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 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 Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae

2001 ◽  
Vol 45 (6) ◽  
pp. 1693-1699 ◽  
Author(s):  
Kimiko Ubukata ◽  
Yumi Shibasaki ◽  
Kentarou Yamamoto ◽  
Naoko Chiba ◽  
Keiko Hasegawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Binding Protein ◽  
Amino Acid Substitutions ◽  
Penicillin Binding Protein ◽  
Gene Encoding ◽  
Group Iii ◽  
Peptidoglycan Synthesis ◽  
Development Of Resistance ◽  
Group I

ABSTRACT The affinity of [3H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of β-lactamase-negative ampicillin (AMP)-resistant (BLNAR)Haemophilus influenzae for which the AMP MIC was ≥1.0 μg/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of thedacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-Thr-Gly (KTG) motif, was substituted for Arg-517 in group I strains (n = 9), and Lys-526 was substituted for Asn-526 in group II strains (n = 12). In group III strains (n = 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of β-lactams forH. influenzae transformants into which the ftsIgene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for β-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in β-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to β-lactams in BLNAR strains.

scholarly journals Diversity of β-Lactam Resistance-Conferring Amino Acid Substitutions in Penicillin-Binding Protein 3 of Haemophilus influenzae

2002 ◽  
Vol 46 (7) ◽  
pp. 2208-2218 ◽  
Author(s):  
Henri Dabernat ◽  
Catherine Delmas ◽  
Martine Seguy ◽  
Roseline Pelissier ◽  
Genevieve Faucon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Binding Protein ◽  
Amino Acid Substitutions ◽  
Penicillin Binding Protein ◽  
Gene Encoding ◽  
Peptidoglycan Synthesis ◽  
Group I ◽  
Adults And Children

ABSTRACT The sequences of the ftsI gene, encoding the transpeptidase domain of penicillin binding protein (PBP) 3A and/or PBP 3B, which are involved in septal peptidoglycan synthesis, were determined for 108 clinical strains of Haemophilus influenzae with reduced susceptibility to β-lactam antibiotics with or without β-lactamase production and were compared to those of the ampicillin-susceptible Rd strain and ampicillin-susceptible clinical isolates. The sequences have 18 different mutation patterns and were classified into two groups on the basis of amino acid substitutions deduced from the nucleotide sequences located between bp 960 and 1618 of the ftsI gene. In group I strains (n = 7), His-517 was substituted for Arg-517. In group II strains (n = 101), Lys-526 was substituted for Asn-526. In subgroup IIa (n = 5; H. influenzae ATCC 49247), the only observed substitution was Lys-526 for Asn-526; in subgroup IIb (n = 56), Val-502 was substituted for Ala-502 (n = 13), along with several other substitutions: Asn-350 for Asp-350 (n = 15), Asn-350 for Asp-350 and Glu-490 for Gly-490 (n = 14), and Asn-350 for Asp-350 and Ser-437 for Ala-437 (n = 5). In subgroup IIc (n = 25), Thr-502 was substituted for Ala-502. In subgroup IId, Val-449 was substituted for Ile-449 (n = 15). The MICs of β-lactam antibiotics for the 108 strains were to 8 to 16 times the MICs for susceptible strains. The strains, isolated from both adults and children, were analyzed for genetic relationship by pulsed-field gel electrophoresis and by determination of ftsI sequence phylogeny. Both analyses revealed the lack of clonality and the heterogeneity of the strains, but some clusters suggest the spread and/or persistence of a limited number of strains of the same pulsotype and pattern of amino acid substitutions. Reduced susceptibility to β-lactam, brought about by mutations of the ftsI gene, is becoming a frequent phenomenon, affecting both strains that produce β-lactamase and those that do not. The level of resistance remains low but opens the way to greater resistance in the future.

scholarly journals Characterization of the Gene Encoding a 26-Kilodalton Protein (OMP26) from Nontypeable Haemophilus influenzae and Immune Responses to the Recombinant Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1935-1942 ◽  
Author(s):  
Wafa El-Adhami ◽  
Jennelle M. Kyd ◽  
David A. Bastin ◽  
Allan W. Cripps
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Amino Acid Sequence ◽  
Haemophilus Influenzae ◽  
Cell Envelope ◽  
Leader Peptide ◽  
Polymorphism Analysis ◽  
Nontypeable Haemophilus Influenzae ◽  
Gene Encoding ◽  
Mucosal Antibody

ABSTRACT A 26-kDa protein (OMP26) isolated and purified from nontypeableHaemophilus influenzae (NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the H. influenzaeRd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.

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 A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals Molecular Evolution of β-Lactam-Resistant Haemophilus influenzae: 9-Year Surveillance of Penicillin-Binding Protein 3 Mutations in Isolates from Japan

2006 ◽  
Vol 50 (7) ◽  
pp. 2487-2492 ◽  
Author(s):  
Yumiko Sanbongi ◽  
Takahisa Suzuki ◽  
Yumi Osaki ◽  
Nami Senju ◽  
Takashi Ida ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Evolution ◽  
Haemophilus Influenzae ◽  
Clavulanic Acid ◽  
Antimicrobial Agents ◽  
Binding Protein ◽  
Amino Acid Substitutions ◽  
Penicillin Binding Protein ◽  
Amoxicillin Clavulanic Acid ◽  
Second Period

ABSTRACT A total of 621 clinical isolates of Haemophilus influenzae collected in Japan between 1995 and 2003 were studied for their susceptibilities to several antimicrobial agents, β-lactamase production, and amino acid substitutions in penicillin-binding protein 3 (PBP 3). Over the four study periods (first period, 1995 to 1996; second period, 1997 to 1998; third period, 2000 to 2001; fourth period, 2002 to 2003), the susceptibilities to β-lactam agents decreased and the incidence of isolates with substitutions at positions 377, 385, 389, 517, and/or 526 in PBP 3 increased from 28.8% to 52.0%. Five hundred seventy-one β-lactamase-nonproducing isolates were grouped into 18 classes, based on the pattern of the five mutations in PBP 3. The Asp526Lys substitution led to 6.0-, 4.3-, 2.4-, and 5.4-fold increases in amoxicillin-clavulanic acid, cefdinir, cefditoren, and faropenem resistance, respectively. PBP 3 with multiple substitutions (Met377Ile, Ser385Thr, and/or Leu389Phe) together with Asp526Lys resulted in increased resistance compared to that for PBP 3 with the Asp526Lys substitution alone. These results indicate that mutations at these five positions increased resistance to most β-lactams. Although a significant change in the prevalence of β-lactamase-producing strains was not observed, the proportions of those possessing both PBP 3 alterations and β-lactamase production have slightly increased (from 1.4% to 5.0%). The ROB-1 β-lactamase was rare, but this is the first report of this β-lactamase in Japan.

scholarly journals Nontypeable Haemophilus influenzae isolates from the lower respiratory tract in Western Sichuan, China: MIC value of ofloxacin and Mutation characteristics of Quinolones Resistance determining region gene

2020 ◽  
Author(s):  
Wang Xiaolei ◽  
Xie Jiang ◽  
Guo YuanBiao ◽  
Shao ZhuJun ◽  
Zhu BingQing ◽  
...  
Keyword(s):  
Amino Acid ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Lower Respiratory Tract ◽  
Amino Acid Sequences ◽  
Quinolone Resistance ◽  
Nontypeable Haemophilus Influenzae ◽  
Western Sichuan ◽  
Tract Infections ◽  
Mic Value

Abstract Background: Fluoroquinolones are one of the most widely used antibiotics in the treatment of respiratory tract infections, and the mechanism of resistance to fluoroquinolones is considered to be related to the amino acid substitution of gyrA, gyrB, parC, and parE, the Quinolone Resistance-Determining Regions (QRDRs) of DNA cyclase type II topoisomerase and IV topoisomerase. The purpose of this study was to explore the molecular mechanism of quinolone resistance of Nontypeable Haemophilus influenzae(NTHi ) isolates and analyze the mutation law of the QRDRs gene. Results: MIC value of ofloxacin of 280 NTHi isolates from lower respiratory tract secretions in children group during 2003~2004 and in whole age group during 2013~2014 in Western Sichuan, China were monitored and the amino acid sequences of gyrA, gyrB, parC and parE gene in QRDRs were detected. The resistance rate of ofloxacin in adult group was 1.92% (n=52), while the NTHi strains with ofloxacin MIC value≥0.5 showed an upward trend in all age groups. No ofloxacin-resistant strains were found in 57 NTHi strains isolated from the children patient. Four amino acid substitutions were found in GyrA genes, four in GyrB, three in parC and nine in parE genes. The results showed that different amino acid replacement patterns of the gyrA , gyrB, parC and parE gene had different effects on ofloxacin MIC values. Conclusions: Ser-84-leu and asp-88-tyr/asn mutation of the gyrA, ser-84-lys/ile and ser -133-ala mutations of the parC and ala-400-val variation of the gyrB were the main factors leading to the increase of MIC value of ofloxacin of NTHi strains in Western Sichuan, China. It can be predicted that with the increase of quinolones exposure, the susceptibility of isolates from children to quinolones will be further reduced.

A New Ioxynil-Resistant Mutant in Synechocystis PCC 6714: Hypothesis on the Interaction of Ioxynil with the D1 Protein

1990 ◽  
Vol 45 (5) ◽  
pp. 436-440 ◽  
Author(s):  
S. Creuzet ◽  
G. Ajlani ◽  
C. Vernotte ◽  
C. Astier
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
D1 Protein ◽  
Resistant Mutant ◽  
Hydroxyl Group ◽  
Amino Acid Substitutions ◽  
Psba Gene ◽  
Molecular Models ◽  
Gene Encoding ◽  
Synechocystis Pcc 6714

A new Synechocystis 6714 mutant, loxIIA, resistant to the phenol-type herbicide ioxynil was isolated and characterized. The mutation found in the psbA gene (encoding the D1 photosystem II protein) is at the same codon 266 as for the first ioxynil-resistant mutant IoxIA previously selected [G. Ajlani. I. Meyer, C. Vernotte. and C. Astier, FEBS Lett. 246, 207-210 (1989)]. In IoxIIA, the change of Asn 266 to Asp gives a 3 × resistance, whereas in IoxIA, the change of the same amino acid to Thr gives a 10 × resistance. The effect of these different amino acid substitutions on the ioxynil resistance phenotype has allowed us to construct molecular models and calculate the hydrogen-bonding energies between the hydroxyl group of ioxynil and the respective amino acids at position 266.

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