scholarly journals Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate ofNeisseria gonorrhoeaePossessing a Mosaic-LikemtrEfflux Pump Locus

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Corinne E. Rouquette-Loughlin ◽  
Jennifer L. Reimche ◽  
Jacqueline T. Balthazar ◽  
Vijaya Dhulipala ◽  
Kim M. Gernert ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antimicrobial Susceptibility ◽  
Dna Sequences ◽  
Efflux Pump ◽  
Spontaneous Mutation ◽  
Content Type ◽  
Transporter Protein ◽  
Single Nucleotide Change ◽  
Clinical Resistance ◽  
Mechanistic Basis

ABSTRACTRecent reports suggest that mosaic-like sequences within themtr(multipletransferableresistance) efflux pump locus ofNeisseria gonorrhoeae, likely originating from commensalNeisseriasp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within themtrRgene encoding the transcriptional repressor (MtrR) of themtrCDEefflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018,https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strongcis-acting transcriptional impact of a single nucleotide change within the −35 hexamer of themtrCDEpromoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-likemtrlocus.IMPORTANCEHistorically, after introduction of an antibiotic for treatment of gonorrhea, strains ofN. gonorrhoeaeemerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of theNeisseriagenus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensalNeisseriathat recombine within the gonococcalmtrefflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.

scholarly journals Mechanistic basis for decreased antimicrobial susceptibility in a clinical isolate ofNeisseria gonorrhoeaepossessing a mosaic-likemtrefflux pump locus

2018 ◽  
Author(s):  
Corinne E. Rouquette-Loughlin ◽  
Jennifer L. Reimche ◽  
Jacqueline T. Balthazar ◽  
Vijaya Dhulipala ◽  
Kim M. Gernert ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antimicrobial Susceptibility ◽  
Dna Sequences ◽  
Efflux Pump ◽  
Spontaneous Mutation ◽  
Gene Encoding ◽  
Transporter Protein ◽  
Single Nucleotide Change ◽  
Clinical Resistance ◽  
Mechanistic Basis

AbstractRecent reports suggest that mosaic-like sequences within themtr(multipletransferableresistance) efflux pump locus ofNeisseria gonorrhoeaelikely originating from commensalNeisseria sp.by transformation can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within themtrRgene encoding the transcriptional repressor (MtrR) of themtrCDEefflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (Wadsworthet al.2018. MBio. doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates including Azi. We report that a strongcis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of themtrCDEpromoter as well gain-of-function amino acid changes at theC-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-likemtrlocus.IMPORTANCEHistorically, after introduction of an antibiotic for treatment of gonorrhea, strains ofN. gonorrhoeaeemerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of theNeisseriagenus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented herein provide a framework for understanding how mosaic-like DNA sequences from commensalNeisseriathat recombine within the gonococcalmtrefflux pump locus function to decrease bacterial susceptibility to antimicrobials including antibiotics used in therapy of gonorrhea.

scholarly journals Carbapenem-NonsusceptibleHaemophilus influenzaewith Penicillin-Binding Protein 3 Containing an Amino Acid Insertion

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Kazuki Kitaoka ◽  
Kouji Kimura ◽  
Hiromitsu Kitanaka ◽  
Hirotsugu Banno ◽  
Wanchun Jin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Efflux Pump ◽  
Binding Protein ◽  
Antimicrobial Susceptibility Testing ◽  
Nucleotide Sequence Analysis ◽  
Penicillin Binding Protein ◽  
Binding Assays ◽  
Content Type

ABSTRACTThe prevalence of β-lactamase-negative ampicillin-resistant (BLNAR)Haemophilus influenzaehas become a clinical concern. In BLNAR isolates, amino acid substitutions in penicillin-binding protein 3 (PBP3) are relevant to the β-lactam resistance. Carbapenem-nonsusceptibleH. influenzaeisolates have been rarely reported. Through antimicrobial susceptibility testing, nucleotide sequence analysis offtsI, encoding PBP3, and the utilization of a collection ofH. influenzaeclinical isolates in our laboratory, we obtained a carbapenem-nonsusceptible clinical isolate (NUBL1772) that possesses an altered PBP3 containing V525_N526insM. The aim of this study was to reveal the effect of altered PBP3 containing V525_N526insM on reduced carbapenem susceptibility. After generating recombinant strains with alteredftsI, we performed antimicrobial susceptibility testing and competitive binding assays with fluorescent penicillin (Bocillin FL) and carbapenems. Elevated carbapenem MICs were found for the recombinant strain harboring the entireftsIgene of NUBL1772. The recombinant PBP3 of NUBL1772 also exhibited reduced binding to carbapenems. These results demonstrate that altered PBP3 containing V525_N526insM influences the reduced carbapenem susceptibility. The revertant mutant lacking the V525_N526insM exhibited lower MICs for carbapenems than NUBL1772, suggesting that this insertion affects reduced carbapenem susceptibility. The MICs of β-lactams for NUBL1772 were higher than those for the recombinant possessingftsIof NUBL1772. NUBL1772 harbored AcrR with early termination, resulting in low-level transcription ofacrBand high efflux pump activity. These findings suggest that the disruption of AcrR also contributes to the reduced carbapenem susceptibility found in NUBL1772. Our results provide the first evidence that the altered PBP3 containing V525_N526insM is responsible for the reduced susceptibility to carbapenems inH. influenzae.

scholarly journals Involvement of Fe Uptake Systems and AmpC β-Lactamase in Susceptibility to the Siderophore Monosulfactam BAL30072 in Pseudomonas aeruginosa

2013 ◽  
Vol 57 (5) ◽  
pp. 2095-2102 ◽  
Author(s):  
Christian van Delden ◽  
Malcolm G. P. Page ◽  
Thilo Köhler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Receptor Gene ◽  
Sufficient Conditions ◽  
Multidrug Resistant ◽  
Content Type ◽  
Single Nucleotide Change ◽  
Gene Coding ◽  
Iron Deficient ◽  
Iron Receptor

ABSTRACTBAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistantPseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 inP. aeruginosaPAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC β-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression ofampCand the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of thefecIpromoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. AfecImutant and afecAmutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 μg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. ThepiuCgene, coding for a Fe(II)-dependent dioxygenase located next to thepiuAiron receptor gene, was also downregulated. The MICs of BAL30072 forpiuCandpiuAtransposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility ofP. aeruginosaPAO1 to BAL30072.

scholarly journals Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam

2015 ◽  
Vol 59 (7) ◽  
pp. 3710-3717 ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Marisa L. Winkler ◽  
Magdalena A. Taracila ◽  
Robert A. Bonomo
Keyword(s):  
Amino Acid ◽  
Molecular Modeling ◽  
Clavulanic Acid ◽  
Amino Acid Position ◽  
Proton Donor ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type ◽  
Mechanistic Basis ◽  
Dynamics Simulations

ABSTRACTKPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling.Escherichia coliDH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; thek2/Kratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M−1s−1to 1.2 M−1s−1). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent againstE. coliDH10B strains possessing all of these variants.

scholarly journals Fermentative Production of Cysteine by Pantoea ananatis

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Kazuhiro Takumi ◽  
Mikhail Kharisovich Ziyatdinov ◽  
Viktor Samsonov ◽  
Gen Nonaka
Keyword(s):  
Amino Acid ◽  
Efflux Pump ◽  
Feedback Inhibition ◽  
Efflux Pumps ◽  
Genetically Engineered ◽  
Efficient Production ◽  
Acid Fermentation ◽  
Pantoea Ananatis ◽  
Content Type ◽  
Fermentative Production

ABSTRACT Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe. IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to establish the fermentative production of cysteine. This study provides crucial insights into the design and construction of cysteine-producing strains, which may play central roles in realizing commercial basis production.

scholarly journals Genomic Analysis of Reduced Susceptibility to Tigecycline in Enterococcus faecium

2014 ◽  
Vol 59 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Vincent Cattoir ◽  
Christophe Isnard ◽  
Thibaud Cosquer ◽  
Arlène Odhiambo ◽  
Fiona Bucquet ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Enterococcus Faecium ◽  
Efflux Pump ◽  
Acquired Resistance ◽  
Ribosomal Subunit ◽  
Genomic Analysis ◽  
Lactate Oxidase ◽  
Content Type

ABSTRACTTigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported inEnterococcus faecium. Using the serial passage method,in vitromutant AusTig andin vitromutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 μg/ml) were obtained from strainsE. faeciumAus0004 and HM1070 (MICs, 0.03 μg/ml), respectively. In addition, two vancomycin-resistantE. faeciumclinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 μg/ml, respectively) were studied. Compared to the wild-type strains, thein vitromutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected therpsJgene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit),efau004_01228(encoding a cation transporter),efau004_01636(coding for a hypothetical protein), andefau004_02455(encoding thel-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci.

scholarly journals The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii

2015 ◽  
Vol 60 (2) ◽  
pp. 1085-1090 ◽  
Author(s):  
Jennifer Nowak ◽  
Thamarai Schneiders ◽  
Harald Seifert ◽  
Paul G. Higgins
Keyword(s):  
Acinetobacter Baumannii ◽  
Antimicrobial Susceptibility ◽  
Efflux Pump ◽  
Response Regulator ◽  
Two Component System ◽  
Content Type ◽  
Susceptibility Data ◽  
Genes Encoding ◽  
Induced Changes

ABSTRACTOverexpression of the resistance-nodulation-cell division-type efflux pump AdeABC is often associated with multidrug resistance inAcinetobacter baumanniiand has been linked to mutations in the genes encoding the AdeRS two-component system. In a previous study, we reported that the Asp20→Asn amino acid substitution in the response regulator AdeR is associated withadeBoverexpression and reduced susceptibility to the antimicrobials levofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. To further characterize the effect of the Asp20→Asn substitution on antimicrobial susceptibility, the expression of the efflux genesadeB,adeJ, andadeG, and substrate accumulation, four plasmid constructs [containingadeR(Asp20)S,adeR(Asn20)S,adeR(Asp20)SABC, andadeR(Asn20)SABC] were introduced into theadeRSABC-deficientA. baumanniiisolate NIPH 60. NeitheradeRSconstruct induced changes in antimicrobial susceptibility or substrate accumulation from that for the vector-only control. TheadeR(Asp20)SABCtransformant showed reduced susceptibility to 6 antimicrobials and accumulated 12% less ethidium than the control, whereas the Asn20 variant showed reduced susceptibility to 6 of 8 antimicrobial classes tested, and its ethidium accumulation was only 72% of that observed for the vector-only construct.adeBexpression was 7-fold higher in theadeR(Asn20)SABCtransformant than in its Asp20 variant. No changes inadeGoradeJexpression or in acriflavine or rhodamine 6G accumulation were detected. The antimicrobial susceptibility data suggest that AdeRS does not regulate any resistance determinants other than AdeABC. Furthermore, the characterization of the Asp20→Asn20 substitution proves that the reduced antimicrobial susceptibility previously associated with this substitution was indeed caused by enhanced efflux activity of AdeB.

scholarly journals Amino Acid Substitution in the Major Multidrug Efflux Transporter Protein AcrB Contributes to Low Susceptibility to Azithromycin in Haemophilus influenzae

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Shoji Seyama ◽  
Takeaki Wajima ◽  
Hidemasa Nakaminami ◽  
Norihisa Noguchi
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Amino Acid Substitution ◽  
Nonsense Mutation ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Content Type ◽  
Transporter Protein ◽  
Stepwise Mutation

ABSTRACT Clarithromycin-resistant Haemophilus influenzae strains with a nonsense mutation in acrR generally exhibited susceptibility to azithromycin, although one strain was found to be nonsusceptible; we aimed to clarify the differences. This strain had an amino acid substitution, Arg327Ser, in AcrB. Introduction of this substitution into H. influenzae Rd caused an increase in the MIC of azithromycin, suggesting that this substitution contributed to nonsusceptibility. These findings indicate that azithromycin-nonsusceptible isolates could occur through stepwise mutation in the acr region.

scholarly journals The Role of Efflux Pumps in the Transition from Low-Level to Clinical Antibiotic Resistance

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 855
Author(s):  
Anna Elisabeth Ebbensgaard ◽  
Anders Løbner-Olesen ◽  
Jakob Frimodt-Møller
Keyword(s):  
Antibiotic Resistance ◽  
Target Genes ◽  
Defense Mechanism ◽  
Efflux Pump ◽  
Spontaneous Mutation ◽  
Efflux Pumps ◽  
Infection Site ◽  
Low Level ◽  
Clinical Resistance ◽  
Level Resistance

Antibiotic resistance is on the rise and has become one of the biggest public health challenges of our time. Bacteria are able to adapt to the selective pressure exerted by antibiotics in numerous ways, including the (over)expression of efflux pumps, which represents an ancient bacterial defense mechanism. Several studies show that overexpression of efflux pumps rarely provides clinical resistance but contributes to a low-level resistance, which allows the bacteria to persist at the infection site. Furthermore, recent studies show that efflux pumps, apart from pumping out toxic substances, are also linked to persister formation and increased spontaneous mutation rates, both of which could aid persistence at the infection site. Surviving at the infection site provides the low-level-resistant population an opportunity to evolve by acquiring secondary mutations in antibiotic target genes, resulting in clinical resistance to the treating antibiotic. Thus, this emphasizes the importance and challenge for clinicians to be able to monitor overexpression of efflux pumps before low-level resistance develops to clinical resistance. One possible treatment option could be an efflux pump-targeted approach using efflux pump inhibitors.

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