Cooperation between active efflux and porin alteration is sufficient to confer high-level resistance to meropenem (MEM) in Pseudomonas aeruginosa (Pa) clinical isolates

ABSTRACT In enterobacteria, the ampG gene encodes a transmembrane protein (permease) that transports 1,6-GlcNAc-anhydro-MurNAc and the 1,6-GlcNAc-anhydro-MurNAc peptide from the periplasm to the cytoplasm, which serve as signal molecules for the induction of ampC β-lactamase. The role of AmpG as a transporter is also essential for cell wall recycling. Pseudomonas aeruginosa carries two AmpG homologues, AmpG (PA4393) and AmpGh1 (PA4218), with 45 and 41% amino acid sequence identity, respectively, to Escherichia coli AmpG, while the two homologues share only 19% amino acid identity. In P. aeruginosa strains PAO1 and PAK, inactivation of ampG drastically repressed the intrinsic β-lactam resistance while ampGh1 deletion had little effect on the resistance. Further, deletion of ampG in an ampD-null mutant abolished the high-level β-lactam resistance that is associated with the loss of AmpD activity. The cloned ampG gene is able to complement both the P. aeruginosa and the E. coli ampG mutants, while that of ampGh1 failed to do so, suggesting that PA4393 encodes the only functional AmpG protein in P. aeruginosa. We also demonstrate that the function of AmpG in laboratory strains of P. aeruginosa can effectively be inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), causing an increased sensitivity to β-lactams among laboratory as well as clinical isolates of P. aeruginosa. Our results suggest that inhibition of the AmpG activity is a potential strategy for enhancing the efficacy of β-lactams against P. aeruginosa, which carries inducible chromosomal ampC, especially in AmpC-hyperproducing clinical isolates.

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Molecular aspects of high-level resistance to sulbactam-cefoperazone in Klebsiella oxytoca clinical isolates.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.9.1988 ◽

1996 ◽

Vol 40 (9) ◽

pp. 1988-1994 ◽

Cited By ~ 7

Author(s):

K Kimura ◽

Y Arakawa ◽

S Ohsuka ◽

H Ito ◽

K Suzuki ◽

...

Keyword(s):

Klebsiella Oxytoca ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Point Of View ◽

Clinical Isolates ◽

Beta Lactamase ◽

Beta Lactamases ◽

Molecular Aspects ◽

High Level ◽

Level Resistance

Nine Klebsiella oxytoca strains which demonstrated resistance to the combination of sulbactam and cefoperazone were isolated from geographically separate hospitals in Japan in 1995. Among them, K. oxytoca SB23 showed high-level resistance to sulbactam-cefoperazone (MIC > 128 micrograms/ml) and aztreonam (MIC, 128 micrograms/ml). The sulbactam-cefoperazone resistance was not transferred from strain SB23 to Escherichia coli CSH2 by conjugation, beta-Lactamase RbiA, produced by strain SB23, was purified, and the molecular mass was estimated to be 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic parameters for RbiA revealed that cefoperazone and aztreonam were hydrolyzed efficiently by this enzyme. Moreover, ceftazidime and imipenem were also hydrolyzed weakly by RbiA, although strain SB23 did not show any resistance to these agents. Clavulanate, sulbactam, and tazobactam failed to block the hydrolysis of cefoperazone by RbiA. The structural gene of RbiA (blaRBI) was cloned and sequenced, and the deduced amino acid sequence of RbiA demonstrated high-level similarities to those of the beta-lactamases found in K. oxytoca D488, E23004, and plasmid-mediated MEN-1, which have been classified into Bush functional group 2be. Although RbiA demonstrates high-level molecular similarity to the enzymes in group 2be, from an enzymological point of view, this enzyme might be differentiated from the enzymes in that group. Hybridization analysis revealed that beta-lactamase genes highly similar to blaRBI were generally encoded on the chromosome of the sulbactam-cefoperazone-resistant clinical isolates of K. oxytoca tested in the study, despite their different derivations. This observation suggests that sulbactam-cefoperazone-resistant A. oxytoca strains which produce RbiA-type beta-lactamases have been proliferating in many hospitals in Japan.

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Rapid Emergence of High-Level Resistance to Quinolones in Campylobacter jejuni Associated with Mutational Changes in gyrA and parC

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.12.3276 ◽

1998 ◽

Vol 42 (12) ◽

pp. 3276-3278 ◽

Cited By ~ 55

Author(s):

Amera Gibreel ◽

Eva Sjögren ◽

Bertil Kaijser ◽

Bengt Wretlind ◽

Ola Sköld

Keyword(s):

Campylobacter Jejuni ◽

Quinolone Resistance ◽

Clinical Isolates ◽

High Level ◽

Chromosomal Mutations ◽

Rapid Emergence ◽

Level Resistance

ABSTRACT Quinolone resistance in clinical isolates of Campylobacter jejuni in Sweden increased more than 20-fold at the beginning of the 1990s. Resistance to 125 μg of ciprofloxacin per ml in clinical isolates was associated with chromosomal mutations in C. jejuni leading to a Thr-86-Ile substitution in thegyrA product and a Arg-139-Gln substitution in theparC product.

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High-level resistance to gentamicin in clinical isolates of Streptococcus (Enterococcus) faecium.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.32.10.1528 ◽

1988 ◽

Vol 32 (10) ◽

pp. 1528-1532 ◽

Cited By ~ 53

Author(s):

G M Eliopoulos ◽

C Wennersten ◽

S Zighelboim-Daum ◽

E Reiszner ◽

D Goldmann ◽

...

Keyword(s):

Enterococcus Faecium ◽

Clinical Isolates ◽

High Level ◽

Level Resistance

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Problematic clinical isolates of Pseudomonas aeruginosa from the university hospitals in Sofia, Bulgaria: current status of antimicrobial resistance and prevailing resistance mechanisms

Journal of Medical Microbiology ◽

10.1099/jmm.0.46986-0 ◽

2007 ◽

Vol 56 (7) ◽

pp. 956-963 ◽

Cited By ~ 17

Author(s):

Tanya Strateva ◽

Vessela Ouzounova-Raykova ◽

Boyka Markova ◽

Albena Todorova ◽

Yulia Marteva-Proevska ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Carbapenem Resistance ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Current Status ◽

University Hospitals ◽

Active Efflux ◽

Genes Encoding

A total of 203 clinical isolates of Pseudomonas aeruginosa was collected during 2001–2006 from five university hospitals in Sofia, Bulgaria, to assess the current levels of antimicrobial susceptibility and to evaluate resistance mechanisms to antipseudomonal antimicrobial agents. The antibiotic resistance rates against the following antimicrobials were: carbenicillin 93.1 %, azlocillin 91.6 %, piperacillin 86.2 %, piperacillin/tazobactam 56.8 %, ceftazidime 45.8 %, cefepime 48.9 %, cefpirome 58.2 %, aztreonam 49.8 %, imipenem 42.3 %, meropenem 45.5 %, amikacin 59.1 %, gentamicin 79.7 %, tobramycin 89.6 %, netilmicin 69.6 % and ciprofloxacin 80.3 %. A total of 101 of the studied P. aeruginosa isolates (49.8 %) were multidrug resistant. Structural genes encoding class A and class D β-lactamases showed the following frequencies: bla VEB-1 33.1 %, bla PSE-1 22.5 %, bla PER-1 0 %, bla OXA-groupI 41.3 % and bla OXA-groupII 8.8 %. IMP- and VIM-type carbapenemases were not detected. In conclusion, the studied clinical strains of P. aeruginosa were problematic nosocomial pathogens. VEB-1 extended-spectrum β-lactamases appear to have a significant presence among clinical P. aeruginosa isolates from Sofia. Carbapenem resistance was related to non-enzymic mechanisms such as a deficiency of OprD proteins and active efflux.

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PmrB Mutations Promote Polymyxin Resistance of Pseudomonas aeruginosa Isolated from Colistin-Treated Cystic Fibrosis Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05829-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 1019-1030 ◽

Cited By ~ 106

Author(s):

Samuel M. Moskowitz ◽

Mark K. Brannon ◽

Nandini Dasgupta ◽

Miyuki Pier ◽

Nicole Sgambati ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lipid A ◽

Clinical Isolates ◽

Gain Of Function ◽

Content Type ◽

Regulatory Systems ◽

Repeated Passage ◽

High Level

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance,P. aeruginosaisolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-functionpmrBalleles that conferred polymyxin resistance to strains with a wild-type orpmrABdeletion background. Double mutantpmrBalleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutantpmrBalleles induced transcription from the promoter of thearnBoperon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate thatpmrBgain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains ofP. aeruginosa.

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Carbapenem-Resistant Pseudomonas aeruginosa in Malaysia Producing IMP-7 β-Lactamase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.10.3286-3287.2002 ◽

2002 ◽

Vol 46 (10) ◽

pp. 3286-3287 ◽

Cited By ~ 23

Author(s):

Siaw Eng Ho ◽

Geetha Subramaniam ◽

Selvi Palasubramaniam ◽

Parasakthi Navaratnam

Keyword(s):

Pseudomonas Aeruginosa ◽

Dna Sequencing ◽

Isoelectric Focusing ◽

Carbapenem Resistant ◽

High Level ◽

Level Resistance

ABSTRACT We have isolated and identified a carbapenem-resistant Pseudomonas aeruginosa strain from Malaysia that produces an IMP-7 metallo-β-lactamase. This isolate showed high-level resistance to meropenem and imipenem, the MICs of which were 256 and 128 μg/ml, respectively. Isoelectric focusing analyses revealed pI values of >9.0, 8.2, and 7.8, which indicated the possible presence of IMP and OXA. DNA sequencing confirmed the identity of the IMP-7 determinant.

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High-Level Pacidamycin Resistance in Pseudomonas aeruginosa Is Mediated by an Opp Oligopeptide Permease Encoded by theopp-fabIOperon

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01198-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5565-5571 ◽

Cited By ~ 19

Author(s):

Anita Mistry ◽

Mark S. Warren ◽

John K. Cusick ◽

RoxAnn R. Karkhoff-Schweizer ◽

Olga Lomovskaya ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

High Frequency ◽

Cross Resistance ◽

Peptide Antibiotics ◽

Content Type ◽

Resistant Mutants ◽

High Level ◽

Level Resistance ◽

Resistance Mutants

ABSTRACTPacidamycins (or uridyl peptide antibiotics) possess selectivein vivoactivity againstPseudomonas aeruginosa. An important limitation for the therapeutic use of pacidamycins withP. aeruginosais the high frequency (10−6to 10−7) at which resistant mutants emerge. To elucidate the mechanism(s) of this resistance, pacidamycin-resistantP. aeruginosamutants were isolated. Two types of mutants were obtained. Type 1, or high-level resistance mutants with a pacidamycin MIC of 512 μg/ml, were more abundant, with a frequency of ∼2 × 10−6, and did not show cross-resistance with other antibiotics. Type 2, low-level resistance mutants, were isolated with a frequency of ∼10−8and had a pacidamycin MIC of 64 μg/ml (the MIC for the wild-type strain was 4 to 16 μg/ml). These mutants were cross-resistant to levofloxacin, tetracycline, and erythromycin and were shown to overexpress either the MexAB-OprM or MexCD-OprJ multidrug resistance efflux pumps. High-level resistant mutants were isolated by transposon mutagenesis and one insertion was localized tooppB, one of two periplasmic binding protein components of an oligopeptide transport system which is encoded by theopp-fabIoperon. The Opp system is required for uptake of pacidamycin across the inner membrane, since variousopp, but notfabI, mutants were resistant to high levels of pacidamycin. Both of the two putative Opp periplasmic binding proteins, OppA and OppB, were required for pacidamycin uptake. Although both impaired uptake into and efflux from the cell can cause pacidamycin resistance inP. aeruginosa, our data suggest that impaired uptake is the primary reason for the high-frequency and high-level pacidamycin resistance.

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Susceptibility trends of zoliflodacin against multidrug-resistant Neisseria gonorrhoeae clinical isolates in Nanjing, China (2014-2018)

10.1101/2020.05.04.078055 ◽

2020 ◽

Author(s):

Wenjing Le ◽

Xiaohong Su ◽

Xiangdi Lou ◽

Xuechun Li ◽

Xiangdong Gong ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Dna Sequencing ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Clinical Isolates ◽

Drug Resistant ◽

Extended Spectrum ◽

High Level ◽

Level Resistance

ABSTRACTPreviously, we reported potent activity of a novel spiropyrimidinetrione, zoliflodacin, against N. gonorrhoeae isolates from symptomatic men in Nanjing, China, collected in 2013. Here, we investigated trends of susceptibilities of zoliflodacin in 986 gonococcal isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in gyrA, gyrB, parC and parE genes were determined by PCR and DNA sequencing. The MIC of zoliflodacin for N. gonorrhoeae ranged from ≤0.002 to 0.25 mg/L; the overall MIC50s and MIC90s were 0.06 mg/L and 0.125mg/L in 2018, increasing two-fold from 2014. However, the percent of isolates with lower zoliflodacin MICs declined in each year sequentially while the percent with higher MICs increased yearly (P≤0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥1 μg/ml); 21.2% (209/986) were resistant to azithromycin (≥1 μg/ml), 43.4% (428/986) were penicillinase-producing (PPNG), 26.9% (265/986) tetracycline-resistant (TRNG) and 19.4% (191/986) were multi-drug resistant (MDR) isolates. Among 143 isolates with higher zoliflodacin MICs (0.125-0.25 mg/L), all had quinolone resistance associated double or triple mutations in gyrA; 139/143 (97.2%) also had mutations in parC. There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; a S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin has excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin and extended spectrum cephalosporins.

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