Role of the AcrAB-TolC Efflux Pump in Determining Susceptibility of Haemophilus influenzae to the Novel Peptide Deformylase Inhibitor LBM415

ABSTRACT Haemophilus influenzae isolates vary widely in their susceptibilities to the peptide deformylase inhibitor LBM415 (MIC range, 0.06 to 32 μg/ml); however, on average, they are less susceptible than gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae. Insertional inactivation of the H. influenzae acrB or tolC gene in strain NB65044 (Rd strain KW20) increased susceptibility to LBM415, confirming a role for the AcrAB-TolC pump in determining resistance. Consistent with this, sequencing of a PCR fragment generated with primers flanking the acrRA region from an LBM415-hypersusceptible H. influenzae clinical isolate revealed a genetic deletion of acrA. Inactivation of acrB or tolC in several clinical isolates with atypically reduced susceptibility to LBM415 (MIC of 16 μg/ml or greater) significantly increased susceptibility, confirming that the pump is also a determinant of decreased susceptibility in these clinical isolates. Examination of acrR, encoding the putative repressor of pump gene expression, from several of these strains revealed mutations introducing frameshifts, stop codons, and amino acid changes relative to the published sequence, suggesting that loss of pump repression leads to decreased susceptibility. Supporting this, NB65044 acrR mutants selected by exposure to LBM415 at 8 μg/ml had susceptibilities to LBM415 and other pump substrates comparable to the least sensitive clinical isolates and showed increased expression of pump genes.

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Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

Eukaryotic Cell ◽

10.1128/ec.00181-13 ◽

2013 ◽

Vol 13 (4) ◽

pp. 438-451 ◽

Cited By ~ 20

Author(s):

Srisuda Pannanusorn ◽

Bernardo Ramírez-Zavala ◽

Heinrich Lünsdorf ◽

Birgitta Agerberth ◽

Joachim Morschhäuser ◽

...

Keyword(s):

Biofilm Formation ◽

Candida Parapsilosis ◽

Clinical Isolates ◽

Wild Type ◽

Content Type ◽

Initial Adhesion ◽

High Level ◽

Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

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Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE

Applied and Environmental Microbiology ◽

10.1128/aem.03741-15 ◽

2015 ◽

Vol 82 (3) ◽

pp. 939-953 ◽

Cited By ~ 53

Author(s):

Jovana Kovacevic ◽

Jennifer Ziegler ◽

Ewa Wałecka-Zacharska ◽

Aleisha Reimer ◽

David D. Kitts ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Quaternary Ammonium ◽

Food Processing ◽

Genomic Island ◽

Efflux Pump ◽

Cell Adherence ◽

Outbreak Strain ◽

Content Type ◽

Increased Susceptibility

ABSTRACTA novel genomic island (LGI1) was discovered inListeria monocytogenesisolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenesemrE[emrELm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion oflmo1851had no effect on theL. monocytogenesstress response, and deletion ofsel1did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion ofemrEresulted in increased susceptibility to QACs (P< 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 μg/ml), 14/16 LGI1 genes were induced, andlmo1861(putative repressor gene) was constitutively expressed at 4°C, 37°C, and 52°C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 μg/ml), upregulation ofemrE(49.6-fold),lmo1851(2.3-fold),lmo1861(82.4-fold), andsigB(4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrELmstrain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance ofL. monocytogenesto QACs viaemrELm. Since QACs are commonly used in the food industry, there is a concern thatL. monocytogenesstrains possessingemrEwill have an increased ability to survive this stress and thus to persist in food processing environments.

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Gene expression profiling identifies the novel role of immunoproteasome in doxorubicin-induced cardiotoxicity

Toxicology ◽

10.1016/j.tox.2015.04.009 ◽

2015 ◽

Vol 333 ◽

pp. 76-88 ◽

Cited By ~ 7

Author(s):

Wen-Jie Zhao ◽

Sheng-Nan Wei ◽

Xiang-Jun Zeng ◽

Yun-Long Xia ◽

Jie Du ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

The Novel

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The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection

Microbiology ◽

10.1099/mic.0.28184-0 ◽

2005 ◽

Vol 151 (10) ◽

pp. 3361-3369 ◽

Cited By ~ 62

Author(s):

Piotr Zaleski ◽

Marek Wojciechowski ◽

Andrzej Piekarowicz

Keyword(s):

Haemophilus Influenzae ◽

Phase Variation ◽

Phage Infection ◽

Phase Variable ◽

Repeat Tract ◽

Insertional Inactivation ◽

Phage Dna ◽

Dam Methylation ◽

Tetranucleotide Repeats

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction–modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation. Phase-variable resistance/sensitivity to phage infection correlates with changes in lipooligosaccharide (LOS) structure and occurs by slippage of tetranucleotide repeats within the gene lic2A, coding for a step in the biosynthesis of LOS. The lack of Dam activity destabilizes the tetranuclotide (5′-CAAT) repeat tract and increases the frequency of switching from sensitivity to resistance to phage infection more than in the opposite direction. The PV of the lgtC gene does not influence resistance or sensitivity to phage infection. Insertional inactivation of lic2A, but not lgtC or lgtF, leads to resistance to phage infection and to the same structure of the LOS as observed among phase-variable phage-resistant variants. This indicates that in the H. influenzae Rd LOS only the first two sugars (Glc-Gal) extending from the third heptose are part of bacterial phage receptors.

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Prevalence and role of efflux pump activity in ciprofloxacin resistance in clinical isolates of Klebsiella pneumoniae

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-010-1147-0 ◽

2011 ◽

Vol 30 (6) ◽

pp. 745-752 ◽

Cited By ~ 15

Author(s):

S. Aathithan ◽

G. L. French

Keyword(s):

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Clinical Isolates ◽

Pump Activity ◽

Ciprofloxacin Resistance

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Genetic and Molecular Characterization of β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae with Unusually High Resistance to Ampicillin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.5.1630-1639.2004 ◽

2004 ◽

Vol 48 (5) ◽

pp. 1630-1639 ◽

Cited By ~ 79

Author(s):

Frank S. Kaczmarek ◽

Thomas D. Gootz ◽

Fadia Dib-Hajj ◽

Wenchi Shang ◽

Shawn Hallowell ◽

...

Keyword(s):

North America ◽

Haemophilus Influenzae ◽

Efflux Pump ◽

Geometric Mean ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Ampicillin Resistance ◽

Group I ◽

Acrab Efflux Pump ◽

High Level

ABSTRACT Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 μg/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 μg/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously (K. Ubukata, Y. Shibasaki, K. Yamamoto, N. Chiba, K. Hasegawa, Y. Takeuchi, K. Sunakawa, M. Inoue, and M. Konno, Antimicrob. Agents Chemother. 45:1693-1699, 2001). Geometric mean ampicillin MICs for several clinical isolates were 8 to 10.56 μg/ml. Replacement of the ftsI gene in H. influenzae Rd with the intact ftsI from several clinical isolates resulted in integrants with typical BLNAR geometric mean ampicillin MICs of 1.7 to 2.2 μg/ml. Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 μg/ml) lowered the ampicillin MIC to 3.67 μg/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.

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Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs inM. tuberculosis

10.1101/249102 ◽

2018 ◽

Author(s):

Jiayun Liu ◽

Wanliang Shi ◽

Shuo Zhang ◽

Gail Cassell ◽

Dmitry A. Maslov ◽

...

Keyword(s):

Drug Resistance ◽

Drug Targets ◽

Drug Exposure ◽

Efflux Pump ◽

Active Form ◽

Point Mutations ◽

Drug Susceptibility ◽

Clinical Isolates

AbstractAlthough drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

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The Novel Role of Cdx2 in Trophectodermal Gene Expression During the Peri-Implantation Period.

Biology of Reproduction ◽

10.1093/biolreprod/81.s1.406 ◽

2009 ◽

Vol 81 (Suppl_1) ◽

pp. 406-406

Author(s):

Toshihiro Sakurai ◽

Hanako Bai ◽

Muroi Yoshikage ◽

Kentaro Nagaoka ◽

James D. Godkin ◽

...

Keyword(s):

Gene Expression ◽

The Novel ◽

Implantation Period

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Differential Expression of Resistant and Efflux Pump Genes in MDR-TB Isolates

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530319666191009153834 ◽

2020 ◽

Vol 20 (2) ◽

pp. 271-287 ◽

Cited By ~ 5

Author(s):

Manaf AlMatar ◽

Işıl Var ◽

Begüm Kayar ◽

Fatih Köksal

Keyword(s):

Drug Resistance ◽

Target Genes ◽

Efflux Pump ◽

Gene Mutations ◽

Clinical Isolates ◽

Pomegranate Juice ◽

Drug Efflux ◽

Mdr Tb ◽

Drug Efflux Pump

Background: Numerous investigations demonstrate efflux as a worldwide bacterial mode of action which contributes to the resistance of drugs. The activity of antibiotics, which subjects to efflux, can be improved by the combined usage of efflux inhibitors. However, the efflux role to the overall levels of antibiotic resistance of clinical M. tuberculosis isolates is inadequately comprehended and is still disregarded by many. Method: Here, we assessed the contribution of resistant genes associated with isoniazid (INH) and rifampin (R) resistance to the levels of drug resistance in the (27) clinical isolates of MDR-TB. Additionally, the role of the resistance for six putative drug efflux pump genes to the antibiotics was investigated. The level of katG expression was down-regulated in 24/27 (88.88%) of MDR-TB isolates. Of the 27 MDR-TB isolates, inhA, oxyR-ahpC, and rpoB showed either overexpression or up-regulation in 8 (29.62%), 4 (14.81 %), and 24 (88.88%), respectively. Moreover, the efflux pump genes drrA, drrB, efpA, Rv2459, Rv1634, and Rv1250 were overexpressed under INH/RIF plus fresh pomegranate juice (FPJ) stress signifying the efflux pumps contribution to the overall levels of the resistance of MDR-TB isolates. Conclusion: These results displayed that the levels of drug resistance of MDR-TB clinical isolates are due to combination among drug efflux pump and the presence of mutations in target genes, a truth which is often ignored by the specialists of tuberculosis in favour of the almost undoubted significance of drug target- gene mutations for the resistance in M. tuberculosis.

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