scholarly journals AdeIJK, a Resistance-Nodulation-Cell Division Pump Effluxing Multiple Antibiotics in Acinetobacter baumannii

2008 ◽  
Vol 52 (2) ◽  
pp. 557-562 ◽  
Author(s):  
Laurence Damier-Piolle ◽  
Sophie Magnet ◽  
Sylvie Brémont ◽  
Thierry Lambert ◽  
Patrice Courvalin
Keyword(s):  
Cell Division ◽  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Northern Blotting ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Membrane Components ◽  
Derivatives Of

ABSTRACT We have identified a second resistance-nodulation-cell division (RND)-type efflux pump, AdeIJK, in clinical isolate Acinetobacter baumannii BM4454. The adeI, adeJ, and adeK genes encode, respectively, the membrane fusion, RND, and outer membrane components of the pump. AdeJ belongs to the AcrB protein family (57% identity with AcrB from Escherichia coli). mRNA analysis by Northern blotting and reverse transcription-PCR indicated that the genes were cotranscribed. Overexpression of the cloned adeIJK operon was toxic in both E. coli and Acinetobacter. The adeIJK genes were detected in all of the 60 strains of A. baumannii tested. The two latter observations suggest that the AdeIJK complex might contribute to intrinsic but not to acquired antibiotic resistance in Acinetobacter. To characterize the substrate specificity of the pump, we have constructed derivatives of BM4454 in which adeIJK (strain BM4579), adeABC (strain BM4561), or both groups of genes (strain BM4652) were inactivated by deletion-insertion. Determination of the antibiotic susceptibility of these strains and of BM4652 and BM4579, in which the adeIJK operon was provided in trans, indicated that the AdeIJK pump contributes to resistance to β-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, and sodium dodecyl sulfate. The chemical structure of these molecules suggests that amphiphilic compounds are the preferred substrates. The AdeABC and AdeIJK efflux systems contributed in a more than additive fashion to tigecycline resistance.

Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1227-1238 ◽  
Author(s):  
Caleb W. Dorsey ◽  
Marcelo E. Tolmasky ◽  
Jorge H. Crosa ◽  
Luis A. Actis
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Phosphopantetheinyl Transferase ◽  
Dihydroxybenzoic Acid ◽  
E Coli ◽  
Bacterial Proteins ◽  
Siderophore Receptors ◽  
Close Proximity ◽  
Genes Encoding ◽  
Chorismic Acid

The Acinetobacter baumannii 8399 clinical isolate secretes dihydroxybenzoic acid (DHBA) and a high-affinity catechol siderophore, which is different from other bacterial iron chelators already characterized. Complementation assays with enterobactin-deficient Escherichia coli strains led to the isolation of a cosmid clone containing A. baumannii 8399 genes required for the biosynthesis and activation of DHBA. Accordingly, the cloned fragment harbours a dhbACEB polycistronic operon encoding predicted proteins highly similar to several bacterial proteins required for DHBA biosynthesis from chorismic acid. Genes encoding deduced proteins related to the E. coli Fes and the Bacillus subtilis DhbF proteins, and a putative Yersinia pestis phosphopantetheinyl transferase, all of them involved in the assembly and utilization of catechol siderophores in other bacteria, were found next to the dhbACEB locus. This A. baumannii 8399 gene cluster also contained the om73, p45 and p114 predicted genes encoding proteins potentially involved in transport of ferric siderophore complexes. The deduced products of the p114 and p45 genes are putative membrane proteins that belong to the RND and MFS efflux pump proteins, respectively. Interestingly, P45 is highly related to the E. coli P43 (EntS) protein that participates in the secretion of enterobactin. Although P114 is similar to other bacterial efflux pump proteins involved in antibiotic resistance, its genetic arrangement within this A. baumannii 8399 locus is different from that described in other bacteria. The product of om73 is a Fur- and iron-regulated surface-exposed outer-membrane protein. These characteristics together with the presence of a predicted TonB box and its high similarity to other siderophore receptors indicate that OM73 plays such a role in A. baumannii 8399. The 184 nt om73–p114 intergenic region contains promoter elements that could drive the expression of these divergently transcribed genes, all of which are in close proximity to almost perfect Fur boxes. This arrangement explains the iron- and Fur-regulated expression of om73, and provides strong evidence for a similar regulation for the expression of p114.

Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters

2020 ◽  
Vol 75 (5) ◽  
pp. 1135-1139 ◽  
Author(s):  
Wuen Ee Foong ◽  
Jochen Wilhelm ◽  
Heng-Keat Tam ◽  
Klaas M Pos
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Drug Susceptibility ◽  
Major Facilitator Superfamily ◽  
Rt Pcr ◽  
E Coli ◽  
Rnd Efflux Pump ◽  
Major Facilitator

Abstract Objectives To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. Methods Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT–PCR. Results Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. Conclusions TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.

scholarly journals Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

2020 ◽  
Author(s):  
Bahare Salehi ◽  
Zohreh Ghalavand ◽  
Abbas Yadegar ◽  
Gita Eslami
Keyword(s):  
Cell Division ◽  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Regulatory Genes ◽  
Clinical Isolates ◽  
Drug Resistant ◽  
Broth Microdilution Method ◽  
Rnd Efflux Pumps ◽  
Multiple Antibiotics

Abstract Background: This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. Methods: Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected. Conclusions: Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms.

scholarly journals Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

2020 ◽  
Author(s):  
Bahare Salehi ◽  
Zohreh Ghalavand ◽  
Abbas Yadegar ◽  
Gita Eslami
Keyword(s):  
Cell Division ◽  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Regulatory Genes ◽  
Clinical Isolates ◽  
Drug Resistant ◽  
Broth Microdilution Method ◽  
Rnd Efflux Pumps ◽  
Multiple Antibiotics

Abstract Background: This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.Methods: Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected.Conclusions: Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms.

scholarly journals Genetic Analysis of the Serratia marcescens N28b O4 Antigen Gene Cluster

1999 ◽  
Vol 181 (6) ◽  
pp. 1883-1891 ◽  
Author(s):  
Francesc Saigí ◽  
Núria Climent ◽  
Núria Piqué ◽  
Cesar Sanchez ◽  
Susana Merino ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Insertion Mutant ◽  
Insertion Element ◽  
E Coli ◽  
Antigen Production ◽  
K 12 ◽  
Export System

ABSTRACT The Serratia marcescens N28b wbbL gene has been shown to complement the rfb-50 mutation ofEscherichia coli K-12 derivatives, and a wbbLmutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigı́, N. Piqué, N. Climent, S. Merino, S. Albertı́, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581–7586, 1997). We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phenotype in E. coli DH5α by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with S. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an E. coli K-12 strain with a deletion of the wb cluster was used, suggesting that the O4wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA mutant E. coli strain, suggesting that O4-antigen production iswecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the S. marcescens N28b wbbAinsertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA), suggesting that an IS4-like element could have been involved in the acquisition of the O4 wb cluster.

scholarly journals Expression of the RND-Type Efflux Pump AdeABC in Acinetobacter baumannii Is Regulated by the AdeRS Two-Component System

2004 ◽  
Vol 48 (9) ◽  
pp. 3298-3304 ◽  
Author(s):  
Isabelle Marchand ◽  
Laurence Damier-Piolle ◽  
Patrice Courvalin ◽  
Thierry Lambert
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Constitutive Expression ◽  
Northern Hybridization ◽  
Two Component System ◽  
Component System ◽  
Reverse Transcription Pcr ◽  
Two Component ◽  
Vitro Analysis

ABSTRACT The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153→Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116→Leu in AdeR at the first residue of the α5 helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system.

scholarly journals Expression of the Fluoroquinolones Efflux Pump Genes acrA and mdfA in Urinary Escherichia coli Isolates

2017 ◽  
Vol 66 (1) ◽  
pp. 25-30 ◽  
Author(s):  
Sarah M. Abdelhamid ◽  
Rania R. Abozahra
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Urinary Tract ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Efflux System ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Tract Infections

Escherichia coli is one of the most frequent causes of urinary tract infections. Efflux system overexpression is reported to contribute to E. coli resistance to several antibiotics. Our aim in this study was to investigate the relation between antibiotic resistance and the expression of the efflux pump genes acrA and mdfA in E. coli by real-time reverse transcription-PCR. We tested the in vitro susceptibilities to 12 antibiotics in 28 clinical isolates of E. coli obtained from urine samples. We also determined the minimum inhibitory concentrations of levofloxacin to these samples. We then revealed significant correlations between the overexpression of both mdfA and acrA and MICs of levofloxacin. In conclusion, we demonstrated that the increased expression of efflux pump genes such as mdfA and acrA can lead to levofloxacin resistance in E. coli. These findings contribute to further understanding of the molecular mechanisms of efflux pump systems and how they contribute to antibiotic resistance.

scholarly journals Sublethal Concentrations of the Aminoglycoside Amikacin Interfere with Cell Division without Affecting Chromosome Dynamics

2006 ◽  
Vol 51 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Christophe Possoz ◽  
Jason Newmark ◽  
Nohemy Sorto ◽  
David J. Sherratt ◽  
Marcelo E. Tolmasky
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Acceptor Site ◽  
Protein Fusion ◽  
E Coli ◽  
Z Ring ◽  
A Site ◽  
Derivatives Of ◽  
Sublethal Concentrations

ABSTRACT Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated Escherichia coli cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome ori and ter loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in E. coli strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells.

scholarly journals Alternative Methodologies for the Determination of Aldehydes by Capillary Electrophoresis

1999 ◽  
Vol 82 (6) ◽  
pp. 1562-1570 ◽  
Author(s):  
Elisabete Alves Pereira ◽  
Marina Franco Maggi Tavares ◽  
Arnaldo Alves Cardoso
Keyword(s):  
Capillary Electrophoresis ◽  
Collection Efficiency ◽  
Catalytic Converter ◽  
Sodium Dodecyl ◽  
Indirect Detection ◽  
Sample Collection ◽  
Sample Stacking ◽  
Sample Preconcentration ◽  
Derivatives Of

Abstract This paper describes 2 alternative methodologies for the determination of selected aldehydes (form-aldehyde, acetaldehyde, propionaldehyde, acrolein, and benzaldehyde) by capillary electrophoresis (CE). The first approach is based on the formation of aldehyde-bisulfite adducts and employs free solution CE with reversed electroosmotic flow and indirect detection, using 10 mmol/L 3,5-dinitrobenzoic acid (pH 4.5) containing 0.2 mmol/L cetyltrimethylammonium bromide as the electrolyte. This novel methodology showed a fairly good sensitivity to concentration, with detection limits with respect to a single aldehyde on the order of 10-40 μg/L, a reasonable analysis time (separation was achieved in <8 min), and no need for sample manipulation. A second approach was proposed in which 2,4-dinitrophenylhydrazine derivatives of the aldehydes were detected in a micellar electrolyte medium (20 mmol/L borate buffer containing 50 mmol/L sodium dodecyl sulfate and 15 mmol/L β-cyclodextrin). This latter methodology included a laborious sample preconcentration step and showed much poorer sensitivity (0.5-2 mg/L detection limit, with respect to a single aldehyde), despite the use of sodium chloride to promote sample stacking. Both methodologies proved adequate to evaluate aldehyde levels in vehicular emissions. Samples from the tailpipe exhaust of a passenger car vehicle without a catalytic converter and operated with an ethanol-based fuel were collected and analyzed; the results showed high levels of formaldehyde and acetaldehyde (0.41-6.1 ppm, v/v). The concentrations estimated by the 2 methodologies, which were not in good agreement, suggest the possibility of striking differences in sample collection efficiency, which was not the concern of this work.

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