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Multidrug resistance (MDR), a ubiquitous phenomenon conserved from bacteria to humans, causes serious problems in the treatment of human cancers and infections of bacterial and fungal origin. The development of MDR in yeast is frequently associated with gain-of-function mutations in the Zn 2 Cys 6 transcription factors activating the expression of several plasma membrane exporters. In the aerobic yeast Kluyveromyces lactis the Zn 2 Cys 6 transcription factor Kl Pdr1p is involved in the control of multidrug resistance. The aim of the present study was to identify the changes in K. lactis proteome of the Klpdr1Δ deletion mutant compared with the wild-type expressing the KlPDR1 gene from a multicopy plasmid. A total of 15 differentially expressed proteins, out of 20 spots with different intensities detected, were identified. In the Klpdr1Δ deletion mutant, the increase in the abundance of proteins involved in carbohydrate metabolism (mainly glycolysis/gluconeogenesis) was observed. Most of the proteins overexpressed in the wild type strain containing the KlPDR1 gene on multicopy plasmid were involved in the stress defence and redox homeostasis. The results indicate a close connection between MDR and oxidative stress response associated with the post-translational mechanisms regulating the levels of active forms of proteins involved in K. lactis MDR.

Introductions:Although randomized clinical trials showed no benefit from combining epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) with standard chemotherapy for advanced non–small-cell lung cancer (NSCLC), better results might be obtained by combining EGFR-TKI with individual agents that are substrates for the adenosine triphosphate binding cassette transporters (ABCTs) because EGFR-TKIs can inhibit their efflux. The combination effects deserved to be further examined in vitro.Methods:The combination effects of gefitinib with three antimicrotubule agents (AMTAs), paclitaxel, docetaxel or vinorelbine, or with gemcitabine were tested in 17 NSCLC cell lines using the tetrazolium colorimetric assay and classical isobole method. The effects of drug combinations, identified by the values of mean combination index (mCIs), were correlated with the expression levels of ABCTs. Dose-versus-log-response curves were analyzed to further evaluate the possible mechanisms of drug interactions.Results:Synergistic gefitinib/AMTA interactions were observed in the tested cell lines. The synergism was more robust in the four lines overexpressing de novo or acquired P-glycoprotein (Pgp; individual mCIs range, 0.484–0.859; all p values were < 0.05), or in 12 cell lines exhibiting no sensitizing EGFR mutations (group mCIs for gefitinib/paclitaxel, gefitinib/docetaxel, and gefitinib/vinorelbine were 0.869, 0.82, and 0.853, respectively. All p values were < 0.02). The synergism could be observed in cells expressing nearly undetectable Pgp and other ABCTs tested in this study. The combination of gefitinib/gemcitabine was additive (mCI = 1.027).Conclusions:Combined gefitinib/AMTAs showed synergism in NSCLC cells harboring no sensitizing EGFR mutations. Gefitinib could enhance AMTA effects through mechanisms not restricted to Pgp blockage.

Purpose: This study assesses the ability of multidrug resistance (MDR)–associated gene expression patterns to predict survival in patients with newly diagnosed carcinoma of the ovary. The scope of this research differs substantially from that of previous reports, as a very large set of genes was evaluated whose expression has been shown to affect response to chemotherapy. Experimental Design: We applied a customized TaqMan low density array, a highly sensitive and specific assay, to study the expression profiles of 380 MDR-linked genes in 80 tumor specimens collected at initial surgery to debulk primary serous carcinoma. The RNA expression profiles of these drug resistance genes were correlated with clinical outcomes. Results: Leave-one-out cross-validation was used to estimate the ability of MDR gene expression to predict survival. Although gene expression alone does not predict overall survival (OS; P = 0.06), four covariates (age, stage, CA125 level, and surgical debulking) do (P = 0.03). When gene expression was added to the covariates, we found an 11-gene signature that provides a major improvement in OS prediction (log-rank statistic P < 0.003). The predictive power of this 11-gene signature was confirmed by dividing high- and low-risk patient groups, as defined by their clinical covariates, into four specific risk groups on the basis of expression levels. Conclusion: This study reveals an 11-gene signature that allows a more precise prognosis for patients with serous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. These 11 new targets offer opportunities for new therapies to improve clinical outcome in ovarian cancer. Clin Cancer Res; 18(11); 3197–206. ©2012 AACR.

Objective To investigate changes in the expression of hypoxia-inducible factor-1 (HIF-1) in different brain regions in a pharmacoresistance model of temporal lobe epilepsy, and explore its relation to the expression of drug resistance gene/protein multidrug-resistance-gene1(MDR1)/P-glycoprotein (Pgp). Methods A kindling model of pharmacoresistant temporal lobe epilepsy was developed by injecting Sprague-Dawley (SD) rats with coriaria lactone (CL). Normal SD rats were injected with normal sodium (NS) served as control group. The expressions of HIF-1, MDR1/Pgp in brain tissues were detected by real-time fluorescence-based quantitative polymerase chain reaction (RQ-PCR), immunohistochemistry (IHC), Western blotting, respectively. Results IHC showed that the expressions of HIF-1 in neurons and glial cells were higher in the hippocampus and temporal cortex than in other brain regions in kindled rats, and the regional distribution of HIF-1 was similar to that of Pgp. RQ-PCR and Western blotting showed that the expressions of HIF-1 in the hippocampus and temporal cortex were higher in kindled group than in control group (P < 0.05), and the expressions of MDR1/Pgp in gene and protein levels were consistent with the alterations of HIF-1 in both groups. Conclusion HIF-1 expressions in hippocampus and temporal cortex in kindled rats are higher than those in control rats, and it corresponds to the regional distribution of Pgp. The correlation of HIF-1 and MDR1/Pgp expressions indicates that HIF-1 may be related to the drug resistance of refractory temporal lobe epilepsy.

The occurrence of sulfonamide and tetracycline resistance and their pollution profile in the aquaculture environment of Tianjin, northern China, were investigated. The presence of antibiotic-resistant bacteria was identified and the corresponding antibiotic resistance genes (ARGs) were quantified at 6 aquaculture farms in Tianjin. Sulfonamide-resistance genes were prevalent and their concentrations were the highest detected (3.0 × 10 −5 to 3.3 × 10 −4 for sul1 /16S rDNA, 2.0 × 10 −4 to 1.8 × 10 −3 for sul2 /16S rDNA) among the various ARGs, most likely because the use of sulfonamides is more prevalent than tetracyclines in this area. Bacillus was the most dominant bacterial genus in both sulfamethoxazole resistant bacteria (63.27% of the total resistant bacteria) and tetracycline-resistant bacteria (57.14% of the total resistant bacteria). At least two of those genes ( tetM , tetO , tetT , tetW , sul1 and sul2 ) were detected in the isolates of Bacillus cereus , Bacillus subtilis , Bacillus megaterium and Acinetobacter lwofii , and all of the above genes were detected in B. cereus , suggesting the occurrence of multi-resistance in the studied area. The genetic transfer of sul1 between intestinal bacteria (e.g., Enterococcus spp.) and indigenous bacteria (e.g., Bacillus spp.) was implied by phylogenetic analysis. Several strains of resistant opportunistic pathogens (e.g., Acinetobacter spp.) were found in indigenous bacteria, which increase the risk of ARGs to public health. Overall, this is the first study to comprehensively investigate the antibiotic resistance profile by analyzing the species of antibiotic-resistant bacteria and adopting qualitative and quantitative methods to investigate ARGs at a typical aquaculture area in northern China.

Dear Editor: We estimated the prevalence of transmitted drug resistance mutations (TDRMs) and drug-relevant polymorphisms (DRPs) in primary HIV infection in a cohort of youth (ages 18–24) and determined whether demographic or clinical characteristics were associated with prevalence of TDRMs or DRPs. We report the results of a substudy of Adolescent Medicine Trials Network for HIV/AIDS Interventions (ATN) 061, an ongoing study of highly active antiretroviral therapy (HAART) initiation in HIV-positive youth who have CD4+ T cells (CD4) of>350 cells/mm3 with subsequent deintensification. Subjects were screened from 15 ATN U.S. and Puerto Rico sites and from 7 International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) U.S. sites. The study was approved by each participating institution's Institutional Review Board. Subjects were eligible for ATN 061 if they were 18–24 years of age, had a CD4 T-cell count>350/mm3, HIV-1 RNA viral load (VL)≥1000 copies/mL, and were HAART-naive with the exception of ARVs for the prevention of mother to child transmission for a single pregnancy with documentation of virologic suppression (n=5). Enrollment began in 2006 and was completed in 2010. All subjects had a screening genotype and those without specific mutations that were exclusion criteria for ATN 061 (I50L; I84V; N88D/S, D30N; V32I; L33I/F/V; M46I/L; I47V/A; G48V; I50V/L; I54V/L/A/M/T/S; L76V; V82A/F/T/S/L; L90M, or major RT mutations Q151M and 69 insertion complex) were eligible. The screening genotypes from all patients were evaluated for protease inhibitor (PI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) major transmitted drug resistance mutations (TDRMs) and drug relevant polymorphisms (DRPs) according to World Health Organization (WHO) and International AIDS Society (IAS) algorithms.1,2 Alone, DRPs may not confer resistance, however, they may lead to compensatory changes that may confer a fitness advantage to the virus. All of the 130 eligible subjects had screening genotypes. The median age was 21.4 years, 86.9% were male, 69.2% black, and 99.2% acquired HIV sexually (Table 1). Twelve of 130 subjects (9.2%) had a total of 24 TDRMs: 5 of 24 (20.8%) NRTI; 9 of 24 (37.5%) NNRTI; and 10 of 24 (41.7%) PI. Two of 12 subjects with TDRMs (16.7%) had single, 8 of 12 (66.7%) double, and 2 of 12 (16.7%) triple class resistance; 10/130 (7.7%) had 2 or more class resistance. No factors were significantly associated with identification of TDRMs. Table 1. Demographic and Clinical Characteristics of Patients with Transmitted Drug Resistance Mutations and Drug Relevant Polymorphisms DRPs were found in 105 of 130 (80.8%) subjects including the 12 subjects with TDRMs who all had additional DRPs (Tables 1 and ​and2).2). Geographic region was significantly associated with having a DRP (Table 2). Due to a significant interaction of VL with the MSM HIV acquisition risk category (approximately 0.3 log10 higher VL in MSM risk group, p=0.0448), two multivariate models were created, one that includes HIV VL with geographic region and a separate model that includes HIV acquisition risk with geographic region. In both models, Midwestern and Western geographic regions were associated with a decreased likelihood of DRPs as compared with the South (adjusted odds ratio [AOR] 0.166 [95% confidence interval {CI} 0.047–0.591] and AOR 0.093 [95% CI 0.024–0.360]), respectively). For each log10 increase in VL, the odds of having a DRP increased by 2.118 (AOR 2.118 [95% CI 1.033–4.344]). Additionally, MSM HIV acquisition risk category versus heterosexual acquisition risk was independently associated with an increased likelihood of having DRPs (AOR 4.016 [95% CI 1.251–12.890]). No other demographic or clinical associations were identified. Table 2. Factors Associated with the Presence of Drug Relevant Polymorphisms at Baseline in the Univariate Analysis In this geographically diverse study of U.S. youth, 9.2% had TDRMs and 7.7% had multiclass resistance. These rates are an underestimation as youth known to have harbored particular resistance mutations were excluded by the parent study and not captured by the substudy. The multiclass resistance rate was higher than previously reported from similar sites.3 Reported rates of DRPs have ranged from 60% to 90%, in line with the 80.8% rate we found.4,5. The significance of these high DRP prevalence rates is unclear, however, it may be important to continue to monitor as there may be an impact on treatment response to both current and future PIs. Due to their potency and higher genetic resistance barrier, PI-based regimens are particularly important for youth who tend to have lower treatment adherence.6 Additionally, as youth are a group where HIV vaccines may be particularly targeted both for treatment and primary prevention, high prevalence rates of polymorphisms may also be of significance. We report that MSM HIV acquisition risk and higher screening VL were associated with an increased risk of having DRPs, while being in the Midwest and West was associated with a decreased risk of having DRPs compared to the South. Other studies of resistance in youth have not shown differences by geographic location.3,7 However, the Southern region encompasses locales (e.g., Florida) with high incidence and prevalence of HIV infection8; the finding of significant regional differences in the DRP rates may be of concern and in need of further evaluation. Increasing drug resistance among newly infected adult MSM has been reported; and 45% of known HIV-positive and previously treated adult men (age 26–64) in a community sample in a large, urban city were found to harbor resistance to at least one ARV9. The increased risk of DRPs among those with MSM HIV acquisition risk in this study is noteworthy as this mode of transmission is responsible for increasing proportions of infections among behaviorally infected youth; and age bridging (having sex with older men) is a known risk factor for HIV acquisition.10 Many of these older individuals may have long treatment histories and thus antiretroviral exposure with risk of transmitting viral variants with resistance. The association of increasing viral load with DRPs in this study is of concern as it may reflect greater fitness of viral variants with resistance mutations, as well as higher or possibly more efficient risk of transmission of resistant variants in those individuals with increased viremia.11 As this study was not designed to ascertain cause and effect behind this association, this instead may also reflect that viral quasispecies with higher replication capacities are likely to be more error prone. Our study is limited by relatively small numbers, which diminishes our power to examine significant associations. Primary drug resistance is commonly found among HIV-infected youth, with a significant proportion acquiring and harboring multi-drug resistant viruses. These findings are worrisome, underscoring the importance of genotypic testing prior to ARV initiation for treatment of HIV infection, particularly if ARV pre- and postexposure prophylaxis becomes more frequently prescribed as part of HIV prevention.12 The significance of the high prevalence of DRPs is unclear and needs further study.

International audience; Salmonella genomic island I (variant SGII-J3) has been previously identified in multi-drug resistant (MDR) Salmonella enterica serovar Virchow isolated from humans in 1994. In this study, antimicrobial resistance, genotypes and genetic relationship were investigated in 96 S. Virchow isolates collected from humans in 2004-2006. XbaI-PFGE analysis separated 96 isolates into two main related-clusters, I and II, which consisted of four major pulsotypes differing in prevalence by year. The majority of isolates were MDR to chloramphenicol, sulfonamide, trimethoprim and tetracyclines associated with antimicrobial resistance genes dfrA1, floR2, sull and tet(G) of variant SGII-J3. Among nine variants, we determined two novel variants SGII-J4 and -J5, which have undergone different homologous recombinational events resulting in partial deletions of the MDR region. The first one contained an empty integron structure and the second presented a deletion extending from the IS6100 element to the adjacent SGII backbone. SGII-J3 is largely encountered in clonally related MDR S. Virchow isolates collected from humans which spread vertically. The genomic island SGII appears to be largely responsible for the diversity of MDR phenotypes among S. Virchow isolates in Taiwan.

Objective: Isoniazid (INH) and rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis. Mutations in several genes determine resistance of M. tuberculosis to INH, with the most common gene target of katG, and resistance to RIF is due to mutation in rpoB gene. The aim of present study was to assess the mutations in the regions related to RIF and INH resistance. Methods: We characterized 80 clinical isolates of confirmed M. tuberculosis to analyze the most commonly observed INH and RIF mutations. PCR analysis and sequencing were used to detect mutations related to RIF and INH resistance. The multiplex allele-specific-PCR (MAS-PCR) was performed as a comparative assay and for evaluation of this method. Results: The sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7% of the M. tuberculosis isolates. The sequencing of the 270-bp central region of the rpoB gene revealed point mutations at 7 different codons in 12 (15%) of the M. tuberculosis isolates. The results obtained with MAS-PCR are in accordance with PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526). Conclusion: The results of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of M. tuberculosis. Both DNA sequencing and MAS-PCR yielded high sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases. Keywords: Drug resistance, Genes, MDR, Mutation, Polymerase chain reaction

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of pain and inflammation. However, this group of drugs is associated with serious adverse drug reactions. Previously, we studied the mechanisms underlying toxicity of the NSAID diclofenac using Saccharomyces cerevisiae as model system. We identified the involvement of several mitochondrial proteins, a transporter and cytochrome P450 activity in diclofenac toxicity. In this study, we investigated if these processes are also involved in the toxicity of other NSAIDs. We divided the NSAIDs into three classes based on their toxicity mechanisms. Class I consists of diclofenac, indomethacin and ketoprofen. Mitochondrial respiration and reactive oxygen species (ROS) play a major role in the toxicity of this class. Metabolism by cytochrome P450s further increases their toxicity, while ABC-transporters decrease the toxicity. Mitochondria and oxidative metabolism also contribute to toxicity of class II drugs ibuprofen and naproxen, but another cellular target dominates their toxicity. Interestingly, ibuprofen was the only NSAID that was unable to induce upregulation of the multidrug resistance response. The class III NSAIDs sulindac, ketorolac and zomepirac were relatively non-toxic in yeast. In conclusion, we demonstrate the use of yeast to investigate the mechanisms underlying the toxicity of structurally related drugs. © 2011 Elsevier Ltd.

Since the discovery of antibiotics, the battle between humans and drug resistant bacteria has never stopped. Bacteria have developed various ways to resist the toxic effects of antibiotics and other drugs. Multidrug efflux pumps are integral membrane proteins that utilize cellular energy to extrude antibiotics or biocides actively out of the cell. In this symposium, I first introduce the post-genomic approach to analyze all putative drug efflux genes. Next, I discuss the regulation of drug efflux pumps responding to environmental signals. I also introduce the physiological roles of drug efflux pumps in virulence, which is an ongoing research area. Multidrug efflux pumps have greater clinical relevance than has previously been thought, because there is now accumulating evidence that certain classes of efflux pumps not only confer resistance to drugs used in therapy but also have a role in bacterial pathogenicity.

Multidrug-resistant (MDR) Acinetobacter baumannii is an opportunistic human pathogen that has become highly problematic in the clinical environment. Novel therapies are desperately required. To assist in identifying new therapeutic targets, the antagonistic interactions between A. baumannii and the most common human fungal pathogen, Candida albicans , were studied. We have observed that the C. albicans quorum-sensing molecule, farnesol, has cross-kingdom interactions, affecting the viability of A. baumannii . To gain an understanding of its mechanism, the transcriptional profile of A. baumannii exposed to farnesol was examined. Farnesol caused dysregulation of a large number of genes involved in cell membrane biogenesis, multidrug efflux pumps (AcrAB-like and AdeIJK-like), and A. baumannii virulence traits such as biofilm formation ( csuA , csuB , and ompA ) and motility ( pilZ and pilH ). We also observed a strong induction in genes involved in cell division ( minD , minE , ftsK , ftsB , and ftsL ). These transcriptional data were supported by functional assays showing that farnesol disrupts A. baumannii cell membrane integrity, alters cell morphology, and impairs virulence characteristics such as biofilm formation and twitching motility. Moreover, we showed that A. baumannii uses efflux pumps as a defense mechanism against this eukaryotic signaling molecule. Owing to its effects on membrane integrity, farnesol was tested to see if it potentiated the activity of the membrane-acting polymyxin antibiotic colistin. When coadministered, farnesol increased sensitivity to colistin for otherwise resistant strains. These data provide mechanistic understanding of the antagonistic interactions between diverse pathogens and may provide important insights into novel therapeutic strategies.

The presence of tumor cells in effusions within serosal cavities is a clinical manifestation of advanced-stage cancer and is generally associated with poor survival. Identifying molecular targets may help to design efficient treatments to eradicate these aggressive cancer cells and improve patient survival. Using a state-of-the-art TaqMan-based qRT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of 32 unpaired ovarian serous carcinoma effusion samples obtained at diagnosis or at disease recurrence following chemotherapy. MDR genes were selected a priori based on an extensive curation of the literature published during the last three decades. We found three gene signatures with a statistically significant correlation with overall survival (OS), response to treatment [complete response (CR) vs other], and progression free survival (PFS). The median log-rank p-values for the signatures were 0.023, 0.034, and 0.008, respectively. No correlation was found with residual tumor status after cytoreductive surgery, treatment (with or without chemotherapy) and stage defined according to the International Federation of Gynecology and Obstetrics. Further analyses demonstrated that gene expression alone can effectively predict the survival outcome of women with ovarian serous carcinoma (OS, log-rank p = 0.0000; and PFS, log-rank p = 0.002). Interestingly, the signature for overall survival is the same in patients at first presentation and those who had chemotherapy and relapsed. This pilot study highlights two new gene signatures that may help in optimizing the treatment for ovarian carcinoma patients with effusions.

The genome sequences of a number of Acinetobacter baumanii strains, including representatives of the main epidemic international lineages, have now been determined, and several others are in progress. The study of A. baumannii genomics has provided an expanded view of the adaptation and virulence capacities of this bacterial species, whilst also presenting novel insights into its intraspecies diversity and genome evolution. Genomic analyses have revealed that the current A. baumannii clinical population consists of low-grade pathogens, whose pathogenicity relies mainly on an ability to persist in the hospital setting and survive antibiotic treatment. A. baumannii has a high capacity to acquire new genetic determinants and displays an open pan genome; this feature may have played a crucial role in the evolution of this human opportunistic pathogen towards clinical success. (C) 2011 IUBMB IUBMB Life, 63(12): 1068-1074, 2011

There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44+CD24- population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10(4)sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.

Co-resistance involves transfer of several genes into the same bacteria and/or the acquisition of mutations in different genetic loci affecting different antimicrobials whereas pleiotropic resistance implies the same genetic event affecting several antimicrobials. There is an increasing prevalence of isolates with co-resistance which are over-represented within the so-called high-risk clones. Compensatory events avoid fitness cost of co-resistance, even in the absence of antimicrobials. Nevertheless, they might be selected by different antimicrobials and a single agent might select co-resistant isolates. This process, named as co-selection, is not avoided with cycling or mixing strategies of antimicrobial use. Co-resistance and co-selection processes increase the opportunity for persistence of the bacteria and resistance genes and should be considered when designing strategies for decreasing antimicrobial resistance.

In this study, we have analyzed the expression of the Pseudomonas syringae pv. tomato DC3000 mexAB-oprM efflux pump operon and of the regulatory gene pmeR, and we have investigated the role of the PmeR protein on transcription from both promoters. We demonstrate that mexAB-oprM and pmeR are expressed in vivo at a relatively high and moderate basal level, respectively, which, in both cases, increases in the presence of different flavonoids and other compounds, such as butyl and methylparaben. We show that PmeR is the local repressor of the mexAB-oprM promoter and is able to regulate its own expression. The mechanism for this regulation includes binding to a pseudopalindromic operator site which overlaps both mexAB-oprM and pmeR promoters. We have also proven that flavonoids are able to interact with PmeR and induce a conformational change that interferes with the DNA binding ability of PmeR, thereby modulating mexAB-oprM and pmeR expression. Finally, we demonstrate by in vivo experiments that the PmeR/MexAB-OprM system contributes to the colonization of tomato plants. These results provide new insight into a transcriptional regulator and a transport system that play essential roles in the ability of P. syringae pv. tomato DC3000 to resist the action of flavonoids produced by the host.

This study evaluated the presence of carbapenem hydrolysing β-lactamase genes and plasmid-mediated quinolone resistance (PMQR) determinants in 22 Klebsiella pneumoniae isolates collected from the Istanbul Medical Faculty, Turkey, which reduced the susceptibility or resistance to carbapenem. The VITEK® 2 system and E-tests were used to determine the minimum inhibitory concentrations needed to inhibit bacterial growth. Genes were screened by polymerase chain reaction, and gene transferability was evaluated by transconjugation. Strain clonality was investigated by pulsed-field gel electrophoresis (PFGE). All strains were OXA-48 β-lactamase producers and three (13.6%) were also positive for the aac(6′)-Ib-cr gene. Most of the strains harboured other b-lactamase (bla) genes such as blaTEM, blaSHV, blaCTX-M and blaVEB-1. The transconjugants mostly harboured blaOXA-48 and other β-lactamases separately. PFGE revealed eight pulsotypes among the isolates. The coexistence of blaOXA-48 and PMQR in K. pneumoniae isolates may present a significant threat to health, especially in the nosocomial setting.

Minireview.-- Free via Open Access., In recent years, the explosive spread of antibiotic resistance determinants among pathogenic, commensal, and environmental bacteria has reached a global dimension. Classical measures trying to contain or slow locally the progress of antibiotic resistance in patients on the basis of better antibiotic prescribing policies have clearly become insufficient at the global level. Urgent measures are needed to directly confront the processes influencing antibiotic resistance pollution in the microbiosphere. Recent interdisciplinary research indicates that new eco-evo drugs and strategies, which take ecology and evolution into account, have a promising role in resistance prevention, decontamination, and the eventual restoration of antibiotic susceptibility. This minireview summarizes what is known and what should be further investigated to find drugs and strategies aiming to counteract the ¿four P¿s,¿ penetration, promiscuity, plasticity, and persistence of rapidly spreading bacterial clones, mobile genetic elements, or resistance genes. The term ¿drug¿ is used in this eco-evo perspective as a tool to fight resistance that is able to prevent, cure, or decrease potential damage caused by antibiotic resistance, not necessarily only at the individual level (the patient) but also at the ecological and evolutionary levels. This view offers a wealth of research opportunities for science and technology and also represents a large adaptive challenge for regulatory agencies and public health officers. Eco-evo drugs and interventions constitute a new avenue for research that might influence not only antibiotic resistance but the maintenance of a healthy interaction between humans and microbial systems in a rapidly changing biosphere., Work in the laboratories of F.B. and T.M.C. is funded by research grants from the European Commission (LSHM-2006-037410, LSHMCT-2008-223031, KBBE-2008-2B-227258, and PAR-241476-FPHEALTH 2009), the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (grants PS09/02381 and PI10/02588; the CIBERESP research network in Epidemiology and Public Health [CB06/02/0053]), and the Regional Government of Madrid (DeRemicrobiana Network [CAM.S-SAL-0246- 2006]). Work in the laboratory of F.D.L.C. is supported by research grants from the Spanish Ministry of Education (BFU2008-00995/ BMC), the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (REIPI network [RD06/0008/1012]) and the European Commission (LSHM-CT- 2005_019023). We are also grateful to the Spanish Network for the Study of Plasmids and Extrachromosomal Elements (REDEEX) for encouraging and funding cooperation among Spanish microbiologists working on the biology of mobile genetic elements (Spanish Ministry of Science and Innovation, reference number BFU 2008-0079-E/ BMC).

A 4-bp deletion mutation associated with multiple drug sensitivity exists in the canine multidrug resistance (MDR1) gene. This mutation has been detected in more than 10 purebred dog breeds as well as in mixed breed dogs. To evaluate the breed distribution of this mutation in Germany, 7378 dogs were screened, including 6999 purebred and 379 mixed breed dogs. The study included dog breeds that show close genetic relationship or share breeding history with one of the predisposed breeds but in which the occurrence of the MDR1 mutation has not been reported. The breeds comprised Bearded Collies, Anatolian Shepherd Dog, Greyhound, Belgian Tervuren, Kelpie, Borzoi, Australian Cattle Dog and the Irish Wolfhound. The MDR1 mutation was not detected is any of these breeds, although it was found as expected in the Collie, Longhaired Whippet, Shetland Sheepdog, Miniature Australian Shepherd, Australian Shepherd, Wäller, White Swiss Shepherd, Old English Sheepdog and Border Collie with varying allelic frequencies for the mutant MDR1 allele of 59%, 45%, 30%, 24%, 22%, 17%, 14%, 4% and 1%, respectively. Allelic frequencies of 8% and 2% were determined in herding breed mixes and unclassified mixed breeds, respectively. Because of its widespread breed distribution and occurrence in many mixed breed dogs, it is difficult for veterinarians and dog owners to recognise whether MDR1-related drug sensitivity is relevant for an individual animal. This study provides a comprehensive overview of all affected dog breeds and many dog breeds that are probably unaffected on the basis of ∼15,000 worldwide MDR1 genotyping data.

Current views of multidrug (MD) recognition focus on large drug-binding cavities with flexible elements. However, MD recognition in BmrR is supported by a small, rigid drug-binding pocket. Here, a detailed description of MD binding by the noncanonical BmrR protein is offered through the combined use of X-ray and solution studies. Low shape complementarity, suboptimal packing, and efficient burial of a diverse set of ligands is facilitated by an aromatic docking platform formed by a set of conformationally fixed aromatic residues, hydrophobic pincer pair that locks the different drug structures on the adaptable platform surface, and a trio of acidic residues that enables cation selectivity without much regard to ligand structure. Within the binding pocket is a set of BmrR-derived H-bonding donor and acceptors that solvate a wide range of ligand polar substituent arrangements in a manner analogous to aqueous solvent. Energetic analyses of MD binding by BmrR are consistent with structural data. A common binding orientation for the different BmrR ligands is in line with promiscuous allosteric regulation.

Bacteria showing antimicrobial resistance (AMR) pose a significant global healthcare problem. Although many mechanisms conferring AMR are understood, the ecological processes facilitating its persistence and spread are less well characterised. Aquatic systems represent an important milieu for the environmental release, mixing, persistence and spread of AMR bacteria and resistance genes associated with horizontally transferable genetic elements. Additionally, owing to the use and discharge of antimicrobials and biocides, and the accumulation and abundance of other pollutants, mechanisms that confer AMR might evolve in aquatic systems. In this review, we hypothesise that aquatic systems have an important ecological and evolutionary role in driving the persistence, emergence and spread of AMR, which could have consequences when attempting to reduce its occurrence in clinical settings.

A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), β-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal sample shared resistance genes at a significant level (χ², p

The contributions of multicomponent-type multidrug efflux pumps to antimicrobial resistance and nodulation ability in Sinorhizobium meliloti were comprehensively analyzed. Computational searches identified genes in the S. meliloti strain 1021 genome encoding 1 pump from the ATP-binding cassette family, 3 pumps from the major facilitator superfamily, and 10 pumps from the resistance-nodulation-cell division family, and subsequently, these genes were deleted either individually or simultaneously. Antimicrobial susceptibility tests demonstrated that deletion of the smeAB pump genes resulted in increased susceptibility to a range of antibiotics, dyes, detergents, and plant-derived compounds and, further, that specific deletion of the smeCD or smeEF genes in a Δ smeAB background caused a further increase in susceptibility to certain antibiotics. Competitive nodulation experiments revealed that the smeAB mutant was defective in competing with the wild-type strain for nodulation. The introduction of a plasmid carrying smeAB into the smeAB mutant restored antimicrobial resistance and nodulation competitiveness. These findings suggest that the SmeAB pump, which is a major multidrug efflux system of S. meliloti , plays an important role in nodulation competitiveness by mediating resistance toward antimicrobial compounds produced by the host plant.

To increase understanding of community-acquired resistance, stool samples from 477 nonhospitalized persons in Maryland and Michigan, from 2004 to 2008, were screened for ceftriaxone resistance. Seven (1.5%) yielded ceftriaxone-resistant Escherichia coli; one isolate was resistant to all eight antimicrobial classes routinely tested: aminoglycosides, β-lactam/β-lactamase inhibitor combinations, cephems, penicillins, folate pathway inhibitors, phenicols, quinolones, and tetracyclines. The extensively resistant isolate was from a 50-year-old woman who denied antimicrobial use, hospitalization, or international travel within 6 months. Meat (beef, chicken, and pork) and eggs were consumed within 1 month before stool collection. Further studies are warranted to understand potential sources, including the food supply, of resistant E. coli.

We genetically analyzed the roles of multidrug efflux pumps on the biofilm formation of Escherichia coli K-12 BW25113. We used 22 mutants missing various genes related to the multidrug efflux pumps and found that the biofilm formation of emrD, emrE, emrK, acrD, acre and mdtE-deleted mutants was extremely decreased. These results indicate that some multidrug efflux pumps significantly contribute to the biofilm formation of E. coli.

The emergence of resistance to all classes of antibiotics in previously susceptible bacterial pathogens is a major challenge to infectious disease medicine. The origin of the genes associated with resistance has long been a mystery. There is a growing body of evidence that is demonstrating that environmental microbes are highly drug resistant. The genes that make up this environmental resistome have the potential to be transferred to pathogens and indeed there is some evidence that at least some clinically relevant resistance genes have originated in environmental microbes. Understanding the extent of the environmental resistome and its mobilization into pathogenic bacteria is essential for the management and discovery of antibiotics.

Regulatory mechanisms for chromosomal genes encoding multidrug resistance (MDR) efflux pumps (EPs) in Staphylococcus aureus are poorly defined. Microbiological, quantitative gene expression, mRNA half-life and genome data for 11 strains of S. aureus combined with bioinformatic analyses were used to identify correlates of increased MDR EP gene expression. The presence of qacA/B and/or increased expression of one to two MDR EP genes were identified in eight strains. Microbiological and gene expression data correlated in four instances, existing knowledge of the substrate specificity of NorC resulted in correlation in two others, and a transcriptional/translational disconnect is possible for the remaining two. In silico analyses and mRNA half-life determinations linked insertions of nucleotide repeats 3' to the -10 motif of the norA promoter with increased promoter activity. Mutations in the 5'-untranslated and/or coding regions were identified that may affect transcription efficiency. Substitutions of residues in the helix-turn-helix (HTH) motif of NorG may augment its positive regulation of norB. The correlations proposed provide a guide for further experimentation leading to a better understanding of MDR EP gene expression in this important pathogen.

For detection of the nt230[del4] MDR1 mutation, a 4-bp deletion in the canine MDR1 (ABCB1) gene, a TaqMan allelic discrimination assay was designed that allows for MDR1 genotyping without post-PCR processing. Directly after completion of the PCR amplification, the MDR1 genotype can be assigned based on selective fluorescence measurement. For primer selection the locus of a potential 265A>G single nucleotide polymorphism was omitted; this locus is covered by the oligonucleotide PCR primers from most of the hitherto established MDR1 genotyping methods. Dogs homozygous for the nt230[del4] MDR1 mutation show highly increased susceptibility to many drugs commonly used in veterinary medicine including ivermectin. As more than 10 dog breeds are predisposed to this mutation, reliable genotyping methods are necessary to identify affected dogs before drug treatment. This study provides a new allelic discrimination method that detects the MDR1 mutation with high specificity and reliability and is useful for routine diagnostics.

The progress of drug resistance of RLS(40) resistant lymphosarcoma and specific features of toxic lesions in the liver in polychemotherapy were studied. After intramuscular injection of tumor cells, the mice received a course of polychemotherapy. The tumor material was then collected and transplanted to intact animals, after which polychemotherapy was carried out. A total of 4 passages of tumor cells and 4 polychemotherapy courses were carried out. The expression of mdr1b, bcl-2, and p53 genes in tumor cells increased by 1.3, 2.3, and 1.6 times after 4 courses of polychemotherapy in comparison with intact tumor. Volume density of apoptoses in tumor tissue after 4 polychemotherapy courses decreased 1.7 times compared to that after single course. The increase in cytostatic load was associated with aggravation of destructive changes in the liver: the volume density of necroses in the liver increased 1.3 times after 4 passages of the tumor.

Staphylococcus aureus is an important human pathogen responsible for life-threatening septicemia, endocarditis, and toxic shock syndrome. Although positive (MRSA; ATCC 33591) and negative (MSSA; ATCC 25923) control strains have been used for various pathogenesis or assay studies, little is known about the genomic structure of the strains, and there has been little genome-wide expression analysis. Phylogenetic analyses revealed that ATCC 33591 and ATCC 25923 are the most genetically diverse strains of the 15 S. aureus genomes studied. Microarray analysis showed that the most significantly upregulated group of MRSA genes was the transport group, which includes ATP-binding cassette (ABC) transporters, the two-component system, and the phosphotransferase system. Analysis of the KEGG pathway showed that ABC transporters and the two-component system were the most significantly altered in MRSA. Transcriptional profiling showed a clear difference in gene expression between MRSA and MSSA due to the great genetic distance between the two control strains. Therefore, we suggest that use of the two control strains in comparative genomics or transcriptomics studies would facilitate the identification of major genes for drug resistance in S. aureus.

The thiopyranopyridine moiety was synthesized as a new heterocyclic base to be inserted at the C-7 position of selected quinolone nuclei followed by a determination of antibacterial activity against strains of Staphylococcus aureus. Selected thiopyranopyridinylquinolones showed significant antimicrobial activity, including strains having mutations in gyrA and grlA as well as other strains overexpressing the NorA multidrug (MDR) efflux pump. Most derivatives did not appear to be NorA substrates. The effect of the thiopyranopyridinyl substituent on making these quinolones poor substrates for NorA was investigated further. Several quinolone ester intermediates, devoid of any intrinsic antibacterial activity, were tested for their abilities to inhibit the activities of NorA (MFS family) and MepA (MATE family) S. aureus MDR efflux pumps. Selected quinolone esters were capable of inhibiting both MDR pumps more efficiently than the reference compound reserpine. Moreover, they also were able to restore, and even enhance, the activity of ciprofloxacin toward some genetically modified resistant S. aureus strains.

Class III multidrug resistance P-glycoproteins, Mdr2 in mice and MDR3 in human, are canalicular phospholipid translocators involved in biliary phospholipid (phosphatidylcholine) excretion. The role of an ABCB4 gene defect in liver disease has been initially proven in a subtype of progressive familial intrahepatic cholestasis called PFIC3, a severe pediatric liver disease that may require liver transplantation. Several ABCB4 mutations have been identified in children with PFIC3 and are associated with low level of phospholipids in bile leading to a high biliary cholesterol saturation index. ABCB4 mutations are associated with loss of canalicular MDR3 protein and /or loss of protein function. There is evidence that a biallelic or monoallelic ABCB4 defect causes or predisposes to several human liver diseases (PFIC3, low phospholipid associated cholelithiasis syndrome, intrahepatic cholestasis of pregnancy, drug-induced liver injury, transient neonatal cholestasis, adult biliary fibrosis, or cirrhosis). Most patients with MDR3 deficiency have a favorable outcome with ursodeoxycholic acid (UDCA) therapy, but some PFIC3 patients who do not respond to UDCA treatment still require liver transplantation. The latter should be good candidates for a targeted pharmacologic approach and/or to cell therapy in the future.

Background/aims Locoregional chemotherapy continues to be the mainstay for the treatment of unresectable hepatocellular carcinoma (HCC). One of the principal obstacles implicated in its unsuccessful therapy is multidrug resistance (MDR). Former studies have identified the multidrug-resistant nature and possible mechanisms of hepatoma cells both in vitro and in vivo. This work aimed to develop an effective strategy for the treatment of HCC with MDR. Methods The treatment was exploited to inhibit the MDR cells by co-administration of the recombinant mutant human tumour necrosis factor-alpha (rmhTNF-alpha), a sublethal dose of chemicals [adriamycin (ADM), mitomycin and 5-FU] and hydroxyapatite nanoparticles (nHAPs). Real-time quantitative reverse transcriptase-polymerase chain reaction and electrochemiluminescence Western blot were used to detect the expression of several related genes. Results The chemicals acted synergistically with rmhTNF-alpha and nHAP in suppressing the growth of hepatoma cells and inducing apoptosis of the cells, with the MDR phenotype reversed, as measured by intracellular ADM retention. Analysis of mRNA and protein revealed that rmhTNF-alpha inhibited the gene expression of XIAP, survivin, Ki67, PCNA, MDR1 and BCRP to some extent. Moreover, the inhibitory effects mentioned above could be as good or better than when nHAP is incorporated into the regimens. Conclusions rmhTNF-alpha was not only able to restore the chemotherapeutic sensitivity to HepG2/ADM, its xenograft model and clinical samples but also further inhibited the growth of these tumours by a combination of nHAP. These results strongly suggested that chemicals in combination with rmhTNF-alpha and nHAP may be beneficial for the local treatment of advanced HCC.

Background. Highly lethal outbreaks of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are increasing. Whole-genome sequencing of KwaZulu-Natal MDR and XDR outbreak strains prevalent in human immunodeficiency virus (HIV)-infected patients by the Broad Institute identified 22 novel mutations which were unique to the XDR genome or shared only by the MDR and XDR genomes and not already known to be associated with drug resistance. Methods. We studied the 12 novel mutations which were not located in highly-repetitive genes to identify mutations that were truly associated with drug resistance or were likely to confer a specific fitness advantage. Results. None of these mutations could be found in a phylogenetically and geographically diverse set of drug-resistant and drug-susceptible Mycobacterium tuberculosis isolates, suggesting that these mutations are unique to the KZN clone. Examination of the 600-basepair region flanking each mutation revealed 26 new mutations. We searched for a convergent evolutionary signal in the new mutations for evidence that they emerged under selective pressure, consistent with increased fitness. However, all but 1 rare mutation were monophyletic, indicating that the mutations were markers of strain phylogeny rather than fitness or drug resistance. Conclusions. Our results suggest that virulent XDR tuberculosis in immunocompromised HIV-infected patients can evolve without generalizable fitness changes or other XDR-specific mutations.

Although the architecture of tripartite multiple drug resistance (MDR) efflux pumps of Gram-negative bacteria has been well characterized, the means by which the components recognize each other and assemble into a functional pump remains obscure. In this study we present evidence that the C-terminal domain of the Pseudomonas aeruginosa OprM and the α-helical hairpin domain of Vibrio cholerae VceA play an important role in the recognition/specificity/recruitment step in the assembly of a functional, VceAB-OprM chimeric efflux pump. To our knowledge, this is the first evidence directly linking the C-terminal domain of an outer membrane efflux protein to its recruitment during the assembly of a tripartite efflux pump.

Please cite this paper as: In vivo synthesis and secretion of erythropoietin by genetically modified primary human keratinocytes grafted onto immunocompromised mice. Experimental Dermatology 2010; 19: 289–297. Abstract Background: The skin is an easily accessible tissue with a high blood flow facilitating the distribution of secreted peptides. These features make it a very intriguing target to serve as a biofactory releasing a systemically needed factor, such as erythropoietin (EPO). Methods: To evaluate the potential of human keratinocytes (KC) to systemically synthesize EPO, EPO-transduced KC were grafted onto immunocompromised mice and EPO secretion was followed by serum ELISA. Furthermore, we assessed if topical colchicine application would select for enriched percentages of KC expressing the multi-drug resistance (MDR) gene as a selectable gene connected to the EPO gene (measured by fluorescence-activated cell sorting (FACS)-analysis) and result in enhanced EPO production (determined by ELISA). Results: Transduced KC showed stable EPO production in vivo during a 6-month observation period, pointing to engraftment of EPO-secreting KC progenitor cells. When adding colchicines the number of EPO/MDR+ KC were significantly enriched, both in skin grafts (in vivo) and in skin equivalents (in vitro). Of note, this did not result in enhanced EPO production. Rather, while EPO secretion was substantially increased in transduced KC grown as monolayers and selected with colchicine, it was reduced by more than 50% in both colchicine-treated skin grafts and skin equivalents. Conclusion: Keratinocytes carry the potential to serve as a genetically modified biofactory synthesizing human EPO. In vivo gene selection does not allow to select for increased EPO secretion, most likely because of altered secretory activity of transduced KC in the stratified, differentiated epidermis. Thus, further studies are necessary to optimize the release of EPO by genetically modified KC.

The human multidrug resistance (MDR1) gene product P-glycoprotein is highly expressed in intestinal epithelial cells, where it constitutes a barrier against xenobiotics, bacterial toxins, drugs and other biologically active compounds, possibly carcinogens. In this study, an association of MDR1 gene polymorphism and the occurrence of colorectal cancer were evaluated. In this case-control-designed 118 unrelated colorectal cancer and 137 sex-and-ages matched healthy controls were enrolled. The C3435T MDR1 gene polymorphism was identified using the polymerase chain reaction-restriction fragment length polymorphism method. Significantly increased frequencies of the 3435T allele and the 3435TT were observed in patients with colorectal cancer compared with controls (P = 0.03; OR, 95% CI; 1.46 for 3435T allele and P = 0.003; OR, 95% CI; 2.2 for 3435TT genotype). In contrast, frequency of genotype TT was significantly higher in controls compared to colorectal cancer (P = 0.006; OR, 95% CI; 0.49 for TC genotype). In this study suggest that C3435T MDR1 polymorphism has an association with colorectal cancer. The results support that the presence of allele C results in decreased susceptibility to colorectal cancer.

Human P-glycoprotein (P-gp), encoded by MDR1 (ABCB1), is an efflux transporter with a wide specificity for substrates/drugs, including HIV protease inhibitors which are commonly used in HIV/AIDS treatment. Three single nucleotide polymorphisms (SNPs) in MDR1 have been shown to affect P-gp expression and function, and may affect HIV/AIDS treatment outcome: 1236C>T [G412G, exon-12], 2677G>T/A [A893S/T, exon-21] and 3435C>T [I1145I, exon-26]. In the present study, our aims were (i) to compare the 3-SNP MDR1 haplotype structure and genetic diversity between North American HIV-positive and HIV-negative individuals belonging to four major ethnic groups and (ii) to determine whether the haplotype structure and genetic diversity observed in these ethnically admixed populations differ from that in ethnically non-admixed populations. For these aims, we analyzed a cohort of 447 HIV/AIDS patients (White [n = 193], Black [n = 235], Hispanic [n = 17], and Asian [n = 2]). Results obtained for these patients were compared with the results for (i) HIV-negative individuals (n = 356) and (ii) various HapMap and Environmental Genome Project populations. We observed that the genetic characteristics of MDR1 were largely consistent between HIV-positive and HIV-negative populations, but there were striking interethnic differences in the genetic characteristics of MDR1 in both populations. Although it appeared that the genetic characteristics of MDR1 were largely consistent between ethnically admixed and non-admixed populations, genetic characterization of the admixed populations remains to be done. Thus, our results provide useful comparative insights about the genetic characteristics of MDR1 that could be extrapolated across population groups worldwide. For a meaningful interpretation of these results regarding HIV/AIDS treatment outcome, MDR1 haplotype/diplotype structure data, genetic characterization of population admixture, and polymorphisms in other relevant drug transporter and/or metabolizing enzyme genes should be considered in future clinical studies.