A Novel Efflux System in Inducibly Erythromycin-Resistant Strains of Streptococcus pyogenes

ABSTRACT Streptococcus pyogenes strains inducibly resistant (iMLS phenotype) to macrolide, lincosamide, and streptogramin B (MLS) antibiotics can be subdivided into three phenotypes: iMLS-A, iMLS-B, and iMLS-C. This study focused on inducibly erythromycin-resistant S. pyogenes strains of the iMLS-B and iMLS-C types, which are very similar and virtually indistinguishable in a number of phenotypic and genotypic features but differ clearly in their degree of resistance to MLS antibiotics (high in the iMLS-B type and low in the iMLS-C type). As expected, the iMLS-B and iMLS-C test strains had the erm(A) methylase gene; the iMLS-A and the constitutively resistant (cMLS) isolates had the erm(B) methylase gene; and a control M isolate had the mef(A) efflux gene. mre(A) and msr(A), i.e., other macrolide efflux genes described in gram-positive cocci, were not detected in any test strain. With a radiolabeled erythromycin method for determination of the intracellular accumulation of the drug in the absence or presence of an efflux pump inhibitor, active efflux of erythromycin was observed in the iMLS-B isolates as well as in the M isolate, whereas no efflux was demonstrated in the iMLS-C isolates. By the triple-disk (erythromycin plus clindamycin and josamycin) test, performed both in normal test medium and in the same medium supplemented with the efflux pump inhibitor, under the latter conditions iMLS-B and iMLS-C strains were no longer distinguishable, all exhibiting an iMLS-C phenotype. In conjugation experiments with an iMLS-B isolate as the donor and a Rifr Fusr derivative of an iMLS-C isolate as the recipient, transconjugants which shared the iMLS-B type of the donor under all respects, including the presence of an efflux pump, were obtained. These results indicate the existence of a novel, transferable efflux system, not associated with mef(A) or with other known macrolide efflux genes, that is peculiar to iMLS-B strains. Whereas the low-level resistance of iMLS-C strains to MLS antibiotics is apparently due to erm(A)-encoded methylase activity, the high-level resistance of iMLS-B strains appears to depend on the same methylase activity plus the new efflux system.

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A transferable IncC-IncX3 hybrid plasmid co-carrying blaNDM-4, tet(X4), and tmexCD3-toprJ3 confers resistance to carbapenem and tigecycline

10.1101/2021.06.30.450641 ◽

2021 ◽

Author(s):

Aki Hirabayashi ◽

Trung Duc Dao ◽

Taichiro Takemura ◽

Futoshi Hasebe ◽

Le Thi Trang ◽

...

Keyword(s):

Efflux Pump ◽

Efflux Pump Inhibitor ◽

Health Crisis ◽

Resistance Factors ◽

Public Health Crisis ◽

Carbapenemase Gene ◽

High Level ◽

Different Origins ◽

First Time ◽

Level Resistance

Tigecycline is a last-resort antimicrobial that exhibits promising activity against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, have emerged in China and have spread possibly worldwide. Tet(X) family proteins, Tet(X3) to Tet(X14), function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, we report a CPE isolate co-harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes NUITM-VK5 was isolated from an urban drainage in Vietnam in 2021 and a plasmid pNUITM-VK5_mdr co-carrying tet(X4) and tmexCD3-toprJ3 along with the carbapenemase gene blaNDM-4 was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor canceled TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different origins. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and environment. Thus, future global spread of multidrug-resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis.

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Characterization of Mechanisms Lowering Susceptibility to Flumequine among Bacteria Isolated from Chilean Salmonid Farms

Microorganisms ◽

10.3390/microorganisms7120698 ◽

2019 ◽

Vol 7 (12) ◽

pp. 698

Author(s):

Christopher Concha ◽

Claudio D. Miranda ◽

Luz Hurtado ◽

Jaime Romero

Keyword(s):

Efflux Pump ◽

Acquired Resistance ◽

16S Rrna Genes ◽

Rrna Genes ◽

Intrinsic Resistance ◽

Efflux Pump Inhibitor ◽

Active Efflux ◽

Resistance Patterns ◽

High Level ◽

Chromosomal Mutations

Despite their great importance for human therapy, quinolones are still used in Chilean salmon farming, with flumequine and oxolinic acid currently approved for use in this industry. The aim of this study was to improve our knowledge of the mechanisms conferring low susceptibility or resistance to quinolones among bacteria recovered from Chilean salmon farms. Sixty-five isolates exhibiting resistance, reduced susceptibility, or susceptibility to flumequine recovered from salmon farms were identified by their 16S rRNA genes, detecting a high predominance of species belonging to the Pseudomonas genus (52%). The minimum inhibitory concentrations (MIC) of flumequine in the absence and presence of the efflux pump inhibitor (EPI) Phe-Arg-β-naphthylamide and resistance patterns of isolates were determined by a microdilution broth and disk diffusion assays, respectively, observing MIC values ranging from 0.25 to >64 µg/mL and a high level of multi-resistance (96%), mostly showing resistance to florfenicol and oxytetracycline. Furthermore, mechanisms conferring low susceptibility to quinolones mediated by efflux pump activity, quinolone target mutations, or horizontally acquired resistance genes (qepA, oqxA, aac(6′)-lb-cr, qnr) were investigated. Among isolates exhibiting resistance to flumequine (≥16 µg/mL), the occurrence of chromosomal mutations in target protein GyrA appears to be unusual (three out of 15), contrasting with the high incidence of mutations in GyrB (14 out of 17). Bacterial isolates showing resistance or reduced susceptibility to quinolones mediated by efflux pumps appear to be highly prevalent (49 isolates, 75%), thus suggesting a major role of intrinsic resistance mediated by active efflux.

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Efflux pump expression in MDR Acinetobacter baumannii strains from a tertiary care hospital in Lima, Peru

10.21203/rs.3.rs-25924/v1 ◽

2020 ◽

Author(s):

Stefany Plasencia-Rebata ◽

Saul Levy-Blitchtein ◽

Isaac Peña-Tuesta ◽

Miguel Angel Aguilar-Luis ◽

William Vicente Taboada ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Tertiary Care Hospital ◽

Tertiary Care ◽

Clinical Information ◽

Care Hospital ◽

Efflux System ◽

Efflux Pump Inhibitor ◽

Active Efflux ◽

Different Sources

Abstract Objetive: To analyze the contribution of the active efflux system to quinolones and aminoglycosides resistance in selected outbreak A. baumannii clinical isolates using the efflux pump inhibitor PAβN.Results: A total of nineteen Acinetobacter baumannii strains were included in the study. All were positive for the blaOXA-51 gene by PCR and had clinical information associated. The samples were non-duplicate and collected from different sources. Non-susceptibility rates were as following: tobramycin 31.6% (6), ciprofloxacin 31.6% (6), levofloxacin 21.1% (4), nalidixic acid 26.3% (5) and amikacin 15.8% (3). A total of eight strains (42,1%) demonstrated an increase in the susceptibility rates and sixteen (84,2%) expressed efflux pumps.

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Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab084 ◽

2021 ◽

Cited By ~ 1

Author(s):

J G E Laumen ◽

S S Manoharan-Basil ◽

E Verhoeven ◽

S Abdellati ◽

I De Baetselier ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Ribosomal Proteins ◽

Efflux Pump ◽

23S Rrna ◽

Rrna Gene ◽

23S Rrna Gene ◽

Evolution Of Resistance ◽

High Level ◽

Azithromycin Resistance ◽

Level Resistance

Abstract Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

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Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00439-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Saeed Sharifi ◽

Bita Bakhshi ◽

Shahin Najar-peerayeh

Keyword(s):

Gene Expression ◽

Point Mutation ◽

Campylobacter Jejuni ◽

Significant Contribution ◽

Efflux Pump ◽

Campylobacter Coli ◽

Rapd Pcr ◽

Resistant Strains ◽

High Level ◽

Level Resistance

Abstract Background Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. Methods A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. Results Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. Conclusion Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

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Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3422-3426.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3422-3426 ◽

Cited By ~ 10

Author(s):

Siddhartha Roychoudhury ◽

Tracy L. Twinem ◽

Kelly M. Makin ◽

Mark A. Nienaber ◽

Chuiying Li ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Sequence Analysis ◽

Efflux Pump ◽

Serial Passage ◽

Efflux Pump Inhibitor ◽

In Vitro Development ◽

Development Of Resistance ◽

High Level ◽

Vitro Development

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

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Overexpression of the MtrC-MtrD-MtrE Efflux Pump Due to an mtrR Mutation Is Required for Chromosomally Mediated Penicillin Resistance in Neisseria gonorrhoeae

Journal of Bacteriology ◽

10.1128/jb.184.20.5619-5624.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5619-5624 ◽

Cited By ~ 113

Author(s):

Wendy L. Veal ◽

Robert A. Nicholas ◽

William M. Shafer

Keyword(s):

Neisseria Gonorrhoeae ◽

Base Pair ◽

Efflux Pump ◽

Penicillin Resistance ◽

Single Base ◽

Base Pair Deletion ◽

Resistance To Penicillin ◽

Single Base Pair ◽

High Level ◽

Level Resistance

ABSTRACT The importance of the mtrCDE-encoded efflux pump in conferring chromosomally mediated penicillin resistance on certain strains of Neisseria gonorrhoeae was determined by using genetic derivatives of penicillin-sensitive strain FA19 bearing defined mutations (mtrR, penA, and penB) donated by a clinical isolate (FA6140) expressing high-level resistance to penicillin and antimicrobial hydrophobic agents (HAs). When introduced into strain FA19 by transformation, a single base pair deletion in the mtrR promoter sequence from strain FA6140 was sufficient to provide high-level resistance to HAs (e.g., erythromycin and Triton X-100) but only a twofold increase in resistance to penicillin. When subsequent mutations in penA and porIB were introduced from strain FA6140 into strain WV30 (FA19 mtrR) by transformation, resistance to penicillin increased incrementally up to a MIC of 1.0 μg/ml. Insertional inactivation of the gene (mtrD) encoding the membrane transporter component of the Mtr efflux pump in these transformant strains and in strain FA6140 decreased the MIC of penicillin by 16-fold. Genetic analyses revealed that mtrR mutations, such as the single base pair deletion in its promoter, are needed for phenotypic expression of penicillin and tetracycline resistance afforded by the penB mutation. As penB represents amino acid substitutions within the third loop of the outer membrane PorIB protein that modulate entry of penicillin and tetracycline, the results presented herein suggest that PorIB and the MtrC-MtrD-MtrE efflux pump act synergistically to confer resistance to these antibiotics.

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Triclosan resistance in clinical isolates of Acinetobacter baumannii

Journal of Medical Microbiology ◽

10.1099/jmm.0.008524-0 ◽

2009 ◽

Vol 58 (8) ◽

pp. 1086-1091 ◽

Cited By ~ 38

Author(s):

Yagang Chen ◽

Borui Pi ◽

Hua Zhou ◽

Yunsong Yu ◽

Lanjuan Li

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Resistance Mechanism ◽

Dilution Method ◽

Agar Dilution Method ◽

Active Efflux ◽

Low Level ◽

High Level ◽

Resistance Rates ◽

Triclosan Resistance

The susceptibility to triclosan of 732 clinical Acinetobacter baumannii isolates obtained from 25 hospitals in 16 cities in China from December 2004 to December 2005 was screened by using an agar dilution method. Triclosan MICs ranged between 0.015 and 16 mg l−1, and the MIC90 was 0.5 mg l−1, lower than the actual in-use concentration of triclosan. Twenty triclosan-resistant isolates (MICs ≥1 mg l−1) were characterized by antibiotic susceptibility, clonal relatedness, fabI mutation, fabI expression, and efflux pump phenotype and expression to elucidate the resistance mechanism of A. baumannii to triclosan. The resistance rates of triclosan-resistant isolates to imipenem, levofloxacin, amikacin and tetracycline were higher than those of triclosan-sensitive isolates. Triclosan resistance was artificially classified as low level (MICs 1–2 mg l−1) or high level (MICs ≥4 mg l−1). High-level triclosan resistance could be explained by a Gly95Ser mutation of FabI, whilst wild-type fabI was observed to be overexpressed in low-level resistant isolates. Active efflux did not appear to be a major reason for acquired triclosan resistance, but acquisition of resistance appeared to be dependent on a background of intrinsic triclosan efflux.

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Resistance to β-Lactam Antibiotics inPseudomonas aeruginosa Due to Interplay between the MexAB-OprM Efflux Pump and β-Lactamase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1301 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1301-1303 ◽

Cited By ~ 49

Author(s):

Taiji Nakae ◽

Akira Nakajima ◽

Toshihisa Ono ◽

Kohjiro Saito ◽

Hiroshi Yoneyama

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Efflux System ◽

High Level

ABSTRACT We evaluated the roles of the MexAB-OprM efflux pump and β-lactamase in β-lactam resistance in Pseudomonas aeruginosa by constructing OprM-deficient, OprM basal level, and OprM fully expressed mutants from β-lactamase-negative, -inducible, and -overexpressed strains. We conclude that, with the notable exception of imipenem, the MexAB-OprM pump contributes significantly to β-lactam resistance in both β-lactamase-negative and β-lactamase-inducible strains, while the contribution of the MexAB-OprM efflux system is negligible in strains with overexpressed β-lactamase. Overexpression of the efflux pump alone contributes to the high level of β-lactam resistance in the absence of β-lactamase.

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Efflux-Related Resistance to Norfloxacin, Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00430-07 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3235-3239 ◽

Cited By ~ 122

Author(s):

Carmen E. DeMarco ◽

Laurel A. Cushing ◽

Emmanuel Frempong-Manso ◽

Susan M. Seo ◽

Tinevimbo A. A. Jaravaza ◽

...

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Efflux Pump ◽

Resistance Mechanism ◽

Efflux Pump Inhibitor ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Environmental Surfaces ◽

Genes Encoding ◽

High Level

ABSTRACT Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux. Strains exhibiting efflux of at least two of the four substrates were identified (“effluxing strains” [n = 114]). For these strains, MICs with or without reserpine for an array of typical substrates and the expression of mepA, mdeA, norA, norB, norC, and qacA/B were determined using quantitative real-time reverse transcription-PCR (qRT-PCR). A fourfold or greater increase in gene expression was considered significant. The most commonly effluxed substrates were ethidium bromide and chlorhexidine (100 and 96% of effluxing strains, respectively). qRT-PCR identified strains overexpressing mepA (5 [4.4%]), mdeA (13 [11.4%]), norA (26 [22.8%]), norB (29 [25.4%]), and norC (19 [16.7%]); 23 strains overexpressed two or more genes. Mutations probably associated with increased gene expression included a MepR-inactivating substitution and norA promoter region insertions or deletions. Mutations possibly associated with increased expression of the other analyzed genes were also observed. Effluxing strains comprised 49% of all strains studied (114/232 strains), with nearly half of these overexpressing genes encoding MepA, MdeA, and/or NorABC (54/114 strains). Reduced susceptibility to biocides may contribute to persistence on environmental surfaces, and efflux of drugs such as fluoroquinolones may predispose strains to high-level target-based resistance.

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