whether the Hg-resistant isolates were also antibiotic resistant. Bacteria in dental plaque and saliva from 41 children with amalgam fillings and 42 children without such fillings were screened for mercury resistance by cultivation on a HgCl(2)-containing medium. Surviving organisms were identified and their susceptibility to mercury and to several antibiotics was determined. Seventy-eight per cent and 74% of children in the amalgam group and amalgam-free group, respectively, harboured Hg-resistant bacteria; this difference was not statistically significant. Nor was there any significant difference between the groups in terms of the proportions of Hg-resistant bacteria in the oral microflora of the children. Of Hg-resistant bacteria, 88% and 92% from the amalgam group and the amalgam-free group, respectively, were streptococci; 41% and 33% were resistant to at least one antibiotic, most frequently tetracycline. The results of this study show that there was no significant difference between children with amalgam fillings and those without such fillings with regard to the prevalence, or the proportion, of Hg-resistant bacteria in their oral microflora. The study also found that Hg-resistant bacteria were common in children regardless of whether or not they had amalgam fillings and that many of these organisms were also resistant to antibiotics.

Adolescent↗
PubMed1998

The M184V mutation in HIV-1 reverse transcriptase (RT) conferring lamivudine resistance does not result in broad cross-resistance to nucleoside analogue RT inhibitors.

OBJECTIVE: To investigate the prevalence and magnitude of M184V-mediated changes in susceptibility to zalcitabine, didanosine, stavudine and abacavir (1592U89 succinate) in a cohort of lamivudine-treated patients. DESIGN AND METHODS: A total of 255 samples from patients treated with lamivudine and zidovudine with or without other nucleoside reverse transcriptase inhibitors (NRTI) were analysed for susceptibility to zidovudine, lamivudine, zalcitabine, didanosine and stavudine using a recombinant virus assay. Seventy-three samples originated from patients exposed to zidovudine and lamivudine only. A subset of 27 samples was investigated for cross-resistance to abacavir. Resistance was defined as a change in median inhibitory concentration more than fivefold compared with wild-type (high-level resistance, > 10-fold). A genotypic analysis of plasma-derived reverse transcriptase coding regions was carried out in samples with cross-resistance. RESULTS: The majority of samples displayed wild-type or greater than wild-type sensitivity to zalcitabine, didanosine and stavudine: resistance was seen in 17.2, 9 and 6.3% of the total sample population, respectively. Of these, 1.2, 2.7 and 2.4%, respectively, showed high-level resistance. The prevalence of resistance to a particular NRTI was lower in samples from patients not pretreated with that NRTI and in samples from patients exposed to zidovudine-lamivudine only. Cross-resistance was more prevalent in samples with high ZDV resistance. There was no obvious correlation between cross-resistance and genotype; all but two samples were mutant at codon 184. There were no consistent changes at positions associated with zidovudine resistance. The majority of samples from a subset (n=27) were four- to eightfold less sensitive to abacavir. There were no other genotypic changes in addition to M184V known to be associated with abacavir resistance. CONCLUSIONS: Cross-resistance was not commonly observed in this lamivudine-treated cohort. M184V per se is not expected to compromise subsequent treatment with NRTI such as didanosine-stavudine or combinations containing abacavir.

Anti-HIV Agents↗
PubMed2005

Modulation of multidrug resistance by andrographolid in a HCT-8/5-FU multidrug-resistant colorectal cancer cell line.

OBJECTIVE: To develop an HCT-8/5-FU multidrug-resistant colorectal cancer cell line and to elucidate the effect of Andrographolid (AG), an extract from Andrographis paniculate, a medicinal herb on the HCT-8/5-FU multidrug-resistant colorectal cancer cell line. METHODS: An HCT-8 colorectal cancer cell line was used and a high concentration of 5-Fluorouracid (5-FU) was introduced at the beginning to induce drug resistance, then the concentration of 5-FU was increased in gradients. Approximately 7 months later, the cells grew stably in 2.0 microg/mL of 5-FU, and the cell line was named HCT-8/5-FU multidrug-resistant colorectal cancer cell line. The resistant index of HCT-8/5-FU cells to 5-FU, adriamycin (ADM), cisplatin (DDP) was checked by MTT test, and a growth curve was drawn. The morphological changes were observed by both light and electron microscope. The function of P-170 was detected by rhodamine staining. After the application of AG and co-administration of 5-FU, ADM and DDP, the growth curves and inhibition rate as well as apoptosis rate of HCT-8/5-FU at different concentrations of AG were evaluated by MTT and flow cytometry. Rhodamine staining was used to investigate the possible mechanism involved by AG. RESULTS: The resistance index of HCT-8/5-FU to 5-FU was 16.6, and a cross-resistance to ADM and DDP was noticed. Compared with parental cells, HCT-8/5-FU cell's growth rate did not change significantly but the cell's morphology was remarkably changed as compared with parental cells. Overexpression of P-170 by HCT-8/5-FU cell was indicated through rhodamine staining. AG at a low concentration showed weak inhibitory effect on HCT-8/5-FU. However, a remarkable inhibitory and apoptosis rate was shown when AG was co-administered with 5-FU, ADM and DDP, respectively. Interestingly AG alone could not induce apoptosis and change the cell cycles. AG might affect the expression of P-170, which was indicated by rhodamine staining. CONCLUSIONS: The HCT-8/5-FU multidrug-resistant colorectal cancer cell line has been successfully developed and because it has cross-resistance to 5-FU, ADM and DDP, it might serve as an ideal multidrug resistance (MDR) model for colorectal cancer research. The mechanism of HCT-8/5-FU resistance to chemotherapeutic agents might be related to the overexpression of P-170. Low concentrations of AG alone have no significant inhibition on HCT-8/5-FU and fail to induce apoptosis and to change cell cycles. AG might act as a chemosensitizer when co-administered with 5-FU, ADM and DDP, and the mechanism of reversal modulation of multidrug resistance by AG in the HCT-8/5-FU resistant cell line might be related to its downregulation of overexpression of P-170.

Adenocarcinoma↗
PubMed2003

High-level beta-lactam resistance associated with acquired multidrug resistance in Helicobacter pylori.

Four clinical Helicobacter pylori isolates with high-level resistance to beta-lactams exhibited low- to moderate-level resistance to the structurally and functionally unrelated antibiotics ciprofloxacin, chloramphenicol, metronidazole, rifampin, and tetracycline. This pattern of multidrug resistance was transferable to susceptible H. pylori by natural transformation using naked genomic DNA from a clinical multidrug-resistant isolate. Acquisition of the multidrug resistance was also associated with a change in the genotype of the transformed multidrug-resistant H. pylori. DNA sequence analyses of the gene encoding penicillin binding protein 1A (PBP 1A) showed 36 nucleotide substitutions resulting in 10 amino acid changes in the C-terminal portion (the putative penicillin binding domain). Acquisition of beta-lactam resistance was consistently associated with transfer of a mosaic block containing the C-terminal portion of PBP 1A. No changes of genes gyrA, rpoB, rrn16S, rdxA, and frxA, and nine other genes (ftsI, hcpA, llm, lytB, mreB, mreC, pbp2, pbp4, and rodA1) encoding putative PBPs or involved in cell wall synthesis were found among the transformed resistant H. pylori. Antibiotic accumulations of chloramphenicol, penicillin, and tetracycline were all significantly decreased in the natural and transformed resistant H. pylori compared to what was seen with susceptible H. pylori. Natural transformation also resulted in the outer membrane protein profiles of the transformed resistant H. pylori becoming similar to that of the clinical resistant H. pylori isolates. Overall, these results demonstrate that high-level beta-lactam resistance associated with acquired multidrug resistance in clinical H. pylori is mediated by combination strategies including alterations of PBP 1A and decreased membrane permeability.

Amino Acid Sequence↗
PubMed2004

Incidence of antibiotic resistance in Campylobacter jejuni isolated in Alberta, Canada, from 1999 to 2002, with special reference to tet(O)-mediated tetracycline resistance.

Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 microg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 microg/ml). In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 microg/ml) were higher than those previously reported in C. jejuni (64 to 128 microg/ml). In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene. Four isolates resistant to high levels of tetracycline (MIC = 512 microg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C. jejuni. The tetracycline MICs for transconjugants were comparable to those of the donors. Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 microg/ml for Escherichia coli DH5alpha. In contrast, transfer to a strain of C. jejuni by using mobilization conferred an MIC of 128 microg/ml. DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 microg/ml) were identical to one other, although the tet(O) genes encoding a 512-microg/ml MIC demonstrated several nucleotide substitutions. The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution. In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3'-aminoglycoside phosphotransferase. Resistance to erythromycin was not detected in 203 isolates. In general, resistance to most antibiotics in C. jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years.

Alberta↗
PubMedDate not supplied

Methicillin-resistant staphylococci and ofloxacin-resistant bacteria from clinically healthy conjunctivas.

We evaluated the existence of methicillin (DMPPC)-resistant staphylococci and ofloxacin (OFLX)-resistant bacteria among preoperative patients at an eye clinic. Bacterial growth was studied in 194 conjunctival specimens of 125 preoperative patients without signs of ocular infection. Specimens were evaluated for growth of bacteria on Drigalski's plates, blood agar plates, or chocolate agar plates for 2 days at 37 degrees C. Susceptibilities of the strain to DMPPX, OFLX, minocycline (MINO), gentamicin (GM), erythromycin (EM), cefmenoxime (CMX), chloramphenicol (CP), and sulbenicillin (SBPC) were determined by the disk diffusion method. When DMPPC-resistant Staphylococcus aureus (MRSA) or DMPPC-resistant coagulase- negative staphylococci (MRCNS) were isolated, the susceptibility of the isolated strains to vancomycin (VCM) was additionally examined by the disk diffusion method. Of the 125 preoperative patients (194 eyes), 109 patients (159 eyes) had positive bacterial growth. Methicillin-resistant Staphylococcus aureus (MRSA) were positive in 1 patient. Methicillin-resistant coagulase-negative staphylococci (MRCNS) were positive in another patient. Two eyes with MRSA or MRCNS growth were treated with topical instillation of VCM, which was more sensitive than MINO and GM. They underwent the planned surgery after two subsequent tests showing negative growth. Of 120 isolated coagulase-negative staphylococci (CNS) 8 (6.7%) were resistant to OFLX. OFLX-resistant CNS were sensitive to MINO, CMX, and CP, but also resistant to SBPC. Of 114 isolated aerobic gram-positive rods (GPR), 55 (48.2%) were resistant to OFLX. OFLX-resistant aerobic GPR were sensitive to MINO, CMX, SBPC, and DMPPC. The 157 eyes with positive bacterial growth other than MRSA were treated with topical instillation of sensitive antibiotics. The present findings indicate that DMPPC-resistant staphylococci and OFLX-resistant bacteria exist in conjunctivas without signs of infection.

Adult↗
PubMed2003

Immunotoxin resistance in multidrug resistant cells.

Multidrug resistance (MDR) can be mediated, in part, by overexpression of P-glycoprotein (P-gp) and is characterized by broad resistance to several structurally, chemically, and pharmacologically distinct chemotherapeutic compounds. It has been hypothesized that immunological approaches to cytolysis may be used to overcome drug resistance. RV+ is a P-gp-expressing variant of the human myeloid leukemic cell line HL60 that displays a typical MDR phenotype. MDR RV+ cells displayed relative resistance to the immunotoxin (IT) HuM195-gelonin and to free rGelonin. K562 leukemia cells retrovirally infected to overexpress P-gp are also resistant to HuM195-gelonin. In addition, a monoclonal antibody capable of inhibiting the function of P-gp was able to partially reverse resistance to the IT. These data indicated that the expression of P-gp may contribute to IT resistance in RV+. Resistance to the IT was not mediated through decreased binding to cells, nor reduced internalization into the cell because the IT displayed similar kinetics of binding and internalization for both the parental HL60 and MDR RV+ cell lines. Comparison of the cytotoxicity of other ribosome-inactivating toxins indicated that RV+ cells were not universally resistant to toxins: RV+ cells were sensitive to the actions of ricin A chain, which acts on precisely the same RNase target as gelonin. Sensitivity of the MDR RV+ cells to the protein synthesis inhibitor cycloheximide, saponin, and Pseudomonas exotoxin A additionally confirmed that the resistance was not mediated through the ribosome and that pathways downstream from the inactivation of protein synthesis leading to cell death were not substantially perturbed in the MDR cells. Resistance could be partially abrogated by bafilomycin A, which inhibits lysosomal function. Moreover, direct visualization by confocal microscopy of the intracellular trafficking route of the IT showed that the IT accumulated preferentially in the lysosome in MDR RV+ cells but not in sensitive cells. These observations implicated the process of increased lysosomal degradation as the most likely basis for resistance. Such pathways of resistance may be important in the therapeutic applications of ITs, now becoming available for human use.

Cell Survival↗
PubMed2003

Multidrug-resistant tumor cells remain sensitive to a recombinant interleukin-4-Pseudomonas exotoxin, except when overexpressing the multidrug resistance protein MRP1.

Tumor cells may become resistant to conventional anticancer drugs through the occurrence of transmembrane transporter proteins such as P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or members of the multidrug resistance-associated protein family (MRP1-MRP5; ABCC1-ABCC5). In this report, we studied whether tumor cells that are cytostatic drug resistant because of overexpression of one of the above mentioned proteins are sensitive to a new anticancer agent, interleukin-4 toxin (IL-4 toxin). IL-4 toxin is a fusion protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE) [IL-4(38-37)-PE38KDEL]. Ninety-six-h cytotoxicity assays and 10-day clonogenic assays showed that drug-selected multidrug resistant (MDR) tumor cells that overexpress P-glycoprotein or breast cancer resistance proteins are still sensitive to IL-4 toxin. Also, tumor cells transfected with cDNA for MRP2-5 showed no resistance, or marginal resistance, only to the toxin as compared with the parent cells. In contrast, MRP1-overexpressing cells, both drug selected and MRP1 transfected, are clearly resistant to IL-4 toxin with resistance factors of 4.3 to 8.4. MRP1-overexpressing cells were not resistant to PE itself. IL-4 toxin resistance in MRP1-overexpressing cells could be reversed by the MRP1 inhibitors probenecid or MK571 and were not affected by glutathione depletion by DL-buthionine-S,R-sulfoximine. In a transport assay using plasma membrane vesicles prepared from MRP1-overexpressing cells, IL-4 toxin and IL-4, but not PE, inhibited the translocation of the known MRP1 substrate 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). These data suggest that MRP1-overexpressing cells are resistant to IL-4 toxin because of extrusion of this agent by MRP1. Still, the results of this study demonstrate that IL-4 toxin effectively kills most MDR tumor cells and, therefore, represents a promising anticancer drug.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Drug-resistant Streptococcus pneumoniae: the beginning of the end for many antibiotics? Australian Group on Antimicrobial Resistance (AGAR)

OBJECTIVE: To determine the levels of antibiotic resistance in Streptococcus pneumoniae in Australia. DESIGN: Prospective, Australia-wide, laboratory-based survey. SETTING: 27 hospital and private laboratories around Australia, from January 1994 to August 1995. SUBJECTS: First 100 patients with clinically significant isolates of S. pneumoniae at each laboratory. OUTCOME MEASURES: Resistance to penicillin (determined from penicillin minimum inhibitory concentration [MIC] measured by the Etest), erythromycin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, cefotaxime and ceftriaxone. RESULTS: A total of 2396 isolates were tested (including 537 invasive isolates and 740 from children). Penicillin resistance was seen in 161 isolates (6.7%), including 17 with high level resistance. Penicillin resistance rates were significantly lower in invasive than in non-invasive strains (3.7% versus 7.6%; odds ratio [OR], 0.47; 95% confidence interval [CI], 0.28-0.77; P = 0.001). There was no significant difference in penicillin resistance rates between children (< 15 years) and adults (7.3% versus 6.5%; OR, 1.14; 95% CI, 0.80-1.63; P = 0.47). Resistance rates were higher for most other antibiotics than for penicillin (chloramphenicol, 6%; erythromycin, 11%; tetracycline, 15%; and trimethoprim-sulfamethoxazole, 42%). No high level resistance was seen to third generation cephalosporins, but 17 of 109 penicillin-resistant isolates tested (16%) displayed intermediate resistance to cefotaxime. Rates of antibiotic resistance varied between States, with the lowest rates in Tasmania. CONCLUSIONS: Antibiotic resistance levels in S. pneumoniae are increasing in Australia and high level penicillin resistance is being encountered for the first time (including in invasive strains). This will lead to an increasing number of therapeutic dilemmas and possible therapeutic failures, especially important in meningitis.

Adolescent↗
PubMed1997

Cellular determinants of resistance to indolocarbazole analogue 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (NB-506), a novel potent topoisomerase I inhibitor, in multidrug-resistant human tumor cells.

Membrane protein-associated alterations in cellular drug accumulation have been recently implicated in resistance to topoisomerase I (TOP-I)-interactive drugs. The present study investigated the cellular determinants of resistance to the indolocarbazole compound NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7(6H)-dione], a structurally novel TOP-I-interactive drug, in parental and multidrug-resistant tumor cells expressing either the P-170 glycoprotein (Pgp170) or multidrug resistance protein (MRP). MRP-expressing 250-fold doxorubicin-resistant human fibrosarcoma HT1080/DR4 tumor cells were drug sensitive to NB-506 and camptothecin (CPT) (resistance factor: 0.7 and 0.8, respectively) with no alterations of TOP-I parameters including DNA relaxation, expression of TOP-I protein and mRNA. In contrast, doxorubicin-resistant human ovarian A2780/Dx5 tumor cells [pgp170 phenotype] were 6.2-fold resistant to NB-506, whereas resistance to CPT was 2.6-fold. HPLC analysis of cellular NB-506 accumulation showed no significant differences between A2780 and A2780/Dx5 cells (peak intracellular concentrations after 120-min exposure to 10 microM NB-506: 400+/-85.0 and 352+/-95.1 nmol NB-506/mg protein, respectively). However, resistant A2780/Dx5 cells expressed a lower amount of TOP-I mRNA and 29% protein levels of TOP-I compared to parental A2780 cells, resulting in decreased TOP-I catalytic activity (3.17+/-0.02 vs. 1.16+/-0.15 rel.U/microg nuclear protein) and reduced induction of NB-506-mediated cleavable complex formation in A2780/Dx5 cells. Furthermore, the lower induction of NB-506-induced protein-linked DNA breaks (PLDB) in A2780/Dx5 cells correlated with significantly decreased DNA 12.2-440 kb size fragmentation in these cells. The present study demonstrates that expression of MRP and Pgp170 does not confer resistance to NB-506. Resistance to indolocarbazole substance NB-506 in A2780/Dx5 cells was only related to downregulation of TOP-I associated with lower induction of cleavable complex formation and DNA fragmentation. The data reported herein may indicate that the new indolocarbazole compound NB-506 has potent antitumor efficacy in membrane-associated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Reduction of doxorubicin resistance by tetrandrine and dauricine in harringtonine-resistant human leukemia (HL60) cells.

AIM: To study whether tetrandrine (Tet) and dauricine (Dau) can reduce doxorubicin (Dox) resistance in the harringtonine (Har)-resistant human leukemia cells. METHODS: The drug cytotoxities were determined by counting cell numbers and colony formation. Cell cycle phases were assayed by flow cytometry, Dox contents were quantified by Dox fluorescence. RESULTS: The non-cytotoxic concentrations of Tet and Dau potentiated the growth-inhibitory actions of Dox in the Har-resistant HL60 cells. The colony formation effiencies were reduced from 60% by Dox to 0.2% by Tet + Dox and 9.2% by Dau + Dox. Retardation of the G2M phase cells was increased. But Tet and Dau did not potentiate Dox cytotoxities in the sensitive HL60 cells. Dox accumulation in the Har-resistant HL60 cells treated by Tet was increased. CONCLUSION: Dox resistance in the Har-resistant HL60 cells treated by Tet or Dau was reduced, due to the increase of Dox accumulation in the cells. One of the mechanisms of multidrug resistance in tumor cells is overexpression of cell membrane glycoproteins, termed P-glycoprotein (PGP). PGP pumps antitumor drugs out of tumor cells, causing drug resistance. Calcium antagonists and some calmodulin inhibitors such as verapamil, nifedepine, trifluorapine have effect on reversion of drug resistance, binding directly to PGP, but side effect of them is intolerable in clinical use. So searching for other potentiators to overcome drug resistance may be another avenue. Tetrandrine (Tet) effectively circumvented the resistance of Chinese hamster ovary cells to doxorubicin (Dox). Dauricine (Dau) is a bisbenzylisoquinoline alkaloid from Stephaia tetrandra. In this paper we studied whether Tet and Dau could reduce Dox resistance in the harringtonine (Har)-resistant human leukemia 60 (HL60) cells.

Alkaloids↗
PubMed1989

Reversal of drug resistance by erythromycin: erythromycin increases the accumulation of actinomycin D and doxorubicin in multidrug-resistant cells.

Development of resistance to one type of lipophilic chemotherapeutic drug often leads to resistance to other, structurally unrelated, lipophilic drugs. This suggests that non-toxic lipophilic agents may interfere with and reverse drug resistance by saturating the pathway through which multidrug-resistant (MDR) cells protect themselves against cytotoxic drugs. The lipophilic antibiotic, erythromycin, can significantly reverse the resistance of MDR WEHI 164 murine fibrosarcoma cells to the chemotherapeutic drugs, doxorubicin and actinomycin-D. The MDR cells showed an approximately 10-fold higher expression of the P-glycoprotein than the drug-sensitive parental cells from which the resistant cells were derived. The accumulation of actinomycin-D and doxorubicin was much lower in the drug-resistant cells than in the sensitive parental cells. The concentrations of erythromycin which reversed the drug resistance of the MDR cells increased the accumulation of actinomycin-D and doxorubicin in these cells to a level comparable to that observed in the sensitive parental cells. Our data suggest that erythromycin reverses drug resistance by saturating the drug-binding sites on the P-glycoprotein, thereby reducing the capacity of this protein to pump drugs out of resistant cells. Some of our MDR cells have also become more resistant to tumour necrosis factor (TNF). However, erythromycin did not reverse TNF resistance, suggesting that the mechanisms of multi-drug and TNF resistance are different. TNF did not influence drug accumulation in MDR cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗