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 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 Subtypes of NanS-p Sialate O-Acetylesterase Encoded by Stx2a Bacteriophages

Proceedings ◽  
2021 ◽  
Vol 66 (1) ◽  
pp. 15
Author(s):  
Stefanía B. Pascal ◽  
Juan R. Lorenzo Lopez ◽  
Paula M. A. Lucchesi ◽  
Alejandra Krüger
Keyword(s):  
Amino Acid ◽  
Foodborne Pathogens ◽  
Dna Sequences ◽  
Query Sequence ◽  
Amino Acid Sequences ◽  
Bacterial Genomes ◽  
Gene Encoding ◽  
Terminal Domain ◽  
Structural Differences ◽  
Blast Program

Shiga toxin (Stx)-producing Escherichia coli strains are foodborne pathogens that can cause severe human diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Stxs are encoded by bacteriophages (Stx phages) which show remarkable variations in genome composition and harbour several genes of unknown function. Recently, a gene encoding a sialate O-acetylesterase (NanS-p) was identified in some relevant Stx2a phages and it was suggested that it could provide advantages for bacterial growth in the gut. The aim of this study was to analyse the presence and sequence of nanS-p genes in available Stx2a phage genomes. A total of 59 DNA sequences of Stx2a phages were extracted from the NCBI GenBank database with the BLAST program using the stx2a sequence from phage 933W as a query sequence, either as complete phage genomes (45) or from bacterial genomes by subsequent analysis with the PHASTER web server (14). Comparative analysis revealed that nanS-p was located downstream of stx2a in all genomes. Twenty different amino acid sequences of NanS-p were identified. Specifically, catalytic esterase domains showed only 11 possible sequences, with differences mainly observed in nine amino acid positions. Sequences corresponding to the N-terminal domain (DUF1737) showed three possible sequences, two of them closely related, while the C-terminal domain was highly variable, with four groups with structural differences. Since sialate O-acetylesterase activity has been determined from particular Stx2a phages, new studies are necessary to evaluate if the NanS-p subtypes identified in the present study also differ in their biological activity.

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 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.

scholarly journals Ampicillin-Resistant Non-β-Lactamase-Producing Haemophilus influenzae in Spain: Recent Emergence of Clonal Isolates with Increased Resistance to Cefotaxime and Cefixime

2007 ◽  
Vol 51 (7) ◽  
pp. 2564-2573 ◽  
Author(s):  
Silvia García-Cobos ◽  
José Campos ◽  
Edurne Lázaro ◽  
Federico Román ◽  
Emilia Cercenado ◽  
...  
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Efflux Pump ◽  
Antibiotic Use ◽  
Resistance Mechanisms ◽  
Amino Acid Substitutions ◽  
Gene Encoding ◽  
Reading Frame ◽  
Recent Emergence ◽  
Acrab Efflux Pump

ABSTRACT The sequence of the ftsI gene encoding the transpeptidase domain of penicillin-binding protein 3 (PBP 3) was determined for 354 nonconsecutive Haemophilus influenzae isolates from Spain; 17.8% of them were ampicillin susceptible, 56% were β-lactamase nonproducing ampicillin resistant (BLNAR), 15.8% were β-lactamase producers and ampicillin resistant, and 10.4% displayed both resistance mechanisms. The ftsI gene sequences had 28 different mutation patterns and amino acid substitutions at 23 positions. Some 93.2% of the BLNAR strains had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%). Amino acid substitutions at positions 377, 385, and 389, which conferred cefotaxime and cefixime MICs 10 to 60 times higher than those of susceptible strains, were found for the first time in Europe. In 72 isolates for which the repressor acrR gene of the AcrAB efflux pump was sequenced, numerous amino acid substitutions were found. Eight isolates with ampicillin MICs of 0.25 to 2 μg/ml showed changes that predicted the early termination of the acrR reading frame. Pulsed-field gel electrophoresis analysis demonstrated that most BLNAR strains were genetically diverse, although clonal dissemination was detected in a group of isolates presenting with increased resistance to cefotaxime and cefixime. Background antibiotic use at the community level revealed a marked trend toward increased amoxicillin-clavulanic acid consumption. BLNAR H. influenzae strains have arisen by vertical and horizontal spread and have evolved to adapt rapidly to the increased selective pressures posed by the use of oral penicillins and cephalosporins.

scholarly journals Contribution of Clinically Derived Mutations in the Gene Encoding the Zinc Cluster Transcription Factor Mrr2 to Fluconazole Antifungal Resistance andCDR1Expression inCandida albicans

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Andrew T. Nishimoto ◽  
Qing Zhang ◽  
Brandon Hazlett ◽  
Joachim Morschhäuser ◽  
P. David Rogers
Keyword(s):  
Amino Acid ◽  
Efflux Pump ◽  
Antifungal Resistance ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Gene Encoding ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Zinc Cluster ◽  
Artificial Activation

ABSTRACTMutations in genes encoding zinc cluster transcription factors (ZCFs) such asTAC1,MRR1, andUPC2play a key role inCandida albicansazole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole. Amino acid substitutions in Mrr2 have recently been reported to contribute to fluconazole resistance in clinical isolates. In the present study, 57 C. albicansclinical isolates with elevated fluconazole MICs were examined for mutations inMRR2and expression ofCDR1. Mutations inMRR2resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 substitutions (S466L, A468G, S469T, T470N) previously reported to reduce fluconazole susceptibility. Three additional, novel amino acid substitutions (R45Q, A459T, V486M) were also discovered in fluconazole-resistant isolates. When introduced into a fluconazole-susceptible background, no change in fluconazole MIC orCDR1expression was observed for any of the mutations found in this collection. However, introduction of an allele leading to artificial activation of Mrr2 increased resistance to fluconazole as well asCDR1expression. Moreover, Mrr2 amino acid changes reported previously to have the strongest effect on fluconazole susceptibility andCDR1expression also exhibited no differences in fluconazole susceptibility orCDR1expression relative to the parent strain. While all known fluconazole resistance mechanisms are represented within this collection of clinical isolates and contribute to fluconazole resistance to different extents, mutations inMRR2do not appear to alterCDR1expression or contribute to resistance in any of these isolates.

scholarly journals Using bioinformatic tools in exploited gene encoding enzyme to decompose lignocellulose from metagenome of free - living bacteria in the gut of the lower termite Coptotermes gestroi

2016 ◽  
Vol 14 (1) ◽  
pp. 39-47
Author(s):  
Nguyễn Minh Giang ◽  
Đỗ Thị Huyền ◽  
Trương Nam Hải
Keyword(s):  
Amino Acid ◽  
Melting Temperature ◽  
Dna Sequences ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Conserved Domains ◽  
Bioinformatic Tools ◽  
Coptotermes Gestroi ◽  
Hydrolysis Of

Microbial metagenome DNA in the guts of Coptotermes gestroi has been extracted and sequenced by metagenomic techniques. In previous studies, we acquired and sequenced more than 5 Gb of DNA metagenome DNA of the termite gut microbiota by next-generation sequencing (Illumina). Software MGA (MetaGeneAnnotator) exploited 125.431 open reading frames with 8508 ORFs related to carbohydrate metabolism, including 587 ORFs coding for enzymes involved in the hydrolysis of lignocellulose. We identified software to reliably predict function, structure and characteristics of proteins corresponding to DNA sequences encoding alkaline enzymes from the metgenome of C gestroi. The online software Alcapred was used to predict alkaline enzymes, Blastp to predict conserved domains of amino acid sequences deduced from ORFs, Phyre2 to predict the three dimentional structure and substrate binding site of the enzymes, TBI to predict melting temperature of the enzyme. We identified 6 ORFs encoding alkaline cellulases (GL0101308, GL0038126) or alkaline hemicellulases (GL0120095, GL0074258, GL0112518, GL0067868). The amino acid sequences deduced from ORFs had 90% coverage and from 44% to 99% identity to the corresponding sequences in NCBI by BLASTP. All of them contained conserved domains with corresponding activities and binding sites of the enzyme to the substrate. The three dimentional structures of amino acid sequences were predicted by Phyre2 with reliability from 98% to 100% to the annotated activities. Among six selected amino acid sequences, two sequences of enzymes had the melting temperature above 65 ℃, three sequences had melting temperature from 55℃ to 65℃ and only one below 55℃.

scholarly journals First Characterization of Heterogeneous Resistance to Imipenem in Invasive Nontypeable Haemophilus influenzae Isolates

2007 ◽  
Vol 51 (9) ◽  
pp. 3155-3161 ◽  
Author(s):  
Marina Cerquetti ◽  
Maria Giufrè ◽  
Rita Cardines ◽  
Paola Mastrantonio
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Regulatory Networks ◽  
Efflux Pump ◽  
Population Analysis ◽  
Amino Acid Substitutions ◽  
Nontypeable Haemophilus Influenzae ◽  
Gene Encoding ◽  
Resistance Phenotype ◽  
Imipenem Resistance

ABSTRACT This study describes the first two reported invasive nontypeable Haemophilus influenzae (NTHI) isolates (strains 183 and 184) with heterogeneous resistance to imipenem. For both isolates, Etest showed imipenem MICs of ≥32 μg/ml. When the two strains were examined by the quantitative method of population analysis, both strain populations were heterogeneously resistant to imipenem and contained subpopulations growing in the presence of up to 32 μg of imipenem/ml at frequencies of 1.7 × 10−5 and 1.5 × 10−7, respectively. By pulsed-field gel electrophoresis analysis, the two isolates appeared to be genetically closely related. The sequencing of the ftsI gene encoding penicillin-binding protein 3 (PBP 3) and comparison with the sequence of the imipenem-susceptible H. influenzae strain Rd identified a pattern of six amino acid substitutions shared between strains 183 and 184; an additional change was unique to strain 183. No relationship between mutations in the dacB gene encoding PBP 4 and imipenem resistance was found. The replacement of the ftsI gene in the imipenem-susceptible strain Rd (for which the MIC of imipenem is 0.38 to 1 μg/ml) with ftsI from strain 183 resulted in a transformant for which the MIC of imipenem ranged from 4 to 8 μg/ml as determined by Etest. The Rd/183 transformant population showed heterogeneous resistance to imipenem; it contained subpopulations growing in the presence of up to 32 μg of imipenem/ml at a frequency of 3.3 ×10−8. The presence of additional resistance mechanisms, such as the overexpression of the AcrAB efflux pump, was investigated and does not seem to be involved. These data indicate that the heterogeneous imipenem resistance phenotype of our NTHI clone depends largely on the PBP 3 amino acid substitutions. We speculated that bacterial regulatory networks may play a role in the control of the heterogeneous expression of the resistance phenotype.

scholarly journals mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae.

1997 ◽  
Vol 41 (10) ◽  
pp. 2251-2255 ◽  
Author(s):  
A Tait-Kamradt ◽  
J Clancy ◽  
M Cronan ◽  
F Dib-Hajj ◽  
L Wondrack ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Substrate Specificity ◽  
Streptococcus Pyogenes ◽  
Dna Sequences ◽  
Efflux Pump ◽  
Erythromycin Resistance ◽  
Gene Encoding

Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.

Sign in / Sign up

Export Citation Format

Share Document