Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

ABSTRACT The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 μM did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters.

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NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

Journal of Bacteriology ◽

10.1128/jb.184.5.1370-1377.2002 ◽

2002 ◽

Vol 184 (5) ◽

pp. 1370-1377 ◽

Cited By ~ 65

Author(s):

Jian-Lin Yu ◽

Leo Grinius ◽

David C. Hooper

Keyword(s):

Escherichia Coli ◽

Expression Vector ◽

Cytoplasmic Membrane ◽

Membrane Vesicles ◽

Aqueous Environment ◽

Multidrug Transporter ◽

Efflux Transporter ◽

Multidrug Efflux ◽

Hoechst 33342 ◽

Experimental Systems

ABSTRACT Overexpression of NorA, an endogenous efflux transporter of Staphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates. The norA gene was amplified by PCR and cloned in the expression vector pTrcHis2. Histidine-tagged NorA (NorA-His) was overexpressed in Escherichia coli cells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His. In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment. Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K+-valinomycin having little effect. Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin. In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition. NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture. NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil. These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter.

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Cell Division Defects in Escherichia coli Deficient in the Multidrug Efflux Transporter AcrEF-TolC

Journal of Bacteriology ◽

10.1128/jb.187.22.7815-7825.2005 ◽

2005 ◽

Vol 187 (22) ◽

pp. 7815-7825 ◽

Cited By ~ 40

Author(s):

Sze Yi Lau ◽

Helen I. Zgurskaya

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Fluorescent Protein ◽

Efflux Transporter ◽

Multidrug Efflux ◽

E Coli ◽

Membrane Fusion Protein ◽

Green Fluorescent ◽

Multiple Drugs ◽

In Cells

ABSTRACT The Escherichia coli chromosome contains several operons encoding confirmed and predicted multidrug transporters. Among these transporters only the inactivation of components of the AcrAB-TolC complex leads to substantial changes in susceptibility to multiple drugs. This observation prompted a conclusion that other transporters are silent or expressed at levels insufficient to contribute to multidrug resistance phenotype. We found that increased expression of AcrA, the periplasmic membrane fusion protein, is toxic only in cells lacking the multidrug efflux transporter AcrEF. AcrEF-deficient cells with increased expression of AcrA have a severe cell division defect that results in cell filamentation (>50 μm). Similar defects were obtained in cells lacking the outer membrane channel TolC, which acts with AcrEF, suggesting that cell filamentation is caused by the loss of AcrEF function. Green fluorescent protein-AcrA fusion studies showed that in normal and filamentous cells AcrA is associated with membranes in a confined manner and that this localization is not affected by the lack of AcrEF. Similarly, the structure and composition of membranes were normal in filamentous cells. Fluorescence microscopy showed that the filamentous AcrEF-deficient E. coli cells are defective in chromosome condensation and segregation. Our results suggest that the E. coli AcrEF transporter is expressed under standard laboratory conditions and plays an important role in the normal maintenance of cell division.

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Genotyping and antimicrobial resistance in Escherichia coli from pig carcasses

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2017001100010 ◽

2017 ◽

Vol 37 (11) ◽

pp. 1253-1260 ◽

Cited By ~ 1

Author(s):

Caroline Pissetti ◽

Gabriela Orosco Werlang ◽

Jalusa Deon Kich ◽

Marisa Cardoso

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Antimicrobial Agents ◽

Commensal Bacteria ◽

Pfge Analysis ◽

Gene Detection ◽

E Coli ◽

Antimicrobial Resistance Profile ◽

Resistant Strains ◽

Pig Carcasses

ABSTRACT: The increasing antimicrobial resistance observed worldwide in bacteria isolated from human and animals is a matter of extreme concern and has led to the monitoring of antimicrobial resistance in pathogenic and commensal bacteria. The aim of this study was to evaluate the antimicrobial resistance profile of Escherichia coli isolated from pig carcasses and to assess the occurrence of relevant resistance genes. A total of 319 E. coli isolates were tested for antimicrobial susceptibility against different antimicrobial agents. Moreover, the presence of extended-spectrum β-lactamase (ESBL) and inducible ampC-β-lactamase producers was investigated. Eighteen multi-resistant strains were chosen for resistance gene detection and PFGE characterization. The study showed that resistance to antimicrobials is widespread in E. coli isolated from pig carcasses, since 86.2% of the strains were resistant to at least one antimicrobial and 71.5% displayed multi-resistance profiles. No ampC-producing isolates were detected and only one ESBL-producing E. coli was identified. Genes strA (n=15), floR (n=14), aac(3)IVa (n=13), tetB (n=13), sul2 (n=12), tetA (n=11), aph(3)Ia (n=8) and sul3 (n=5) were detected by PCR. PFGE analysis of these multi-resistant E. coli strains showed less than 80% similarity among them. We conclude that antimicrobial multi-resistant E. coli strains are common on pig carcasses and present highly diverse genotypes and resistance phenotypes and genotypes.

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Quinolone-resistant mutants of escherichia coli DNA topoisomerase IV parC gene.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.710 ◽

1996 ◽

Vol 40 (3) ◽

pp. 710-714 ◽

Cited By ~ 53

Author(s):

Y Kumagai ◽

J I Kato ◽

K Hoshino ◽

T Akasaka ◽

K Sato ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Gyrase ◽

Mutant Gene ◽

Missense Mutations ◽

Topoisomerase Iv ◽

Dna Topoisomerase ◽

E Coli ◽

A Subunit ◽

Resistant Strains ◽

Mutant Genes

Escherichia coli quinolone-resistant strains with mutations of the parC gene, which codes for a subunit of topoisomerase IV, were isolated from a quinolone-resistant gyrA mutant of DNA gyrase. Quinolone-resistant parC mutants were also identified among the quinolone-resistant clinical strains. The parC mutants became susceptible to quinolones by introduction of a parC+ plasmid. Introduction of the multicopy plasmids carrying the quinolone-resistant parC mutant gene resulted in an increase in MICs of quinolones for the parC+ and quinolone-resistant gyrA strain. Nucleotide sequences of the quinolone-resistant parC mutant genes were determined, and missense mutations at position Gly-78, Ser-80, or Glu-84, corresponding to those in the quinolone-resistance-determining region of DNA gyrase, were identified. These results indicate that topoisomerase IV is a target of quinolones in E. coli and suggest that the susceptibility of E. coli cells to quinolones is determined by sensitivity of the targets, DNA gyrase and topoisomerase IV.

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Synergism of the Combination of Traditional Antibiotics and Novel Phenolic Compounds against Escherichia coli

Pathogens ◽

10.3390/pathogens9100811 ◽

2020 ◽

Vol 9 (10) ◽

pp. 811

Author(s):

Md. Akil Hossain ◽

Hae-Chul Park ◽

Sung-Won Park ◽

Seung-Chun Park ◽

Min-Goo Seo ◽

...

Keyword(s):

Escherichia Coli ◽

Rattus Norvegicus ◽

Inhibitory Concentration ◽

Epicatechin Gallate ◽

Antibiotic Resistant ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

Resistant Strains ◽

Novel Approaches ◽

Rapid Emergence

Pathogenic Escherichia coli (E. coli)-associated infections are becoming difficult to treat because of the rapid emergence of antibiotic-resistant strains. Novel approaches are required to prevent the progression of resistance and to extend the lifespan of existing antibiotics. This study was designed to improve the effectiveness of traditional antibiotics against E. coli using a combination of the gallic acid (GA), hamamelitannin, epicatechin gallate, epigallocatechin, and epicatechin. The fractional inhibitory concentration index (FICI) of each of the phenolic compound-antibiotic combinations against E. coli was ascertained. Considering the clinical significance and FICI, two combinations (hamamelitannin-erythromycin and GA-ampicillin) were evaluated for their impact on certain virulence factors of E. coli. Finally, the effects of hamamelitannin and GA on Rattus norvegicus (IEC-6) cell viability were investigated. The FICIs of the antibacterial combinations against E. coli were 0.281–1.008. The GA-ampicillin and hamamelitannin-erythromycin combinations more effectively prohibited the growth, biofilm viability, and swim and swarm motilities of E. coli than individual antibiotics. The concentration of hamamelitannin and GA required to reduce viability by 50% (IC50) in IEC-6 cells was 988.54 μM and 564.55 μM, correspondingly. GA-ampicillin and hamamelitannin-erythromycin may be potent combinations and promising candidates for eradicating pathogenic E. coli in humans and animals.

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Prevalence of antibiotic-resistant Escherichia coli isolated from urban and agricultural streams in Canterbury, New Zealand

FEMS Microbiology Letters ◽

10.1093/femsle/fnz104 ◽

2019 ◽

Vol 366 (8) ◽

Cited By ~ 2

Author(s):

Sophie Van Hamelsveld ◽

Muyiwa E Adewale ◽

Brigitta Kurenbach ◽

William Godsoe ◽

Jon S Harding ◽

...

Keyword(s):

Escherichia Coli ◽

New Zealand ◽

Pathogenic Bacteria ◽

Freshwater Ecosystems ◽

Urban Setting ◽

Antibiotic Resistant ◽

E Coli ◽

Mesophilic Bacteria ◽

Resistant Strains ◽

Population Counts

Abstract Baseline studies are needed to identify environmental reservoirs of non-pathogenic but associating microbiota or pathogenic bacteria that are resistant to antibiotics and to inform safe use of freshwater ecosystems in urban and agricultural settings. Mesophilic bacteria and Escherichia coli were quantified and isolated from water and sediments of two rivers, one in an urban and one in an agricultural area near Christchurch, New Zealand. Resistance of E. coli to one or more of nine different antibiotics was determined. Additionally, selected strains were tested for conjugative transfer of resistances. Despite having similar concentrations of mesophilic bacteria and E. coli, the rivers differed in numbers of antibiotic-resistant E. coli isolates. Fully antibiotic-susceptible and -resistant strains coexist in the two freshwater ecosystems. This study was the first phase of antibiotic resistance profiling in an urban setting and an intensifying dairy agroecosystem. Antibiotic-resistant E. coli may pose different ingestion and contact risks than do susceptible E. coli. This difference cannot be seen in population counts alone. This is an important finding for human health assessments of freshwater systems, particularly where recreational uses occur downstream.

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In Vitro Activity of a Novel Siderophore-Cephalosporin, GT-1 and Serine-Type β-Lactamase Inhibitor, GT-055, against Escherichia coli, Klebsiella pneumoniae and Acinetobacter spp. Panel Strains

Antibiotics ◽

10.3390/antibiotics9050267 ◽

2020 ◽

Vol 9 (5) ◽

pp. 267 ◽

Cited By ~ 3

Author(s):

Le Phuong Nguyen ◽

Naina Adren Pinto ◽

Thao Nguyen Vu ◽

Hyunsook Lee ◽

Young Lag Cho ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Vitro Activity ◽

Intrinsic Activity ◽

In Vitro Activity ◽

E Coli ◽

Acinetobacter Spp ◽

Resistant Strains ◽

Lactamase Inhibitor

This study investigates GT-1 (also known as LCB10-0200), a novel-siderophore cephalosporin, inhibited multidrug-resistant (MDR) Gram-negative pathogen, via a Trojan horse strategy exploiting iron-uptake systems. We investigated GT-1 activity and the role of siderophore uptake systems, and the combination of GT-1 and a non-β-lactam β-lactamase inhibitor (BLI) of diazabicyclooctane, GT-055, (also referred to as LCB18-055) against molecularly characterised resistant Escherichia coli, Klebsiella pneumoniae and Acinetobacter spp. isolates. GT-1 and GT-1/GT-055 were tested in vitro against comparators among three different characterised panel strain sets. Bacterial resistome and siderophore uptake systems were characterised to elucidate the genetic basis for GT-1 minimum inhibitory concentrations (MICs). GT-1 exhibited in vitro activity (≤2 μg/mL MICs) against many MDR isolates, including extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing E. coli and K. pneumoniae and oxacillinase (OXA)-producing Acinetobacter spp. GT-1 also inhibited strains with mutated siderophore transporters and porins. Although BLI GT-055 exhibited intrinsic activity (MIC 2–8 μg/mL) against most E. coli and K. pneumoniae isolates, GT-055 enhanced the activity of GT-1 against many GT-1–resistant strains. Compared with CAZ-AVI, GT-1/GT-055 exhibited lower MICs against E. coli and K. pneumoniae isolates. GT-1 demonstrated potent in vitro activity against clinical panel strains of E. coli, K. pneumoniae and Acinetobacter spp. GT-055 enhanced the in vitro activity of GT-1 against many GT-1–resistant strains.

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Intestine and Environment of the Chicken as Reservoirs for Extraintestinal Pathogenic Escherichia coli Strains with Zoonotic Potential

Applied and Environmental Microbiology ◽

10.1128/aem.01324-08 ◽

2008 ◽

Vol 75 (1) ◽

pp. 184-192 ◽

Cited By ~ 133

Author(s):

Christa Ewers ◽

Esther-Maria Antï¿½o ◽

Ines Diehl ◽

Hans-C. Philipp ◽

Lothar H. Wieler

Keyword(s):

Escherichia Coli ◽

Virulence Gene ◽

Infection Model ◽

E Coli ◽

Gene Profiles ◽

Pathogenic Escherichia Coli ◽

Zoonotic Risk ◽

Resistant Strains ◽

Genetic Pool

ABSTRACT Although research has increasingly focused on the pathogenesis of avian pathogenic Escherichia coli (APEC) infections and the “APEC pathotype” itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (n = 121) with nonoutbreak strains, including fecal E. coli strains from clinically healthy chickens (n = 211) and strains from their environment (n = 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic E. coli (ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal E. coli strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.

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Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.65 ◽

1998 ◽

Vol 42 (1) ◽

pp. 65-71 ◽

Cited By ~ 119

Author(s):

Ramakrishnan Srikumar ◽

Tatiana Kon ◽

Naomasa Gotoh ◽

Keith Poole

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Crystal Violet ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Efflux ◽

Efflux System ◽

E Coli ◽

Multidrug Efflux Pumps

ABSTRACT The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

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Genetic Characterization of Highly Fluoroquinolone-Resistant Clinical Escherichia coli Strains from China: Role ofacrR Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.5.1515-1521.2001 ◽

2001 ◽

Vol 45 (5) ◽

pp. 1515-1521 ◽

Cited By ~ 181

Author(s):

Hui Wang ◽

Joann L. Dzink-Fox ◽

Minjun Chen ◽

Stuart B. Levy

Keyword(s):

Escherichia Coli ◽

Blot Analysis ◽

Genetic Basis ◽

Efflux Pump ◽

Pcr Amplification ◽

Fluoroquinolone Resistance ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

E Coli ◽

High Level

ABSTRACT The genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coliclinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes (gyrA/B, parC) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of soxS RNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. colitested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type acrR gene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrR are an additional genetic basis for fluoroquinolone resistance.

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