EVI1 upregulates PTGS1 (COX1) and decreases the action of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia cells.

Tyrosine kinase inhibitors (TKIs) are highly effective in treating chronic myelogenous leukemia (CML). However, primary and acquired drug resistance to TKIs have been reported. In this study, we used RNA sequencing followed by RQ-PCR to show that the proto-oncogene EVI1 targets the drug-metabolizing gene PTGS1 in CML. The PTGS1 promoter element had an EVI1 binding site, and CHIP assay confirmed its presence. Data from a publicly available CML microarray dataset and an independent set of CML samples showed a significant positive correlation between EVI1 and PTGS1 expression in CML. Downregulation of EVI1 in K562 cells and subsequent treatment with TKIs resulted in a lower IC50 in the control cells. Furthermore, combined inhibition of BCR-ABL with imatinib and PTGS1 with FR122047 (PTGS1 inhibitor) synergistically reduced the viability of imatinib-resistant K562 cells. We conclude that elevated EVI1 expression contributes to TKIs resistance and that combined inhibition of PTGS1 and BCR-ABL may represent a novel therapeutic approach.

Involvement of topoisomerase mutations and qnr and aac(6')Ib-cr genes in conferring quinolone resistance to clinical isolates of Vibrio and Shigella spp. from Kolkata, India (1998-2009).

OBJECTIVES: Quinolone antibiotics have been widely used to treat diarrhoeal diseases caused by bacterial agents such as those belonging to the genera Vibrio and Shigella. As these pathogens are accumulating quinolone resistance, treating infections caused by them has become complicated. METHODS: In this study, Vibrio and Shigella spp. isolates obtained from diarrhoeal patients from Kolkata, India, over a period of 12 years (1998-2009) were analysed for quinolone resistance. A total of 27 Vibrio spp. (9 Vibrio cholerae, 11 Vibrio fluvialis and 7 Vibrio parahaemolyticus) and 10 Shigella spp. isolates (7 Shigella flexneri, 2 Shigella dysenteriae and 1 Shigella sonnei) showing reduced susceptibility to quinolones were studied to unravel the genetic factors responsible for quinolone resistance. RESULTS: Antimicrobial susceptibility testing showed a wide spectrum and varying degree of resistance to different generations of quinolones. Genotypic characterisation revealed the involvement of GyrA(S83I) and ParC(S85L) mutations in V. cholerae and V. fluvialis, whereas Shigella spp. isolates showed the mutations S83L and/or D87N/Y in GyrA and S80I or E84K in ParC. Analysis of plasmid-mediated quinolone resistance genes showed that qnrVC5 was detected in three V. fluvialis isolates, aac(6')-Ib-cr in one V. fluvialis isolate and qnrS1 in a S. flexneri isolate. CONCLUSIONS: These results emphasise that quinolone resistance is widespread and therefore quinolones should be used prudently. To the best of our knowledge, this is the first study where resistance to various generations of quinolones in Vibrio and Shigella spp. has been examined in terms of detailed genotype-phenotype correlation.

Characterization of Vibrio fluvialis qnrVC5 Gene in Native and Heterologous Hosts: Synergy of qnrVC5 with other Determinants in Conferring Quinolone Resistance.

Resistance of various pathogens toward quinolones has emerged as a serious threat to combat infections. Analysis of plethora of genes and resistance mechanisms associated with quinolone resistance reveals chromosome-borne and transferable determinants. qnr genes have been found to be responsible for transferable quinolone resistance. In the present work, a new allele qnrVC5 earlier reported in Vibrio fluvialis from this laboratory was characterized in detail for its sequence, genetic context and propensity to decrease the susceptibility for quinolones. The study has revealed persistence of qnrVC5 in clinical isolates of V. fluvialis from Kolkata region through the years 2002-2006. qnrVC5 existed in the form of a gene cassette with the open reading frame being flanked by an upstream promoter and a downstream V. cholerae repeat region suggestive of its superintegron origin. Sequence analysis of different qnrVC alleles showed that qnrVC5 was closely related to qnrVC2 and qnrVC4 and these alleles were associated with V. cholerae repeats. In contrast, qnrVC1, qnrVC3, and qnrVC6 belonging to another group were associated with V. parahaemolyticus repeats. The gene manifested its activity in native V. fluvialis host as well as in Escherichia coli transformants harboring it by elevating the MIC toward various quinolones by twofold to eightfold. In combination with other quinolone resistance factors such as topoisomerase mutations and aac(6')-Ib-cr gene, qnrVC5 gene product contributed toward higher quinolone resistance displayed by V. fluvialis isolates. Silencing of the gene using antisense peptide nucleic acid sensitized the V. fluvialis parent isolates toward ciprofloxacin. Recombinant QnrVC5 vividly demonstrated its role in conferring quinolone resistance. qnrVC5 gene, its synergistic effect and global dissemination should be perceived as a menace for quinolone-based therapies.

Bacterial quorum sensing inhibitors: attractive alternatives for control of infectious pathogens showing multiple drug resistance.

Quorum sensing (QS) is a bacterial communication process that depends on the bacterial population density. It involves small diffusible signaling molecules which activate the expression of myriad genes that control diverse array of functions like bioluminescence, virulence, biofilm formation, sporulation, to name a few. Since QS is responsible for virulence in the clinically relevant bacteria, inhibition of QS appears to be a promising strategy to control these pathogenic bacteria. With indiscriminate use of antibiotics, there has been an alarming increase in the number of antibiotic resistant pathogens. Antibiotics are no longer the magic bullets they were once thought to be and therefore there is a need for development of new antibiotics and/or other novel strategies to combat the infections caused by multidrug resistant organisms. Quorum sensing inhibition or quorum quenching has been pursued as one of such novel strategies. While antibiotics kill or slow down the growth of bacteria, quorum sensing inhibitors (QSIs) or quorum quenchers (QQs) attenuate bacterial virulence. A large body of work on QS has been carried out in deadly pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio fischeri, V. harveyi, Escherichia coli and V. cholerae etc to unravel the mechanisms of QS as well as identify and study QSIs. This review describes various aspects of QS, QSI, different model systems to study these phenomena and recent patents on various QSIs. It suggests QSIs as attractive alternatives for controlling human, animal and plant pathogens and their utility in agriculture and other industries.

Triplex PCR assay for the rapid identification of 3 major Vibrio species, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio fluvialis.

A triplex PCR assay was developed for the identification of 3 major Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio fluvialis by targeting their haemolysin, haem-utilizing, and central regulatory genes, respectively. This simple, rapid, sensitive, and specific assay using cell lysates from 227 samples established its usefulness in research and epidemiology.

Synergistic effect of various virulence factors leading to high toxicity of environmental V. cholerae non-O1/ non-O139 isolates lacking ctx gene : comparative study with clinical strains.

BACKGROUND: Vibrio cholerae non-O1/ non-O139 serogroups have been reported to cause sporadic diarrhoea in humans. Cholera toxins have been mostly implicated for hypersecretion of ions and water into the small intestine. Though most of the V. cholerae non-O1/ non-O139 strains lack these cholera toxins, several other innate virulence factors contribute towards their pathogenicity. The environmental isolates may thus act as reservoirs for potential spreading of these virulence genes in the natural environment which may cause the emergence of epidemic-causing organisms. RESULTS: The environmental isolates of vibrios were obtained from water samples, zooplanktons and phytoplanktons, from a village pond in Gandhinagar, Gujarat, India. They were confirmed as Vibrio cholerae non-O1/ non-O139 using standard biochemical and serotyping tests. PCR experiments revealed that the isolates lacked ctxA, ctxB, tcpA, zot and ace genes whereas other pathogenicity genes like toxR, rtxC, hlyA, hapA and prtV were detected in these isolates. Compared with epidemic strain V. cholerae O1 El Tor N16961, culture supernatants from most of these isolates caused higher cytotoxicity to HT29 cells and higher hemolytic, hemagglutinin and protease activities. In rabbit ileal loop assays, the environmental isolates showed only 2-4 folds lesser fluid accumulation in comparison to N16961 and a V. cholerae clinical isolate IDH02365 of 2009. Pulsed Field Gel electrophoresis and Random amplification of Polymorphic DNA indicated that these isolates showed considerable diversity and did not share the same clonal lineage even though they were derived from the same water source. All the isolates showed resistance to one or more antibiotics. CONCLUSION: Though these environmental isolates lacked the cholera toxins, they seem to have adopted other survival strategies by optimally utilising a diverse array of several other toxins. The current findings indicate the possibility that these isolates could cause some gastroenteric inflammation when ingested and may serve as progenitors for overt disease-causing organisms.

Clinical isolates of Vibrio fluvialis from Kolkata, India, obtained during 2006: plasmids, the qnr gene and a mutation in gyrase A as mechanisms of multidrug resistance.

Resistance profiles and their correlation with genetic factors were investigated in 12 isolates of Vibrio fluvialis obtained from hospitalized patients in Kolkata, India, in 2006. All the strains displayed drug resistance with varying antibiograms. However, resistance to ampicillin and neomycin was common to all of them. Three isolates harboured plasmids carrying drug-resistance genes that could be transferred to recipient strains by conjugation and transformation. PCR results indicated the absence of class 1 integrons and SXT elements in these isolates. A mutation in gyrase A (serine 83→isoleucine) and the presence of the qnrVC-like [corrected] gene were found to contribute towards quinolone resistance. In the 12 isolates, the qnrVC-like [corrected] gene was associated only with two plasmid-bearing isolates, L10734 and L9978, which displayed resistance to quinolones. The gene was transferable during transformation and conjugation, indicating that it was plasmid-borne. Taken together, these data indicate that plasmids, the qnrVC-like [corrected] gene and a mutation in gyrase A were responsible for the observed drug resistance in these strains. To the best of our knowledge, this is the first report of the presence of the qnrVC-like [corrected] allele in V. fluvialis isolates from India.