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PubMed2004

Comparative in vitro activity of antiribosomal agents on penicillin-susceptible and -resistant Streptococcus pneumoniae in relation to their resistance genotypes.

A collection of 326 strains of Streptococcus pneumoniae isolated from blood, cerebrospinal fluid, bronchoalveolar lavage, transtracheal aspirate or sputum from January 1996-June 2002 were included in this study. The activity of clarithromycin, clindamycin, telithromycin, linezolid and quinupristin/dalfopristin against penicillin G and erythromycin A susceptible and resistant pneumococci were determined; the erythromycin A resistance phenotypes and genotypes were identified and susceptibilities of these agents were assessed according to the resistance genotypes. MICs were determined for all strains of pneumococci using an agar dilution method. MLS(B) resistance phenotypes were determined by the double disk (erythromycin A and clindamycin) diffusion method. Genetic determinants for macrolide resistance were identified by PCR using primers specific for erm(B) and mef(A). Erythromycin A resistance was detected in 13.8% of the strains. MLS(B) resistance phenotype was observed in 82% of these (60% being cMLS(B) and 40% being iMLS(B)), and M type resistance in about 18%. All the MLS(B) phenotype strains except four, revealed the presence of erm(B) gene and all except one M phenotype strains revealed the mef(A) gene. Of the erythromycin A resistant pneumococci about 49% were also resistant to clindamycin. No strains were resistant to telithromycin, quinupristin/dalfopristin and linezolid. Telithromycin had the lowest MIC values for both erythromycin A resistant and susceptible strains of all the antiribosomal agents tested. The most prevalent mechanism of macrolide resistance was mediated by the erm(B) gene leading to the expression of MLS(B) phenotype. Telithromycin was the most active antiribosomal agent, regardless of the macrolide resistance genotype of the pneumococci tested.

Drug Resistance, Microbial↗
PubMed2002

Biochemical changes associated with a multidrug-resistant phenotype of a human glioma cell line with temozolomide-acquired resistance.

Temozolomide (TMZ) is a newly approved alkylating agent for the treatment of malignant gliomas. To investigate resistance mechanisms in a multidrug therapeutic approach, a TMZ-resistant human glioma cell line, SF188/TR, was established by stepwise exposure of human SF188 parental cells to TMZ for approximately 6 months. SF188/TR showed 6-fold resistance to TMZ and cross-resistance to a broad spectrum of other anticancer agents that included 3-5-fold resistance to melphalan (MEL), gemcitabine (GEM), paclitaxel (PAC), methotrexate (MTX), and doxorubicin (DOX), and 1.6-2-fold resistance to cisplatin (CDDP) and topotecan (TPT). Alkylguanine alkyltransferase (AGT) activity was increased significantly in the resistant cell line compared with the parental cell line (P<0.05), whereas no significant differences occurred in the cellular uptake of TMZ and PAC between resistant and parental cells. Depletion of AGT by O(6)-benzylguanine significantly increased the cytotoxicity of TMZ in both the sensitive and resistant cell lines, but did not influence the cytotoxicity of the other drugs tested. Treatment with TMZ caused SF188 cells to accumulate in S phase, whereas SF188/TR cells were unaffected. Expression of Bcl-2 family members in SF188/TR cells compared with SF188 cells indicated that the pro-apoptotic proteins (i.e. Bad, Bax, Bcl-X(S)) were reduced 2-4-fold in the resistant cell line, whereas the anti-apoptotic proteins Bcl-2 and Bcl-X(L) were expressed at similar levels in both cell lines. In conclusion, the mechanism of resistance of SF188/TR cells to TMZ involved increased activity of AGT, a primary resistance mechanism, whereas the broad cross-resistance pattern to other anticancer drugs was due to a common secondary resistance mechanism related to alterations in the relative expression of the pro-apoptotic and anti-apoptotic proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1989

N-benzyladriamycin-14-valerate versus progressively doxorubicin-resistant murine tumours: cellular pharmacology and characterisation of cross-resistance in vitro and in vivo.

N-Benzyladriamycin-14-valerate (AD198) is a novel lipophilic anthracycline with greater in vivo antitumour activity than doxorubicin (DOX) in experimental model systems. Using sensitive and progressively DOX-resistant L1210 mouse leukaemia and B16-BL6 mouse melanoma lines, we have determined the cellular pharmacokinetics and cytotoxic response in vitro and in vivo of AD198. In the L1210 leukaemia model following 3 h drug exposure in vitro, the IC50 for AD198 was approximately 0.35 microgram ml-1 for the sensitive and 10-fold DOX resistant cells and 1.0 microgram ml-1 for the 40-fold DOX resistant cells. A similar pattern of cross-resistance to AD198 was also observed with the B16-BL6 melanoma, with and IC50 for AD198 with the sensitive and 10-fold DOX-resistant cells being similar, and about 2-fold higher with the 40-fold resistant cells. In the L1210 leukaemia model, cellular pharmacokinetics of AD198 revealed the following: (a) accumulation of AD198 was concentration but not time dependent, and cellular drug levels in the sensitive and resistant sublines were similar when treated with equimolar concentrations; (b) retention of AD 198 was 60% of the initial drug uptake and, in cells treated with the IC50 of AD198, cellular levels in the 40-fold DOX-resistant line were, as expected, 2-fold higher than in sensitive or 10-fold DOX-resistant cells; (c) in vitro biotransformation of AD 198 in the sensitive and resistant sublines was comparable. Studies in vivo with i.p. L1210 leukaemia (disseminating) and B16-BL6 melanoma (non-disseminating) tumour models evaluating therapeutic efficacy of DOX vs AD 198 in mice implanted with tumour i.p. on day 0 and treated i.p. on days 1-4 indicated: (a) DOX at 3 mg kg-1 administered once daily on days 1-4 resulted in a 55% ILS and 104% ILS with parent-sensitive B16-BL6 melanoma and L1210 leukaemia models respectively; however, similar doses of DOX in the resistant sublines were ineffective, with survival similar to the untreated control; (b) AD198 at 10-12.5 mg kg-1 day-1 for 4 days was extremely effective in the sensitive L1210 (189% ILS), and similar to DOX (61% ILS) in the sensitive B16-BL6; (c) AD198 (10-12.5 mg kg-1) was ineffective (survival similar to untreated control) in the 10-and 40-fold DOX-resistant L1210 leukaemia and 40-fold DOX resistant B16-BL6 melanoma, but produced a 76% ILS in the 10-fold DOX resistant B16-BL6 melanoma.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

P388 leukaemia cells resistant to the anthracycline menogaril lack multidrug resistant phenotype.

Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.

Animals↗
PubMed1999

Mupirocin resistance in staphylococci: development and transfer of isoleucyl-tRNA synthetase-mediated resistance in vitro.

Mupirocin resistance could be transferred from highly resistant clinical isolates of Staphylococcus aureus to highly sensitive recipients of Staph. aureus, Staph. epidermidis and Staph. haemolyticus. Transconjugants of the latter two organisms could transfer this resistance into mupirocin-sensitive Staph. aureus. Moderately resistant strains did not transfer this resistance to sensitive recipients, nor did strains with high-level mupirocin resistance developed by serial transfer or habituation. The inhibitory effects of mupirocin on crude isoleucyl-tRNA synthetases (IRS) isolated from mupirocin-sensitive and -resistant strains of Staph. aureus have been determined. Drug concentrations needed to produce 50% inhibition, I50 values, were very low against IRS from a highly sensitive strain, somewhat higher against IRS from moderately resistant strains, much higher against enzyme from strains trained in vitro to high-level resistance, and considerably higher still against IRS extracted from clinical isolates possessing high-level mupirocin resistance and from the transconjugates of such strains resulting from crosses with mupirocin-sensitive strains. It is concluded that high-level resistance in clinical isolates is plasmid-mediated involving a second, mupirocin-resistant IRS whereas in moderately resistant strains, and in strains trained in vitro to high-level resistance, chromosomal mutations are likely to be responsible for decreasing IRS sensitivity.

Anti-Bacterial Agents↗
PubMed2002

Genetic basis of quinolone resistance and epidemiology of resistant and susceptible isolates of porcine Campylobacter coli strains.

AIMS: The aims of this study were to investigate the epidemiology of quinolone-resistant and -susceptible porcine isolates of Campylobacter coli and to characterize the genetic basis of quinolone resistance. METHODS AND RESULTS: Penner serotyping and flagellin gene sequence polymorphisms were used to investigate the epidemiology of the C. coli isolates. A total of 55 isolates were included, of which 30 were paired resistant and susceptible isolates from 15 pigs. Amplification of gyrA, gyrB and parC, followed by direct sequencing of amplicons was used to identify mutations in the targets of quinolones. Overall, 31 of the isolates were resistant to ciprofloxacin (minimum inhibitory concentrations (MIC), 2- >or = 32 microg x ml(-1)). Thirteen DdeI-flaA profiles were observed and resistant and susceptible strains were identified for nine profiles. The majority of resistant strains exhibited either profile 1 or 6. While profile 1 comprised susceptible and resistant strains, all of the strains with profile 6 were resistant to ciprofloxacin. The serogroup (O:24) of the profile 6 strains was identical. The only other serogroup to be uniformly associated with quinolone resistance was O:5. Strains with this phenotype comprised a number of genotypes, including profile 1. Only four of the paired isolates from individual pigs had the same profile. The genetic basis of quinolone resistance was investigated in two strains with ciprofloxacin MICs of 2 and > or = 32 miccrog x ml(-1), respectively. The amino acid substitution of isoleucine for threonine at position 86 was identified in the GyrA proteins from both strains. No mutations were identified in the GyrB proteins. CONCLUSIONS: There was an association between two of the genotypes, serotypes 5 and 24, and quinolone resistance. The association between genotype, serotype and resistance in C. coli isolates has not been reported previously. Only the mutation in GyrA associated with quinolone resistance was identified. No mutations in GyrB were identified. Amplification products of parC were not obtained and it may be that this gene is not present in some Campylobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on the distribution of ciprofloxacin resistance between subtypes of C. coli.

Animals↗
PubMed2005

Clinical epidemiology of ciprofloxacin resistance and its relationship to broad-spectrum cephalosporin resistance in bloodstream infections caused by Enterobacter species.

OBJECTIVES: To evaluate the clinical features of ciprofloxacin-resistant Enterobacter bacteremia and to examine the risk factors for ciprofloxacin resistance in Enterobacter species isolates causing bacteremia. DESIGN: A case-control study. SETTING: A 1,500-bed, tertiary-care university hospital and referral center. PATIENTS: All patients older than 16 years with Enterobacter species isolated from blood were enrolled. The medical records of 183 patients with clinically significant Enterobacter bacteremia from January 1998 to December 2002 were identified. We compared patients with bacteremia caused by ciprofloxacin-susceptible isolates with patients with bacteremia caused by ciprofloxacin-resistant isolates. RESULTS: Twenty-three (12.6%) of the patients had bacteremia caused by isolates resistant to ciprofloxacin. There were no significant differences in age, gender, underlying diseases, primary site of infection, or Acute Physiology and Chronic Health Evaluation II score between the ciprofloxacin-resistant and the ciprofloxacin-susceptible groups. Broad-spectrum cephalosporin resistance, defined as resistance to cefotaxime or ceftazidime in vitro, was detected in 21 (91.3%) of 23 ciprofloxacin-resistant isolates compared with 65 (40.6%) of 160 ciprofloxacin-susceptible isolates (P < .001). Multivariate analysis revealed that independent risk factors for ciprofloxacin resistance were the prior receipt of fluoroquinolones (P < .001) and broad-spectrum cephalosporin resistance (P < .001). CONCLUSIONS: In Enterobacter species isolates causing bacteremia, ciprofloxacin resistance was closely associated with the prior receipt of fluoroquinolones and broad-spectrum cephalosporin resistance. The close relationship between ciprofloxacin resistance and broad-spectrum cephalosporin resistance is particularly troublesome because it severely restricts the therapeutic options for Enterobacter species infection.

Adolescent↗
PubMed2006

Antimicrobial resistance of coagulase-negative staphylococci from bovine subclinical mastitis with particular reference to macrolide-lincosamide resistance phenotypes and genotypes.

OBJECTIVES: The aim of this study was to analyse coagulase-negative staphylococci (CoNS) for their resistance to antimicrobial agents approved for the control of pathogens involved in bovine mastitis, with particular reference to macrolide and/or lincosamide (ML) resistance and the resistance genes involved. METHODS: A total of 298 CoNS collected between 2003 and 2005 in Germany from cases of subclinical mastitis in dairy cows were identified to the species level and investigated for their MICs by broth microdilution. ML-resistant isolates were subjected to plasmid profiling and electrotransformation experiments. The ML resistance genes were detected using PCR and hybridization. Selected PCR products were cloned and sequenced. RESULTS: The CoNS isolates used in this study showed a low level of resistance to all antimicrobial agents tested (0-7.4%) except ampicillin (18.1%). In the erythromycin-resistant and/or pirlimycin-resistant isolates, the ML resistance genes erm(B), erm(C), msr(A), mph(C) and lnu(A) were present, either alone or in different combinations. Isolates carrying erm methylase genes or the exporter gene msr(A) showed higher MICs than those harbouring only the genes mph(C) or lnu(A) coding for inactivating enzymes. Most of the ML resistance genes were found on plasmids. CONCLUSIONS: This is the first report of pirlimycin MICs for CoNS collected from cases of bovine subclinical mastitis in Germany. After 3-5 years of veterinary therapeutic use, pirlimycin resistance was rarely detected among CoNS. The finding that five different resistance genes--present in various combinations--were responsible for ML resistance underlines the heterogeneous character of this resistance trait.

Animals↗
PubMed1991

Multidrug resistance phenotype associated with selection of an aminopterin resistant dog kidney cell line.

A determination of the mechanisms of drug resistance in tumour cells is important for developing strategies to combat such resistance in persons receiving chemotherapy. This report describes a combined cellular, biochemical, and molecular analysis of a dog kidney cell line selected for resistance to increasing levels of the hydrophilic antifolate, aminopterin. Three distinct drug resistance phenotypes were observed in cells exhibiting high levels of aminopterin resistance. Two of these phenotypes were decreased aminopterin accumulation and increased levels of dihydrofolate reductase specific activity. The third drug resistance phenotype was noted initially as cross resistance to a variety of hydrophobic drugs indicating multidrug resistance. Biochemical assays demonstrated reduced accumulation of the hydrophobic fluorescent drug daunorubicin and of 3H-colchicine in the aminopterin resistant cells. These results were then correlated with increased levels of the multidrug resistance (mdr) gene product, P-glycoprotein, and mdr mRNA levels in the aminopterin resistant cells. However, experiments designed to prove a role for expression of the mdr gene in providing a degree of aminopterin resistance were unsuccessful. It is concluded that aminopterin selection in these dog kidney cells resulted in expression of at least three distinct drug resistance phenotypes and that one of these phenotypes, multidrug resistance, represented a secondary response to the aminopterin selection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1975

Transfer of drug resistance to myxococcus from bacteria carrying drug-resistance factors.

Resistance to chloramphenicol was successfully transferred from strains of Escherichia coli carrying R factors representative of compatibility groups F, W, S and N to strains of Myxococcus xanthus and M. fulvus. Resistance to kanamycin was transferred from an R factor in group S, and to neomycin from an R factor of group P. Myxobacterial strains differed in their capacity to take up the resistances and also in the stability of the resistance character. strains of M. fulvus were obtained that acquired resistance to chloramphenicol without exposure to R plus eubacterial strains. Cell-free preparations of all the chloramphenicol-resistant strains catalysed the acetylation of the drug. Cholramphenicol resistance was successfully transferred from the presumed R plus strains of Myxococcus and also from the spontaneously occurring chloramphenicol-resistant M. fulvus to other Myxococcus strains. Moreover, recombinants resistant to both rifampicin and 5-fluorouracil were obtained, though infrequently, by mixing Myococcus strains resistant to rifampicin and chloramphenicol with other myxococci resistant to 5-fluorouracil, both when the chloramphenicol resistance was derived from S-a (group W) and when it was the endogenous M fulvus resistance. Thus it appears that S-a and a new chloramphenicol resistance factor from M. fulvus will mobilize a chromosomal genetic marker in Myxococcus.

Acetylation↗
PubMed2002

Prediction of abacavir resistance from genotypic data: impact of zidovudine and lamivudine resistance in vitro and in vivo.

Abacavir is frequently used in antiretroviral combination therapies as a potent nucleoside reverse transcriptase inhibitor (NRTI). Four mutations are selected for by abacavir in vitro and in vivo: K65R, L74V, Y115F, and M184V. Abacavir resistance has also been observed in NRTI multidrug-resistant samples. Furthermore, abacavir resistance has been described in the context of zidovudine resistance. To evaluate the genetic basis of abacavir resistance, the viral genotype and phenotypic resistance were analyzed for 307 patient samples. Low- and high-level resistances were defined as 2.5- to 5.5-fold- and >5.5-fold-reduced susceptibility, respectively. If all samples with abacavir-selected and NRTI multidrug resistance-associated mutations were scored as resistant, 27.6% of the samples were misclassified, mainly due to samples falsely scored as susceptible. Therefore, the relative frequencies of other mutations were evaluated. Mutations at codons 44 and 118 were rarely detected in abacavir-susceptible samples but were overrepresented in resistant samples. Site-directed mutagenesis of E44D, V118I, and M184V resulted in low-level resistance for the double mutant 44/184 and the triple mutant. Low-level abacavir resistance was also detected for a viral clone carrying zidovudine mutations only. Additional insertion of M184V into the zidovudine background doubled the resistance, whereas 44/118 did not lead to a further increase. Incorporating combinations of zidovudine mutations and M184V into the scoring system markedly reduced the number of misclassified samples, whereas 44/118 did not improve the prediction. In conclusion, the combination of M184V with zidovudine mutations gives rise to high-level abacavir resistance, which may be clinically relevant. Thus, options for useful sequential combinations of NRTI are limited.

Algorithms↗
PubMed2005

Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance.

The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

ATP-Binding Cassette Transporters↗
PubMed1998

Variable cross-resistance to Cry11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to single or multiple toxins of Bacillus thuringiensis subsp. israelensis.

A novel mosquitocidal bacterium, Bacillus thuringiensis subsp. jegathesan, and one of its toxins, Cry11B, in a recombinant B. thuringiensis strain were evaluated for cross-resistance with strains of the mosquito Culex quinquefasciatus that are resistant to single and multiple toxins of Bacillus thuringiensis subsp. israelensis. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC95) between B. thuringiensis subsp. jegathesan and the different B. thuringiensis subsp. israelensis-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the B. thuringiensis subsp. israelensis Cry toxin mixtures used to select the resistant mosquito strains. The LC95 RR obtained with the mosquito strains were as follows: 53.1 against Cq4D, which was resistant to Cry11A; 80.7 against Cq4AB, which was resistant to Cry4A plus Cry4B; and 347 against Cq4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in Cq4D but resulted in synergism factors of 4.8 and 11.2 against strains Cq4AB and Cq4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of B. thuringiensis subsp. jegathesan and B. thuringiensis subsp. israelensis, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.

Animals↗
PubMed2006

High prevalence of ceftazidime-resistant Klebsiella pneumoniae and increase of imipenem-resistant Pseudomonas aeruginosa and Acinetobacter spp. in Korea: a KONSAR program in 2004.

A nationwide antimicrobial resistance surveillance has been conducted since 1997 in Korea. In this study, susceptibility test data generated in 2004 by KONSAR group hospitals were analyzed and compared to those at a commercial laboratory. In hospitals, the rank orders of organisms in 2004 were identical to those in 2003. The most prevalent species was Staphylococcus aureus (20.2%) in hospitals, but Escherichia coli (29.7%) in the commercial laboratory. The proportions of Enterococcus faecium to all isolates of Enterococcus faecalis plus E. faecium were 47.2% in hospitals and 24.9% in the commercial laboratory. The mean resistance rates of significant antimicrobial-organism combinations in hospitals were: oxacillin-resistant S. aureus (68%), oxacillin-resistant (penicillin- nonsusceptible) Streptococcus pneumoniae (68%), vancomycin-resistant E. faecium (25%), cefotaxime-resistant E. coli (14%), ceftazidime- and cefoxitin-resistant Klebsiella pneumoniae (34% and 32%, respectively), and imipenem-resistant Acinetobacter spp. and Pseudomonas aeruginosa (17% and 24%, respectively). In conclusion, oxacillin-resistant staphylococci, expanded-spectrum cephalosporin-resistant K. pneumoniae, and imipenem-resistant Acinetobacter spp. and P. aeruginosa were prevalent in 2004. Increasing trends were observed for vancomycin-resistant E. faecium, cefoxitin- resistant E. coli and K. pneumoniae, and imipenem-resistant Acinetobacter spp. and P. aeruginosa. Certain antimicrobial- organism combinations were also prevalent among the commercial laboratory-tested strains.

Acinetobacter↗
PubMed2000

Surveillance of drug-resistant tuberculosis and molecular evaluation of transmission of resistant strains in refugee and non-refugee populations in North-Eastern Kenya.

SETTING: Three refugee camp complex clinics and an adjacent non-refugee treatment centre in North-Eastern Kenya. OBJECTIVES: To use conventional and molecular epidemiology tools to determine: 1) the prevalence of drug resistance in newly diagnosed patients with smear-positive pulmonary tuberculosis in refugee and non-refugee populations; 2) risk factors for resistance in the two populations; and 3) whether IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping showed similarities in DNA fingerprinting patterns of drug-resistant isolates that could infer transmission within and between the two populations. RESULTS: Of 241 isolates from the camps, 44 (18.3%) were resistant to one or more drugs, seven of which (2.9%) were multidrug-resistant TB (MDR-TB). Of 88 isolates from the non-refugees, five (5.7%) were resistant to one or more drugs without MDR-TB. Drug resistance was higher in the camps than in the non-refugee population (OR = 3.7; 95%CI 1.42-9.68; P < 0.007). Resistance was significantly higher in one camp compared with the other two, despite a comparable ethnic distribution. Unusually, females were more associated with drug resistance than their male counterparts in both populations (OR = 2.3; 95%CI 1.2-4.8; P = 0.008). There was evidence of transmission of streptomycin-resistant strains in the refugee population. DNA fingerprints of resistant strains from the non-refugee population were unique and different from those in the refugee camps. CONCLUSION: The observed high levels of drug resistance and MDR-TB, combined with evidence of transmission of strains resistant to streptomycin in the refugee population, suggest a need for strengthened TB control programmes in settings with a high risk of developing drug-resistant strains.

Adolescent↗
PubMed2004

[Mechanisms of the drug resistance of a 2', 2-difluorodeoxycytide (gemcitabine)-resistant variant of the human lung adenocarcinoma cell line].

OBJECTIVE: To investigate the mechanisms of the drug resistance of gemcitabine resistance variant of the human lung adenocarcinoma cell line A549-Gem. METHOD: Immunohistochemistry and RT-PCR were used to tested the expression of P-glycoprotein and transcription of mRNA of multidrug resistance gene and deoxycytidine kinase. Using a cDNA microarray compared the expression profiles between the resistant A549-Gem and the parent cell line A549. RESULTS: The A549-Gem shown slight positive expression of P-glycoprotein compared with positive control by immunohistochemistry, but A549 was negative for P-gp. RT-PCR amplified mRNA, using specific dCK and multidrug resistant gene 1 (mdr1) primers, demonstrated that A549-Gem express amplicon of mdr1 but no products of mdr1 was detected in A549. In the parent cell line, dCK mRNA amplication product could be detected, but did not found in resistant cells. About 18.8% of the total DNA elements had substantially altered level of expression in resistant A549-Gem compared with A549 by cDNA microarray. The biochemical functions of the different expressed genes are diverse and include oncogene and tumor suppressor gene, cell cycle regulator, heat shock protein, apoptotic and antiapoptotic factors, DNA transcription factors, DNA repair and recombination factors, signal transduction genes, protein translation genes, as well as a large number of metabolic genes. Several cluster genes overexpressed in resistant cell line, including ubiquitin-proteasome system, zinc finger protein, glutaredoxin and heat shock protein, may be related to the mechanisms of gemcitabine resistance. CONCLUSION: The mechanisms of resistance to gemcitabine are multifactorial, may include multidrug resistance and dCK deficiency. Differential expression genes monitored by cDNA microarray between A549-Gem and A549 may be related to the mechanisms of gemcitabine resistance in human lung cancer and potentially suggest the prevalence of expression of these genes in drug resistance. The use of cDNA microarray has provided a global view of the response of lung cancer cells to gemcitabine at the genomic level, it should be suitable for examining the development of drug resistance in cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

A novel mechanism for acquired cisplatin-resistance: suppressed translation of death-associated protein kinase mRNA is insensitive to 5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells.

The molecular mechanism for cisplatin (CDDP)-resistance of cancer cells has not yet been clarified, despite extensive studies. Here, we investigated whether death-associated protein (DAP) kinase, an apoptosis modulator, was involved in CDDP-resistance by examining the ME180 human cervical squamous cancer cell line and 6 monoclonal ME180-derived CDDP-resistant subclones. Co-treatment with CDDP and 5-aza-2'-deoxycytidine (5-aza-CdR), a demethylating agent, significantly enhanced the CDDP-sensitivities of the parent cells and CDDP-resistant subclones. Subsequent removal of 5-aza-CdR rapidly reversed the CDDP-sensitivity of the CDDP-resistant subclones to their original levels, whereas the parent cells retained the enhanced CDDP-sensitivity for at least 24 h. Quantitative RT-PCR revealed that the CDDP-resistant subclones expressed higher DNA methyltransferase (DNMT) mRNA levels than the parent cells, suggesting that increased DNMT expressions easily restored the CDDP-resistance of the CDDP-resistant subclones following 5-aza-CdR removal. Although the parent cells showed hypermethylation in the DAP kinase promoter region, corresponding methylated bands were not detected in 2 of the 6 CDDP-resistant subclones by methylation-specific PCR. All 6 CDDP-resistant subclones expressed higher DAP kinase mRNA levels than the parent cells, as evaluated by quantitative RT-PCR. Although DAP kinase protein expression was strongly suppressed in the parent cells and CDDP-resistant subclones, 5-aza-CdR treatment of the parent cells dose-dependently stimulated the DAP kinase protein expression, and this was synergistically enhanced by inhibiting histone deacetylation via trichostatin treatment in addition to 5-aza-CdR. However, DAP kinase protein expression in the CDDP-resistant subclones was not stimulated by treatment with 5-aza-CdR and/or trichostatin. These results indicate that post-transcriptional translation of DAP kinase mRNA is strongly suppressed and insensitive to treatment with 5-aza-CdR and trichostatin in the CDDP-resistant subclones established from ME180 human cervical squamous cancer cells. This CDDP-resistance is accompanied by molecular changes that disturb the post-transcriptional translation of the DAP kinase mRNA, and these molecular changes are transiently restored by demethylation.

Antineoplastic Agents↗
PubMed1988

Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia.

Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established, including P388/ADR/3 and P388/ADR/7 that are 5- and 10-fold more resistant than the cloned sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). A time course of ADR-induced DNA double-strand breaks revealed that in sensitive P388/4 cells, evidence of DNA repair was noted 4 h after removal of drug, whereas in resistant clone 3 and 7 cells repair was observed 1 h after drug removal. The earlier onset of DNA repair was statistically significant (p = 0.0154 for clone 3 cells, and p = 0.0009 for clone 7 cells). By contrast, once the repair process was initiated, the rate of repair was similar for all three cell lines. The level of glutathione transferase activity was determined in whole cell extracts. Enzyme activity (mean +/- SE) in sensitive cells was 9.49 +/- 1.00 nmol/min/mg protein, that in resistant clone 3 cells was 13.36 +/- 1.03 nmol/min/mg, and that in clone 7 cells was 13.96 +/- 1.44 nmol/min/mg; the 1.44-fold increase in enzyme activity in resistant cells was statistically significant (p = 0.01). Further evidence of induction of glutathione transferase was provided by Northern blot analysis using a 32P-labeled cDNA for an anionic glutathione transferase, which demonstrated approximately a twofold increase in mRNA in resistant clone 7 cells. Western blot analysis with a polyvalent antibody against anionic glutathione transferase also revealed a proportionate increase in gene product in resistant cells. Dose-survival studies showed that ADR-resistant cells were cross-resistant to actinomycin D, daunorubicin, mitoxantrone, colchicine, and etoposide, but not to the alkylating agent melphalan; this finding provided evidence that these cells are multidrug resistant. Using a cDNA probe for P-glycoprotein, a phenotypic marker for multidrug resistance, Northern blot analysis showed an increase in the steady state level of mRNA of approximately twofold in resistant clone 3 and 7 cells. Southern analysis with the same cDNA probe showed no evidence of gene amplification or rearrangement. Western blot analysis with monoclonal C219 antibody demonstrated a distinct increase in P-glycoprotein in resistant cells. Efflux of Adriamycin as measured by the efflux rate constant was identical in all three cell lines. Furthermore, the metabolic inhibitors azide and dinitrophenol did not augment drug uptake in either sensitive or resistant cells. These findings suggest that despite the increase in P-glycoprotein, an active extrusion pump was not operational in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗