Analysis of Pneumocystis jirovecii DHPS alleles implicated in sulfamethoxazole resistance using an Escherichia coli model system

Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP). Drug treatment failure has been associated epidemiologically with point mutations in the gene for dihydropteroate synthase which is part of a gene that encodes three covalently linked enzymes involved in folic acid synthesis (FAS). The evaluation of whether mutations found in P. jirovecii FAS lead to sulfa drug resistance is hampered by the lack of a culture system for P. jirovecii as well as the failure of P. jirovecii FAS to complement in a heterologous system. Therefore, we chose to model the P. jirovecii mutations in the Saccharomyces cerevisiae FAS protein (encoded by FOL1) via its expression in Escherichia coli. An optimized drug diffusion assay was used to evaluate the FAS mutants against 15 sulfa drugs. It was established that the single amino acid substitution, P599S, in the (DHPS) domain of FAS led to sulfa drug resistance, whereas the T597A substitution led to increased sensitivity. The presence of both mutations (T597A and P599S) was cooperative and led to increased sulfa drug resistance. Analysis of a novel double mutant, (T597V P599S) was found to have significantly higher sulfa drug resistance than the T597A P599S mutant. These data suggest that further amino acid substitutions may lead to the evolution of higher sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine and sulfathiazole) were identified that had higher inhibitory potential than sulfamethoxazole, which is currently the preferred treatment for PCP.     

Inhibition studies of sulfonamide-containing folate analogs in yeast

In the folate biosynthetic pathway, sulfa drugs (sulfonamides and sulfones) compete with the natural substrate, para-aminobenzoate (pABA) causing depletion of dihydrofolate (DHF) and subsequent growth inhibition. The sulfa drugs condense with 2-amino-4-hydroxy-6-hydroxymethyl-7,8 dihydropteridine pyrophosphate (DHPPP) forming sulfa-dihydropteroate (sulfa-DHP). Here evidence is presented using yeast that such dihydropteroate (DHP) analogs are inhibitory through competition with DHF. Two folate synthesis mutants, with respective dihydrofolate synthase (DHFS) and dihydropteroate synthase (DHPS) deletions and requiring DHF for growth were exposed to sulfa drugs. The DHFS knockout mutant was inhibited, but the DHPS knockout mutant that was incapable of forming sulfa-DHP was insensitive. Such sulfa-DHP compounds were chemically synthesized and shown to be inhibitory in vivo by competing with DHF, but in vitro assays with double the concentration of the sulfa-DHP to DHF showed no inhibition of dihydrofolate reductase (DHFR). Sequence analysis of resistant mutants obtained in the presence of sulfa drugs showed no changes in DHFR, or DHPS, unlike previously found antifolate-resistant mutants. The diamino derivatives, which are precursors of the sulfa-DHP, were found to be DHFR inhibitors. These results suggest that a new class of drugs, based on DHP analogs, could be investigated.     

Pneumocystis jirovecii dihydropteroate synthase genotypes in French patients with pneumocystosis: a 1998-2001 prospective study.

Dihydropteroate synthase gene (DHPS) mutations at codons 55 and 57 have been associated with sulfa/sulfone resistance in Pneumocystis jirovecii strains from patients who previously received prophylaxis. To evaluate the prevalence of these mutations, a portion of P. jirovecii DHPS gene was analysed using PCR combined with restriction fragment length polymorphism (RFLP) analysis in 92 bronchoalveolar fluid samples collected between January 1998 and September 2001 from French patients with pulmonary pneumocystosis (PCP). Seventy-six samples contained the wild-type DHPS genotype (82.6%) and 16 contained a mutant genotype (17.4%). Twelve out of the 16 isolates with a mutant DHPS genotype corresponded to patients who had never received sulfa or sulfone prophylaxis, suggesting that DHPS mutants may be acquired de novo. There was no significant difference in favourable or adverse outcome in PCP caused by the wild or mutant DHPS genotypes (P = 0.34).     

Absence of mutations associated with sulfa resistance in Pneumocystis carinii dihydropteroate synthase gene from non-human primates.

The dihydropteroate synthase (DHPS) gene from Pneumocystis carinii isolated from non-human primates was amplified using a polymerase chain reaction (PCR) and sequenced to analyse point mutations associated with sulfa resistance. P. carinii DHPS gene amplification was obtained from eight lung samples from five New World primate species and one Old World primate species. None of the animals had been exposed to sulfa drugs and only the wild-type P. carinii DHPS sequence at codons 55 and 57 was observed. These data support the hypothesis that high rates of DHPS mutants in P. carinii f. sp. hominis have arisen with increased use of sulfa drugs for P. carinii pneumonia prophylaxis.     

Linking Pneumocystis jiroveci sulfamethoxazole resistance to the alleles of the DHPS gene using functional complementation in Saccharomyces cerevisiae

Curative and prophylactic therapy for Pneumocystis jiroveci pneumonia relies mainly on co-trimoxazole, an association of trimethoprim and sulfamethoxazole (SMX). SMX inhibits the folic acid pathway through competition with para-aminobenzoic acid (pABA), one of the two substrates of the dihydropteroate synthase (DHPS), a key enzyme in de novo folic acid synthesis. The most frequent non-synonymous single nucleotide polymorphisms (SNPs) in P. jiroveci DHPS are seen at positions 165 and 171, the combination leading to four possible different genetic alleles. A number of reports correlate prophylaxis failure and mutation in the P. jiroveci DHPS but, because of the impossibility of reliably cultivating P. jiroveci, the link between DHPS mutation(s) and SMX susceptibility is not definitively proven. To circumvent this limitation, the yeast Saccharomyces cerevisiae was used as a model. The introduction of the P. jiroveci DHPS gene, with or without point mutations, directly amplified from a clinical specimen and cloned in a centromeric plasmid into a DHPS-deleted yeast strain, allowed a fully effective complementation. However, in the presence of SMX at concentrations >250 mg/L, yeasts complemented with the double mutated allele showed a lower susceptibility compared with strains complemented with either a single mutated allele or wild-type alleles. These results confirm the need for prospective study of pneumocystosis, including systematic determination of the DHPS genotype, to clarify further the impact of mutations on clinical outcome. Additionally, the S. cerevisiae model proves to be usefulfor the study of still uninvestigated biological properties of P. jiroveci.     

Relationship between mutations in dihydropteroate synthase of Pneumocystis carinii f. sp. hominis isolates in Japan and resistance to sulfonamide therapy

We examined mutations in the dihydropteroate synthase (DHPS) genes of Pneumocystis carinii f. sp. hominis (P. carinii) strains isolated from 24 patients with P. carinii pneumonia (PCP) in Japan. DHPS mutations were identified at amino acid positions 55 and/or 57 in isolates from 6 (25.0%) of 24 patients. The underlying diseases for these six patients were human immunodeficiency virus type 1 infection (n = 4) or malignant lymphoma (n = 2). This frequency was almost the same as those reported in Denmark and the United States. None of the six patients whose isolates had DHPS mutations were recently exposed to sulfa drugs before they developed the current episode of PCP, suggesting that DHPS mutations not only are selected by the pressure of sulfa agents but may be incidentally acquired. Co-trimoxazole treatment failed more frequently in patients whose isolates had DHPS mutations than in those whose isolates had wild-type DHPS (n = 4 [100%] versus n = 2 [11.1%]; P = 0.002). Our results thus suggest that DHPS mutations may contribute to failures of co-trimoxazole treatment for PCP.     

Genetic basis for sulfonamide resistance in Bacillus anthracis

Natural resistance of field strains of Bacillus anthracis to drugs from the sulfonamide class of antimicrobials that act by inhibiting dihydropteroate synthase (DHPS) has been reported. Though the structure of B. anthracis DHPS has been determined, its connection to the apparent intrinsic sulfonamide resistance of the bacterium has not been established. The aim of this study was to determine if a connection exists between DHPS and the observed sulfonamide resistance of B. anthracis. Microdilution broth assays verified that B. anthracis Sterne is highly resistant to a variety of sulfonamides with minimum inhibitory concentrations (MICs) exceeding 1250 microg/ml. A putative gene encoding DHPS (folP) was amplified from B. anthracis Sterne chromosomal DNA by polymerase chain reaction (PCR) and cloned. Sequence comparisons showed 100% identity with DHPSs from published genome sequences for various strains of B. anthracis. Additionally, expression of folP in B. anthracis Sterne was confirmed. Functionality of the B. anthracis DHPS was confirmed by complementation of an Escherichia coli folP deletion mutant as well as a standard enzyme assay. Concomitant transfer of high level sulfonamide resistance to this mutant along with increased sulfonamide IC(50)values for purified B. anthracis DHPS links DHPS to sulfonamide resistance in B. anthracis. These findings lay the groundwork that will aid future development of antimicrobics that target DHPS to treat anthrax infections.     

Genotyping of Pneumocystis jiroveci pneumonia in Italian AIDS patients. Clinical outcome is influenced by dihydropteroate synthase and not by internal transcribed spacer genotype

BACKGROUND: Two Pneumocystis jiroveci independent genomic regions, internal transcribed spacer (ITS) 1 and ITS2, and dihydropteroate synthase (DHPS) gene have been used for typing a cohort of HIV-infected Italian patients with P jiroveci pneumonia (PcP). METHODS: Bronchoalveolar lavage samples isolated from 207 HIV-infected adults were ITS and DHPS genotyped by DNA sequencing and by restriction fragment length polymorphism analysis, respectively. Mutant DHPS samples were cloned and ITS typed. Data on severity, treatment, and outcome of PcP were obtained by chart review. RESULTS: High diversity with 46 different ITS genotypes was observed. At the DHPS locus, 9.1% of samples analyzed were found to be mutated. A correlation was observed between DHPS mutants and greater severity of PcP, as defined by higher lactate dehydrogenase (P = 0.015) and need for intubation (P = 0.002), and worse outcomes, as defined by failure of sulfa treatment (P = 0.04), death, and/or relapse of PcP (P = 0.008). There was a significant difference in ITS genotype patterns between DHPS wild-type and mutants (P = 0.028). CONCLUSIONS: The present data suggest the absence of a correlation between P jiroveci ITS types and specific clinical characteristics. DHPS mutations correlate with possible failure of anti-P jiroveci sulfa therapy, and a trend of association is shown between DHPS mutations and some clinical PcP features.     

High Prevalence of Dihydropteroate Synthase Mutations in Pneumocystis jirovecii Isolated from Patients with Pneumocystis Pneumonia in South Africa

Pneumocystis jirovecii pneumonia (PCP) is an important cause of morbidity and mortality in immunocompromised patients. Sulfa-containing drugs are used for the treatment and prophylaxis of PCP. Mutations in the P. jirovecii fas gene, which encodes dihydropteroate synthase (DHPS), are associated with prior exposure to sulfa drugs, and their appearance suggests the emergence of variants with reduced sulfa susceptibility. The present study examined the prevalence of DHPS mutations in P. jirovecii strains isolated from South African patients with PCP. P. jirovecii infection was investigated by immunofluorescence microscopy and quantitative real-time PCR with respiratory specimens from 712 patients (93% of whom were >15 years of age) with suspected PCP consecutively received for the detection of P. jirovecii over 1 year. PCR amplification and sequencing of the DHPS fas gene was attempted with DNA from the P. jirovecii-positive samples. P. jirovecii infection was confirmed by immunofluorescence microscopy in 168/712 (24%) of the patients. Carriage of the fungus was revealed by real-time PCR in 17% of the patients with negative microscopy results. The P. jirovecii fas gene was successfully amplified from specimens from 151 patients and sequenced. Mutations resulting in the Thr55Ala and/or Pro57Ser amino acid substitution were detected in P. jirovecii strains from 85/151 (56%) patients. The high frequency of PCP episodes with P. jirovecii harboring DHPS mutations in South Africa indicates that populations of this fungus are evolving under the considerable selective pressure exerted by sulfa-containing antibiotics. These results, similar to previous observations of sulfa drug resistance in bacterial populations, underscore the importance of the rational use of sulfa medications either prophylactically against PCP or for the treatment of other infections.Pneumocystis pneumonia (PCP), a major opportunistic infection in immunocompromised patients, is caused by the fungus Pneumocystis jirovecii. The incidence of PCP, which increased dramatically with the advent of the HIV/AIDS pandemic, has decreased in the industrialized world owing to the widespread use of sulfa drug prophylaxis and the introduction of highly active antiretroviral therapy (HAART). However, PCP remains an important cause of morbidity and mortality in HIV/AIDS patients, as well as in immunocompromised non-HIV-infected patients, in whom its incidence is increasing (17, 35). In South Africa, which has a population of 48.5 million, an estimated 5.7 million people were living with HIV in 2007, and 350,000 deaths were attributed to AIDS during the same year (20). The South African government initiated the provision of HAART to the public sector in April 2004, but prior to that, the HIV epidemic was largely untreated. By the end of 2006, the rate of HAART coverage was estimated to be 21% among those needing antiretroviral therapy (20). Studies from African countries report variable incidences of PCP in adult patients with HIV/AIDS and generally higher rates in children (1, 3, 27, 28, 43, 47, 49). In South Africa, where a limited number of laboratories offer testing for P. jirovecii, the vast majority of PCP cases are diagnosed clinically and radiologically.Sulfonamides, usually combined with trimethoprim, as in trimethoprim-sulfamethoxazole (TMP-SMX), and dapsone are used for the treatment and prophylaxis of PCP. There are few alternative drugs for the treatment of this infection. Sulfonamides inhibit the enzyme dihydropteroate synthase (DHPS), an essential component of the folate synthesis pathway (36). In P. jirovecii, two nonsynonymous point mutations in the fas gene, which encodes the DHPS enzyme, are associated with prior exposure to sulfa drugs (5, 15, 16, 22, 26, 32), and concerns have been raised about the possible emergence of resistance to sulfa drugs (38). These mutations, at nucleotide positions 165 and 171, cause the amino acid substitutions Thr55Ala and Pro57Ser in the DHPS protein, respectively. Point mutations in the DHPS-encoding genes of microorganisms such as Plasmodium falciparum, Staphylococcus aureus, Mycobacterium leprae, and Escherichia coli have been shown to confer resistance to sulfonamides (6, 14, 21, 45). As P. jirovecii cannot yet be cultured, conventional in vitro susceptibility tests cannot be utilized; therefore, studies of drug resistance in this organism rely on the use of genetic markers and suitable models. Functional complementation of either DHPS-disrupted E. coli with a mutant P. jirovecii fas gene or FOL1-disrupted Saccharomyces cerevisiae with the fol1 gene mutated at positions analogous to positions 165 and 171 in fas results in the loss of susceptibility to sulfamethoxazole and other sulfa-containing drugs (19, 29).The prevalence of P. jirovecii DHPS mutations reported from countries in the developed world ranges widely, from 4% to 81% (2, 5, 15, 18, 32, 39, 41, 42). In South Africa, a few studies that screened specimens from limited numbers of adults and children have reported mostly low mutation rates (8, 12, 34, 48). Here we present the results of a large laboratory-based study aimed at ascertaining the prevalence in South Africa of P. jirovecii strains harboring mutations at positions 165 and 171 in the fas gene.     

Mechanisms of sulfadoxine resistance in Plasmodium falciparum

Three possible mechanisms of resistance to sulfadoxine were investigated in resistant Plasmodium falciparum: drug uptake, metabolism and alternate pathways. Uptake of [35S] sulfadoxine was markedly reduced in resistant plasmodia. By Thin Layer Radiochromatography it could be demonstrated that plasmodia do not metabolize sulfadoxine to pharmacologically inactive forms. Metabolism of sulfadoxine to the toxic analog of dihydropteroate is reduced in resistant plasmodia. Para-aminobenzoic acid (pABA) is not an essential nutrient for sulfonamide-resistant plasmodia. Instead, they seem to be able to synthesize pABA de novo. Four enzymes of the respective biosynthetic chain were demonstrated in isolated plasmodia: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase (EC 4.2.1.15), shikimate dehydrogenase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71) and pABA synthetase. We conclude that these three effects account for the reduced sulfonamide stress observed in the resistant parasite.     

Absence of Btn1p in the yeast model for juvenile Batten disease may cause arginine to become toxic to yeast cells

Lymphoblast cell lines established from individuals with juvenile Batten disease (JNCL) bearing mutations in CLN3 and yeast strains lacking Btn1p (btn1-Delta), the homolog to CLN3, have decreased intracellular levels of arginine and defective lysosomal/vacuolar transport of arginine. It is important to establish the basis for this decrease in arginine levels and whether restoration of arginine levels would be of therapeutic value for Batten disease. Previous studies have suggested that synthesis and degradation of arginine are unaltered in btn1-Delta. Using the yeast model for the Batten disease, we have determined that although btn1-Delta results in decreased intracellular arginine levels, it does not result from altered arginine uptake, arginine efflux or differences in arginine incorporation into peptides. However, expression of BTN1 is dependent on arginine and Gcn4p, the master regulator of amino acid biosynthesis. Moreover, deletion of GCN4 (gcn4-Delta), in combination with btn1-Delta, results in a very specific growth requirement for arginine. In addition, increasing the intracellular levels of arginine through overexpression of Can1p, the plasma membrane basic amino acid permease, results in increased cell volume and a severe growth defect specific to basic amino acid availability for btn1-Delta, but not wild-type cells. Therefore, elevation of intracellular levels of arginine in btn1-Delta cells is detrimental and is suggestive that btn1-Delta and perhaps mutation of CLN3 predispose cells to keep arginine levels lower than normal.     

Flow cytometry of Candida albicans for investigations of surface marker expression and phagocytosis

Several cytoplasmic virulence factors of Candida albicans are altered in the presence of estrogen and this fact may imply the existence of a global virulence regulatory system in this organism. The response of virulence-associated surface markers to estrogen, however, has not been studied. We exploited flow cytometry methods for assessment of the iC3b receptor analog and mannoproteins on 2 clinical yeast strains selected for their different rates of growth in the presence of estradiol 17beta. Although, as expected, iC3b receptor analog expression increased in the presence of glucose, growth in the presence of estradiol did not increase the levels of iC3b receptor analog on either organism. Exposure to human serum caused massive conversion to mycelial growth, but cells examined by flow cytometry did not show increased levels of iC3b receptor analog expression, possibly due to inability of the flow cytometer to sample the mycelial forms of Candida. In contrast, estradiol increased expression of mannoproteins as evidenced by concanavalin A binding to yeast. This increase occurred in both yeast strains but was less pronounced with strain GT188, which also showed limited growth in estradiol compared to strain GT142. Effective phagocytosis by human neutrophils required exposure of yeast to human serum. Yeast grown in the presence of estradiol were ingested by human PMN but not at a significantly greater rate than yeast grown without estradiol. While flow cytometry appears to be useful in determining estrogen-enhanced concanavalin A binding to yeast, it probably does not reflect the surface markers on large mycelial masses. Consequently, the results of this study are applicable to Candida primarily in its yeast form.     

p-Aminobenzoic acid transport by normal and Plasmodium falciparum-infected erythrocytes.

De novo folate biosynthesis is required for the growth of malarial parasites and is inhibited by several important antimalarial agents. We show here that exogenous p-aminobenzoic acid (pABA) can be utilized by malaria parasites to synthesize folates. The transport of pABA into parasite infected red cells was therefore characterized. Normal red cells transport pABA in a saturable and energy-dependent manner, with a dissociation constant of 83 nM. pABA transport in parasite-infected red cells may use the same mechanism, as demonstrated by similarities in time course, concentration-response, and dissociation constant (111 nM). The transport capacity of red cells is temperature-, energy- and pH-dependent. It is inhibited by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), but not by the sodium ionophores nigericin and monensin. p-Aminosalicylic acid (PAS) inhibits pABA transport competitively, with a inhibition constant of 378 nM. Phloritin, flufanamic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DITS), which are inhibitors of the anion transporter (band 3), and oxalic acid, a substrate of this transporter, partially inhibit pABA transport into both normal and infected red cells. Interestingly, in both normal and infected red cells, the inhibitory effects of PAS and the anion transport inhibitors are additive, suggesting the involvement of 2 independent mechanisms.     

Generation of genetic diversity in herpes simplex virus: an antimutator phenotype maps to the DNA polymerase locus

We have measured the spontaneous production of mutants in derivatives of herpes simplex virus type 1 resistant to phosphonoacetic acid. Six such derivatives produced 9- to 123-fold fewer iododeoxycytidine (ICdR-)-resistant progeny (i.e., thymidine kinase deficient) than their wild-type parents. To locate the mutation which controls mutant production in one of the strains (PAAr-5), we constructed phosphonoacetic acid-resistant, recombinant viruses by marker transfer, using wild-type viral DNA and DNA restriction fragments conferring the resistance phenotype. The resultant recombinants also produced very low levels of ICdR-resistant progeny during growth, indicating a close linkage (within 1.1 kilobase pairs) between the drug resistance locus and the sequences controlling production of mutant progeny. Evidence is presented that the low mutant yield in PAAr-5 is not due to abnormal expression of mutants, hypersensitivity to ICdR, altered thymidine kinase activity, or slow replication rates. Since the locus conferring resistance to phosphonoacetic acid in PAAr-5 has been shown previously to be the DNA polymerase gene, we hypothesize that the reduced yield of mutants results from enhanced replication fidelity by the altered DNA polymerase. The existence of antimutator derivatives of herpes simplex indicates that the observed high mutation rate for wild-type strains is an intrinsic property of the virus and may provide a selective advantage during growth in animal hosts.     

Phenotypic and genotypic evaluation of fluconazole resistance in vaginal Candida strains isolated from HIV-infected women from Brazil.

We conducted a study to determine the antifungal susceptibility of vaginal Candida isolates from HIV-infected Brazilian women. Among 127 women enrolled, positive cultures for yeast were obtained from 31 of 38 (81%) women with symptomatic vulvovaginitis, and from 41 of 89 (46%) asymptomatic women. Susceptibility testing demonstrated 11 of the 72 isolates had either resistance or dose-dependent susceptibility to azole drugs, including four Candida albicans strains. Expression of the MDR1, CDR1, CDR2 and ERG11 genes was evaluated in all of the C. albicans isolates, and all four of the strains with reduced susceptibility to fluconazole had increased expression of CDR1 as compared to the fluconazole-sensitive strains. No increased expression of the other genes was identified. This large survey of Candida isolates from HIV-infected women from Brazil demonstrates that reduced susceptibility to azoles occurs at a low frequency among vaginal yeast isolates, and when present in C. albicans, azole resistance is associated with increased expression of CDR1.     

Mutations and horizontal transmission have contributed to sulfonamide resistance in Streptococcus pyogenes

Two variants of dihydropteroate synthase (DHPS) were found among sulfonamide-resistant Streptococcus pyogenes, one of which was characterized by a 2-amino-acid addition in a conserved part of the enzyme. The enzyme kinetics of both variants was compared with the kinetics of DHPS from a sulfonamide-susceptible S. pyogenes. The most striking difference was a substantially elevated Ki for both variants, but variations in Km for both of its substrates p-aminobenzoic acid (p-AB) and dihydropteridine-pyrophosphate (pteridine) were also found. In the resistance variant lacking additions, the amino acid at position 213 was changed by site-directed mutagenesis from a Gly to an Arg, which resulted in a lower Ki. The corresponding change from an Arg to a Gly in the DHPS from a susceptible isolate led to a substantially increased Ki, confirming the importance of this amino acid difference for the resistance. Nucleotide sequence determinations of the complete folate operon revealed in some isolates a mosaic pattern of differences compared to the wild type, not only in the genes coding for DHPS and GTP cyclohydrolase (GTPCH) noted earlier but also in genes coding for dihydroneopterin aldolase (DHNA) and hydroxymethylpterin pyrophosphokinase (HPPK). Regions of sequence differences were interspersed with regions of complete identity in a mosaic pattern, indicating a dispersed pattern of uptake of foreign DNA in the resistant isolates.     

Antibiotic resistance in Enterotoxigenic and non-enterotoxigenic Escherichia coli.

Antibiotic disk susceptibility tests were done on 220 strains of Escherichia coli belonging to serotypes reported in the literature to be associated with the production of enterotoxin. A total of 128 (58%) were resistant to one or more antibiotics, sulfa drugs, or chemotherapeutic agents. An analysis of these strains revealed primary, secondary, and tertiary drug resistance patterns that indicated a selective pattern in the formation of multiple drug resistance in E. coli. Resistances to certain antibiotics were more likely to occur in pairs and triads (secondary resistance patterns) that were often combined or coexisted in a single strain of E. coli to produce tertiary drug resistance patterns, conferring drug resistance to five or six different antibiotics. Among enterotoxin-associated serotypes, single and multiple drug resistance was less frequently associated with enterotoxin-produced strains than with strains from the same serotype that were not enterotoxigenic. Within the enterotoxigenic E. coli, single and multiple resistance to antibiotics was more frequent in strains producing only heat-stable enterotoxin (ST) than in strains producing only heat-labile enterotoxin (LT) or both. The number of resistances to different antibiotics per resistant strain averaged approximately 1.4 for LT plus ST or LT strains, and 3.9 for ST strains and nonenterotoxigenic strains. Phenotypic characterization of 170 strains for four usually plasmid-mediated characteristics showed that the number of antibiotics to which a strain was directly resistant varied with the type and number of plasmid-mediated characteristics present.     

血液肿瘤细胞株细胞survivin基因表达以及ATRA对NB4细胞株survivin基因表达的调节

目的:检测survivin基因在血液肿瘤细胞株细胞的表达。方法:应用RT-PCR法检测survivin基因mRNA表达。结果:10种血液肿瘤细胞株和K562/ADM细胞株细胞均有survivinmRNA表达,不同的血液肿瘤细胞株survivinmRNA表达有明显的差异性,K562/ADM细胞株与K562细胞株细胞相比,survivinmRNA表达上调了1.6倍。外周血单个核细胞survivinmRNA表达阴性。经ATRA处理后,NB4细胞survivinmRNA表达随着时间的延长而呈逐渐下降趋势,在72h几乎检测不到survivinmRNA的表达。结论:survivin基因在血液肿瘤细胞株中广泛表达,它是肿瘤细胞抗凋亡和耐药的重要机制之一,survivin基因有望成为肿瘤治疗新靶点。     

Genotypes and in vivo resistance of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> isolates in an endemic region of Iran

Mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum have been correlated with and used to detect antifolate treatment failure, such as sulfadoxine–pyrimethamine (SP), in regions endemic for malaria. To determine the association between molecular markers of SP resistance and in vivo drug resistance, a quick and simple technique that detects single nucleotide polymorphisms in the DHFR and DHPS genes, using PCR–ELISA and sequence-specific oligonucleotide probes, was applied to 53 isolates obtained from an in vivo study in Sistan and Baluchistan Province, in southeastern Iran. Overall, 11.3% of these isolates were obtained from patients with SP treatment failure. Four DHFR polymorphisms (codons 51, 59, 108, and 164) and five DHPS polymorphisms (codons 436, 437, 540, 581, and 613) were investigated. Mutations DHFR Asn-108, DHFR Arg-59, and DHPS 436-Ala/Phe were very common (100, 81.1, and 85%, respectively). Plasmodium falciparum was isolated from 96% of patients with at least two DHFR/DHPS mutations. All resistant isolates had at least three mutations. The high prevalence of mutation associated with antifolate resistance may point toward low drug efficacy in the future.