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Objectives: Characterization of non-Escherichia coli NDM-carrying Enterobacteriaceae in the poultry production environment., Methods: A total of 36 NDM-positive Enterobacteriaceae (22 Klebsiella pneumoniae, 13 Enterobacter cloacae and 1 Salmonella enterica) were isolated from a chicken farm and WGS was conducted using Illumina Hiseq2500. The genomic characterization of the isolates acquired through WGS analysis included the genomic context-flanking blaNDM genes, MLST, the antibiotic resistance genes (ARGs) and replicon types of plasmids. WGS information for another 73 K. pneumoniae isolates from different sources was retrieved from GenBank and then combined with isolates in this study for comparative genomic and phylogenetic analysis., Results: Three types of genetic environment carrying blaNDM were identified in 36 non-E. coli Enterobacteriaceae isolates. Sequence comparison analysis indicated these genetic environments were completely identical to our previous findings. WGS further revealed three major types of plasmids (IncFIB, IncX3 and IncFII) from these isolates and the phylogenetic analysis suggested several K. pneumoniae isolates with ST11, ST37 and ST147 from the commercial chicken farm that were closely related to isolates of human origin., Conclusions: The blaNDM-harbouring genetic contexts were identified not only in E. coli, but also in K. pneumoniae, E. cloacae and S. enterica, which may indicate that blaNDM has been widely disseminated to non-E. coli Enterobacteriaceae species in animal farms. The close relationship of K. pneumoniae isolates from different origins suggests they could serve as a key vehicle for the transfer of ARGs between humans and food animal production environments., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A Cryptococcus neoformans var. grubii strain, NUBS14020, was the first fluconazole (FLZ)-resistant strain isolated from a feline cryptococcosis. Subsequent work demonstrated that multi-azole-resistant strains are readily isolated from FLZ-resistant strains by culturing in medium containing voriconazole (VRZ). The resulting clones were assessed for mutation and expression of known target genes, including the loci encoding lanosterol 14-α demethylase (ERG11), an ATP-binding cassette (ABC) transporter (AFR1), or a multidrug efflux pump (MEP); mutation and/or overexpression of these genes is known to be associated with azole resistance. We also examined the interaction between an efflux blocker (FK506, calcineurin inhibitor) and VRZ in the multi-azole-resistant strains. The ERG11 genes from multi-azole-resistant strains encoded a protein with a G344S substitution. Expression levels of AFR1, ERG11, and MEP in the multi-azole-resistant strains were not higher than those in the VRZ-susceptible parent strain (NUBS14020) when cultured in Sabourad's dextrose broth containing VRZ. Synergistic effects between FK506 and VRC were observed in all of the multi-azole-resistant strains. The minimal inhibitory concentrations (MICs) of the combination of VRZ and FK506 in multi-azole-resistant strains were 4 to 8 times lower that the MICs of VRZ alone. To the best of our knowledge, this work represents the first report that multi-azole-resistant strains of C. neoformans encode a G344S substitution in Erg11p. Further investigation will be needed to determine the mechanism of multi-azole resistance in C. neoformans, given that feline cryptococcosis due to multi- azole-resistant strains is readily transmitted from cats to humans., (© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: CmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics in Campylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA)., Methods: Eighteen PNAs were designed to bind to the translational initiation regions of cmeABC, spanning the ribosome-binding site (RBS) and the start codon of the cmeABC genes. Campylobacter jejuni was treated with CmeABC-specific PNAs (CmeABC-PNAs) at various concentrations and subjected to western blotting to measure changes in the level of CmeABC expression. The MICs of ciprofloxacin and erythromycin were measured to evaluate the impact of CmeABC knockdown on antibiotic susceptibility., Results: While antisense PNA significantly affected CmeA and CmeB expression, interestingly, CmeC expression was not altered by any of the CmeC-PNAs used in this study. A CmeA-PNA targeting the RBS of cmeA and its upstream region reduced CmeA expression most efficiently, and CmeB expression was most significantly decreased by PNA binding to the RBS of cmeB and its downstream region. CmeA- and CmeB-PNAs increased the susceptibility of C. jejuni to ciprofloxacin and erythromycin in proportion to the inhibition levels observed in western blotting., Conclusions: The cmeA gene is the best target to knockdown CmeABC with antisense PNA. The RBS is the major target for the PNA-mediated antisense inhibition of CmeABC. However, regions in its vicinity also significantly influence the effectiveness of the PNA-based knockdown of CmeABC.

Background: Doxycycline is used in combination with quinine for malaria treatment or alone for malaria chemoprophylaxis. However, the occurrence of malaria after doxycycline chemoprophylaxis has been reported. Identification of genetic determinants that contribute to the susceptibility of Plasmodium falciparum to doxycycline will be important for the detection and surveillance of doxycycline resistance., Methods: Sequence analysis of 11 genes (pftufA, pfEF-TS, pfmdt, pftetQ, pfrps3, pfrps7, pfrps8, pfrps9, pfrps11, pfrps14, and pfrps17) and evaluation of pfmdt and pftetQ copy numbers by quantitative real-time polymerase chain reaction were conducted in 90 African P. falciparum isolates that were obtained from 14 countries and that belonged to phenotypic groups differing in their doxycycline median inhibitory concentrations., Results: We found that pfmdt copy number of >1 (adjusted odds ratio [OR], 7.09 [95% confidence interval {CI}, 1.58-31.82]; P=.011), pftetQ copy number of >1 (adjusted OR, 5.23 [95% CI, 1.06-25.77]; P=.042), and KYNNNN amino acid motif repeats of <3 (adjusted OR, 3.00 [95% CI, 1.02-8.86]; P=.046) were independently associated with decreased susceptibility to doxycycline., Conclusions: Our findings suggest that pfmdt and pftetQ copy numbers and pftetQ sequence polymorphisms are potential molecular markers of decreased in vitro susceptibility to doxycycline in African P. falciparum isolates.

We describe here the results of an analysis of Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene copy number from 440 field isolates from Papua New Guinea. No multiple copies of the gene were found, which corresponds to the lack of usage of mefloquine. These data extend regional knowledge about the distribution of multidrug-resistant P. falciparum.

Amodiaquine/artesunate and artemether/lumefantrine are currently implemented as antimalarial drugs in Africa. In this study, we assessed Plasmodium falciparum pfmdr1 alleles suggesting reduced sensitivity to amodiaquine/artesunate (86Y-184Y-1246Y) and artemether/lumefantrine (184F and 86N-184F-1246D) in 111 African isolates imported to Germany between 1997 and 2005. pfmdr1 86Y-184Y-1246Y occurred less frequently in West African isolates (2.5%) than in East African parasites (45.8%) (P<0.001) and less frequently in infections contracted in countries currently implementing amodiaquine/artesunate (5.7%) compared with countries with an artemether/lumefantrine policy (20.8%) (P=0.03). pfmdr1 184F showed an opposite pattern. pfmdr1 alleles reveal significant differences between West and East Africa, supporting the current implementation of first-line antimalarials.

The global dissemination of drug-resistant Plasmodium falciparum is spurring intense efforts to implement artemisinin (ART)-based combination therapies for malaria, including mefloquine (MFQ)-artesunate and lumefantrine (LUM)-artemether. Clinical studies have identified an association between an increased risk of MFQ, MFQ-artesunate, and LUM-artemether treatment failures and pfmdr1 gene amplification. To directly address the contribution that pfmdr1 copy number makes to drug resistance, we genetically disrupted 1 of the 2 pfmdr1 copies in the drug-resistant FCB line, which resulted in reduced pfmdr1 mRNA and protein expression. These knockdown clones manifested a 3-fold decrease in MFQ IC(50) values, compared with that for the FCB line, verifying the role played by pfmdr1 expression levels in mediating resistance to MFQ. These clones also showed increased susceptibility to LUM, halofantrine, quinine, and ART. No change was observed for chloroquine. These results highlight the importance of pfmdr1 copy number in determining P. falciparum susceptibility to multiple agents currently being used to combat malaria caused by multidrug-resistant parasites.

The 3435C-->T polymorphism at the multidrug resistance gene 1 (MDR1) was examined in 74 patients with human immunodeficiency virus who initiated atazanavir therapy. The MDR1 genotype distribution at position 3435 was 28% CC, 45% CT, and 27% TT. Plasma levels of atazanavir were significantly higher in patients with genotype CC than in those with CT or TT, and bilirubin levels correlated with atazanavir concentrations.

The aim of this study was to examine the effect and mechanism of green tea polyphenols (TP) on reversal of multidrug resistance (MDR) in a carcinoma cell line. Using the MTT assay, TP was examined for its modulating effects on the drug-resistant KB-A-1 cells and drug-sensitive KB-3-1 cells. When 10 microg mL(-1) (-)-epigallocatechin gallate (EGCG) or 40 microg mL(-1) TP were present simultaneously with doxorubicin (DOX), the IC50 of DOX on KB-A-1 cells decreased from 10.3 +/- 0.9 microg mL(-1) to 4.2 +/- 0.2 and 2.0 +/- 0.1 microg mL(-1), respectively. TP and EGCG enhanced the DOX cytotoxicity on KB-A-1 cells by 5.2- and 2.5-times, respectively, but did not show a modulating effect on KB-3-1 cells. This indicated that both TP and EGCG had reversal effects on the MDR phenotype in-vitro. A KB-A-1 cell xenograft model was established, and the effect of TP on reversing MDR in-vivo was determined. Mechanistic experiments were conducted to examine the uptake, efflux and accumulation of DOX. Cloning and expression of the nucleotide binding domain of the human MDR1 gene in Escherichia coli was established, and by using colorimetry to examine the activity of ATPase to hydrolyse ATP, the ATPase activity of target nucleotide binding domain protein was determined. TP exerted its reversal effects through the inhibition of ATPase activity, influencing the function of P-glycoprotein, and causing a decreased extrusion of anticancer drug and an increased accumulation of anticancer drug in drug resistant cells. Using reverse transcription-polymerase chain reaction, the inhibitory effect of TP on MDR1 gene expression was investigated. Down-regulation of MDR1 gene expression was the main effect, which resulted in the reversal effect of TP on the MDR phenotype. TP is a potent MDR modulator with potential in the treatment of P-glycoprotein mediated MDR cancers.

LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA&#95;2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA&#95;2.T-A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA&#95;2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50-100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA&#95;2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.

Nonphysiological overexpression of the ABC transporter P-glycoprotein (P-gp), which is encoded by MDR1, has been associated with reduced susceptibility to human immunodeficiency virus (HIV) type 1 infection in vitro. We analyzed (1) the expression and genotype of MDR1 and their relationship to HIV-1 permissiveness of CD4+ T cells from 128 healthy blood donors and (2) the role that alleles of MDR1 exons 21 and 26 play in modifying disease progression in 411 HIV-1-infected individuals. Differences in physiological levels of MDR1 expression did not modify HIV-1 infection in vitro, nor did MDR1 alleles and haplotypes significantly influence either permissiveness to infection in vitro or disease progression in vivo before the initiation of treatment.

Objectives: To investigate possible molecular mechanisms of azole resistance among fluconazole-susceptible bloodstream isolates of Candida albicans that displayed the trailing growth phenomenon, and to compare these isolates with bloodstream and mucosal isolates that showed reduced susceptibilities to fluconazole., Methods: Twelve C. albicans isolates-seven trailing and five susceptible dose dependent (SDD) or resistant (R)-were screened for ERG11 mutations by DNA sequencing and quantification of ERG11, CDR1 and MDR1 expression by RT-PCR using the LightCycler high-speed PCR system., Results: SDD and R isolates possessed more homozygous ERG11 mutations than did the trailing isolates. Two of these, V404I and V509M, have not been described previously and were found exclusively in fluconazole SDD and R isolates. Quantification of ERG11 expression revealed that both trailing and SDD and R isolates were capable of ERG11 up-regulation in response to fluconazole, although the SDD and R isolates showed maximal up-regulation at higher fluconazole concentrations. Quantification of CDR1 and MDR1 revealed that all isolates, regardless of in vitro fluconazole response, were capable of CDR1 and MDR1 up-regulation following fluconazole exposure. Furthermore, the SDD and R isolates expressed higher constitutive levels of CDR1 and MDR1 or CDR1, respectively, in the absence of drug compared with trailing isolates., Conclusions: Trailing isolates, although susceptible to fluconazole, express the same molecular mechanisms as SDD and R isolates following fluconazole exposure but regulate them differently.

Aims: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype., Materials and Methods: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined., Results: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n = 20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P < 0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n = 20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (P < or = 0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline., Conclusions: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.

Intratumoral injection of controlled-release microsphere formulations of anticancer compounds has the potential to selectively increase tumour exposure to drugs. This work aimed to evaluate the therapeutic effect and toxicity of microsphere formulations containing the anticancer drug, doxorubicin, in a murine tumour model. The effect of co-administration of verapamil, a P-glycoprotein modulator or chemosensitizer, was investigated. Initial in-vitro studies confirmed the ability of verapamil to enhance the accumulation of both doxorubicin and [(99mTc)]sestamibi, also a P-glycoprotein substrate, in EMT6 murine breast sarcoma cells and a doxorubicin-selected multidrug-resistant variant, EMT6/AR1.0. Ex-vivo studies using confocal microscopy demonstrated release of doxorubicin from microspheres and diffusion of the drug through tissue. For in-vivo studies, EMT6 and EMT6/AR1.0 cells were grown in BALB/c mice. Following intratumoral injection of doxorubicin-loaded microspheres, alone or in combination with verapamil-loaded microspheres, the tumour diameter was measured serially as an indication of therapeutic effect, while the weight, appearance, and behaviour of the mice were monitored as an indication of general toxicity. Intratumoral injections of doxorubicin-loaded microspheres were tolerated much better than systemic administration of equivalent drug concentrations. There was a modest (up to 34%) delay of tumour growth compared with groups receiving no treatment or blank microspheres. Co-injection of verapamil microspheres with doxorubicin microspheres produced a moderate increase in toxicity but no further delay in tumour growth. Controlled-release microsphere formulations of anticancer agents administered intratumorally were an efficient way to deliver high drug doses to the tumour with little systemic toxicity.

We describe a mutant of Streptococcus pyogenes NCTC 8198 with a multidrug efflux phenotype. A mutant selected with ethidium bromide showed a four-fold rise in MIC of norfloxacin, a 16-fold rise in MIC of ethidium bromide and an eight-fold rise in MIC of acriflavine when compared with the parent strain. The MICs were unaffected by the efflux pump inhibitors reserpine, rescinnamine and verapamil. The mutant's ethidium bromide MIC was reduced two-fold by norfloxacin. Ethidium bromide accumulation after 10 min was 58% lower in the mutant compared with the parent. This difference was not affected by carbonyl cyanide m-chlorophenylhydrazone.

Study of the multidrug resistance phenomenon in tumor cell lines has led to the discovery of the product of the multidrug resistance (MDR) type 1 genes, the plasma membrane P-glycoprotein (P-gp) that functions as an energy-dependent pump for the efflux of diverse anticancer drugs. P-gp was also recently identified in normal epithelial cells with secretory/excretory functions and in the endothelial cells of the capillary blood vessels in the brain and the testis. These endothelial cells are key elements of the blood-brain and blood-testis barriers, respectively. The aim of this study, in the rat, mouse, guinea pig, and human, was to determine whether testicular cells other than the capillary endothelial cells could express MDR type I genes. Immunohistochemistry on testicular sections revealed that P-gp is present in interstitial cells in the mouse, rat, and human testes, in early and late spermatids in guinea pig testis, and in late spermatids in the rat, mouse, and human. Reverse transcription-polymerase chain reaction analysis on isolated mouse, rat, and human cells showed that all somatic testicular cells (Leydig cells, macrophages, peritubular cells, and Sertoli cells) and the cytoplasmic lobes from rat late spermatids expressed MDR type I mRNAs, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. An ontogenesis study in the mouse reveals that type I MDR gene expression begins at 13.5 days postcoitum at the time when the seminiferous cords and the blood vessels appear and are maintained thereafter. Finally, two functional tests on isolated rat cells, the doxorubicin and rhodamine uptake assays, demonstrated that rat testicular macrophages, Leydig cells, peritubular cells, and Sertoli cells displayed a multidrug-resistance activity, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. Western blot experiments have revealed that a P-gp of 175 kDa is present in the human testis as well as in the rat Leydig cells, testicular macrophages, peritubular cells, and Sertoli cells, but is absent in spermatogonia, spermatocytes, and early spermatids. We conclude that P-gp is involved in the self-protection of the somatic cells and is most probably one of the molecules that confers its functionality to the blood-testis barrier. The absence of expression of MDR type I genes in mitotic and meiotic germ cells probably explains their particular vulnerability to various anticancer drugs. In contrast, expression of the P-gp in the haploid cells most likely reflects the ability of spermatozoa to assume their own antidrug defense.

Renal transplant recipients treated with cyclosporine A (CsA) and FK506 (tacrolimus) develop signs of hypoaldosteronism despite normal plasma aldosterone levels, suggesting a relative resistance of the distal nephron to aldosterone action. To examine the effects of immunosuppressants on human MR (hMR) function, we established the M cell model, renal tubular cells stably transfected with hMR. Upon CsA and FK506 administration, hMR mRNA levels and aldosterone binding in M cells remained unchanged (maximum number of sites, approximately 80 fmol/mg protein; K(d) = approximately 1 nM). Aldosterone-dependent intracellular localization of green fluorescent protein-hMR was not affected by immunosuppressants. A major impact of CsA or FK506 on the multidrug resistance gene product in cellular accumulation of aldosterone was also excluded. In contrast, aldosterone-stimulated hMR transcriptional activity was reduced to 53 +/- 11.2% (P < 0.03) after pretreatment of M cells for 3 d with CsA and to 71 +/- 9.6% (P < 0.05) after pretreatment with FK506. These effects were both time and concentration dependent (IC(50) of CsA, 10(-6) M; IC(50) of FK506, 10(-5) M) and needed at least 2 d to develop. Such an inhibitory effect does not depend on the N-terminal part of hMR, as CsA also reduced transcriptional activity of a 1-453 deletion mutant of hMR. Our results demonstrate that immunosuppressants inhibit hMR transcriptional activity without affecting hMR expression, aldosterone binding properties, and hMR nucleocytoplasmic trafficking. They suggest that ion transport alterations in renal graft recipients are in part induced by impaired hMR function.

Naturally occurring hammerhead ribozymes are produced by rolling circle replication followed by self-cleavage. This results in monomer-length catalytic RNAs which have self-complementary sequences that can occupy their trans -binding domains and potentially block their ability to cleave other RNA strands. Here we show, using small self-processed ribozymes, that this self-binding does not necessarily inhibit trans -cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt circular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Rolling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of complementarity or bind itself with 4+5 nt of self-complementarity by folding back its ends to form hairpins. Kinetics of trans -cleavage of short complementary and mismatched RNAs were measured under multiple turnover conditions, in comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-complementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k (cat)/ K (m)almost the same as MDR40, but with discrimination against mismatches up to 20 times greater. Based on folding predictions, a second self-processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for increasing sequence specificity of ribozymes.

Multidrug resistance-associated protein gene MRP/MRP1, and its family genes, including MRP2/cMOAT, have been isolated and characterized. These ATP-binding cassette (ABC) superfamily transporter genes are differentially expressed in various normal tissues and multidrug-resistant cell lines. Transfection of MRP/MRP1 and MRP2/cMOAT cDNA confers drug resistance on different spectra of anticancer agents from that of MDR1 coding P-glycoprotein. Although it remains unclear how MRP/MRP1 and related family genes are specifically involved in drug resistance in clinical cancers, current knowledge of the MRP subfamily suggests the importance of this class of transporters as a molecular target for drug sensitivity to anticancer agents.

We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans.

A series of newly developed paclitaxel analogues have been tested for their growth inhibitory activity on two human breast cancer cell lines, one of which expresses the MDR (multidrug resistance) phenotype. Paclitaxel (taxol) was used as a reference compound. Three new classes of taxanes were analyzed: the cephalomannine compounds, the pyrazoline derivatives and the seco-derivatives. Our results demonstrated an increased antiproliferative activity of pyrazoline derivatives on drug-resistant cancer cells with respect to paclitaxel. These compounds were able to block MDR-bearing MCF-7 ADRr cells in the G2/M phase of cell cycle and, consequently, induce programmed cell death. In keeping with the antiproliferative effects, cells treated with paclitaxel derivatives showed a more pronounced cell cycle arrest than the parent compound paclitaxel. Also, apoptotic cell death, calculated as a percent of DNA fragmentation, occurred to a greater extent in cells exposed to pyrazoline derivatives. The development of new paclitaxel analogues with greater antitumour activity on MDR-positive cells may be useful in selecting new taxanes effective on resistant tumors.

To assess the level of resistance to chloroquine (CQ) of Plasmodium falciparum in The Gambia in 1995-1996 we measured susceptibility in vivo by quantifying parasitaemia of children with mild malaria on days 4 and 8 after treatment. Pretreatment blood samples were used for susceptibility testing in vitro by the World Health Organization microtest and the prevalence of the tyrosine (tyr)86 allele of the Pfmdr1 gene was assessed by the polymerase chain reaction and restriction fragment length polymorphism analysis. Seven of 42 children (17%) treated with CQ remained parasitaemic on day 4 and required a change of antimalarial treatment. Susceptibility assays in vitro were performed on 50 P. falciparum isolates obtained from eligible children before treatment; 36 (72%) were resistant to CQ (> or = 1.6 mumol/L). The median minimum inhibitory concentration (MIC) of artemether was 3.38 nmol/L (range 0.42-13.51 nmol/L) and the median MIC of dihydroartemisinin was 0.88 nmol/L (range 0.22-14.04 nmol/L). Susceptibility in vitro to CQ and the Pfmdr1 genotype were determined for 31 fresh isolates. The allele was present in 12 of 22 isolates found to be resistant to CQ in vitro, but in none of the 9 isolates which were susceptible (Fisher's exact test, P = 0.005). All P. falciparum isolates with the tyr86 allele were CQ resistant in vitro, but since only half of all resistant isolates contained the allele, additional explanations for CQ resistance are required.