Using the genetic characteristics of Neisseria gonorrhoeae strains with decreased susceptibility to cefixime to develop a molecular assay to predict cefixime susceptibility

Background: In the last two decades, gonococcal strains with decreased cefixime susceptibility and cases of clinical treatment failure have been reported worldwide. Gonococcal strains with a cefixime minimum inhibitory concentration (MIC) ≥0.12 µg mL−1 are significantly more likely to fail cefixime treatment than strains with an MIC <0.12 µg mL−1. Various researchers have described the molecular characteristics of gonococcal strains with reduced cefixime susceptibility, and many have proposed critical molecular alterations that contribute to this decreased susceptibility. Methods: A systematic review of all published articles in PubMed through 1 November 2018 was conducted that report findings on the molecular characteristics and potential mechanisms of resistance for gonococcal strains with decreased cefixime susceptibility. The findings were summarised and suggestions were made for the development of a molecular-based cefixime susceptibility assay. Results: The penicillin-binding protein 2 (PBP2) encoded by the penA gene is the primary target of cefixime antimicrobial activity. Decreased cefixime susceptibility is conferred by altered penA genes with mosaic substitute sequences from other Neisseria (N.) species (identifiable by alterations at amino acid position 375–377) or by non-mosaic penA genes with at least one of the critical amino acid substitutions at positions 501, 542 and 551. Based on this review of 415 international cefixime decreased susceptible N. gonorrhoeae isolates, the estimated sensitivity for an assay detecting the aforementioned amino acid alterations would be 99.5% (413/415). Conclusions: Targeting mosaic penA and critical amino acid substitutions in non-mosaic penA are necessary and may be sufficient to produce a robust, universal molecular assay to predict cefixime susceptibility.

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First Case in Korea of Group B Streptococcus With Reduced Penicillin Susceptibility Harboring Amino Acid Substitutions in Penicillin-Binding Protein 2X

Annals of Laboratory Medicine ◽

10.3343/alm.2019.39.4.414 ◽

2019 ◽

Vol 39 (4) ◽

pp. 414-416 ◽

Cited By ~ 4

Author(s):

Ahram Yi ◽

Chang-Ki Kim ◽

Kouji Kimura ◽

Yoshichika Arakawa ◽

Mina Hur ◽

...

Keyword(s):

Amino Acid ◽

Binding Protein ◽

Group B Streptococcus ◽

Amino Acid Substitutions ◽

Penicillin Binding Protein ◽

First Case ◽

Group B

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Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3470 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3470-3480 ◽

Cited By ~ 98

Author(s):

E Moran ◽

B Zerler ◽

T M Harrison ◽

M B Mathews

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Point Mutations ◽

Apparent Molecular Weight ◽

Amino Acid Position ◽

Cysteine Residue ◽

Transformation Function ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

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MOLECULAR AND GENETIC CHARACTERISTICS OF SURFACE AND NONSTRUCTURE PROTEINS OF PANDEMIC INFLUENZA VIRUSES A(H1N1)PDM09 IN 2015-2016 EPIDEMIC SEASON

Bulletin of Taras Shevchenko National University of Kyiv Series Biology ◽

10.17721/1728_2748.2016.72.44-48 ◽

2016 ◽

Vol 72 (2) ◽

pp. 44-48

Author(s):

O. Smutko ◽

L. Radchenko ◽

A. Mironenko

Keyword(s):

Amino Acid ◽

Pandemic Influenza ◽

Influenza A ◽

Phylogenetic Trees ◽

Influenza Viruses ◽

Amino Acid Substitutions ◽

Ns1 Protein ◽

Genetic Characteristics ◽

Epidemic Season ◽

Influenza A H1n1

The aim of the present study was identifying of molecular and genetic changes in hemaglutinin (HA), neuraminidase (NA) and non-structure protein (NS1) genes of pandemic influenza A(H1N1)pdm09 strains, that circulated in Ukraine during 2015-2016 epidemic season. Samples (nasopharyngeal swabs from patients) were analyzed using real-time polymerase chain reaction (RTPCR). Phylogenetic trees were constructed using MEGA 7 software. 3D structures were constructed in Chimera 1.11.2rc software. Viruses were collected in 2015-2016 season fell into genetic group 6B and in two emerging subgroups, 6B.1 and 6B.2 by gene of HA and NA. Subgroups 6B.1 and 6B.2 are defined by the following amino acid substitutions. In the NS1 protein were identified new amino acid substitutions D2E, N48S, and E125D in 2015-2016 epidemic season. Specific changes were observed in HA protein antigenic sites, but viruses saved similarity to vaccine strain. NS1 protein acquired substitution associated with increased virulence of the influenza virus.

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Amino Acid Substitutions in Mosaic Penicillin-Binding Protein 2 Associated with Reduced Susceptibility to Cefixime in Clinical Isolates of Neisseria gonorrhoeae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00626-06 ◽

2006 ◽

Vol 50 (11) ◽

pp. 3638-3645 ◽

Cited By ~ 68

Author(s):

Sho Takahata ◽

Nami Senju ◽

Yumi Osaki ◽

Takuji Yoshida ◽

Takashi Ida

Keyword(s):

Amino Acid ◽

Neisseria Gonorrhoeae ◽

Molecular Mechanisms ◽

Binding Protein ◽

Complete Sequence ◽

Clinical Isolates ◽

Amino Acid Substitutions ◽

Penicillin Binding Protein ◽

Fourfold Increase ◽

Genes Encoding

ABSTRACT The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.

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Cefotaxime-non-susceptibility of Haemophilus influenzae induced by additional amino acid substitutions of G555E and Y557H in altered penicillin-binding protein 3

Journal of Infection and Chemotherapy ◽

10.1016/j.jiac.2019.02.010 ◽

2019 ◽

Vol 25 (7) ◽

pp. 509-513 ◽

Cited By ~ 4

Author(s):

Ayako Mizoguchi ◽

Shigemi Hitomi

Keyword(s):

Amino Acid ◽

Haemophilus Influenzae ◽

Binding Protein ◽

Amino Acid Substitutions ◽

Penicillin Binding Protein ◽

Additional Amino Acid

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Diversity of Substitutions within or Adjacent to Conserved Amino Acid Motifs of Penicillin-Binding Protein 2X in Cephalosporin-Resistant Streptococcus pneumoniaeIsolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1252 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1252-1255 ◽

Cited By ~ 60

Author(s):

Yasuko Asahi ◽

Yasuo Takeuchi ◽

Kimiko Ubukata

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Three Dimensional ◽

Crystallographic Structure ◽

Amino Acid Substitutions ◽

Penicillin Binding Protein ◽

Amino Acid Motif ◽

Dna Fragment ◽

High Level ◽

Level Resistance

ABSTRACT The sequence of an approximately 1.1-kb DNA fragment of thepbp2x gene, which encodes the transpeptidase domain, was determined for 35 clinical isolates of Streptococcus pneumoniae for which the cefotaxime (CTX) MICs varied. Strains with substitutions within a conserved amino acid motif changing STMK to SAFK and a Leu-to-Val change just before the KSG motif were highly resistant to CTX (MIC, ≧2 μg/ml). Strains with substitutions adjacent to SSN or KSG motifs had low-level resistance. The amino acid substitutions were plotted on the three-dimensional crystallographic structure of the transpeptidase domain of PBP2X. Transformants containing pbp2x from strains with high-level CTX resistance increased the CTX MIC from 0.016 μg/ml to 0.5 to 1.0 μg/ml.

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Contribution of Specific Amino Acid Changes in Penicillin Binding Protein 1 to Amoxicillin Resistance in ClinicalHelicobacter pyloriisolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00545-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 101-109 ◽

Cited By ~ 18

Author(s):

Nadia N. Qureshi ◽

Dimitrios Morikis ◽

Neal L. Schiller

Keyword(s):

Amino Acid ◽

Homology Modeling ◽

Binding Protein ◽

Site Directed Mutagenesis ◽

Amino Acid Substitutions ◽

Peptic Ulcers ◽

Penicillin Binding Protein ◽

Specific Amino Acid ◽

Binding Cleft ◽

H Pylori

ABSTRACTAmoxicillin is commonly used to treatHelicobacter pylori, a major cause of peptic ulcers, stomach cancer, and B-cell mucosa-associated lymphoid tissue lymphoma. Amoxicillin resistance inH. pyloriis increasing steadily, especially in developing countries, leading to treatment failures. In this study, we characterize the mechanism of amoxicillin resistance in the U.S. clinical isolate B258. Transformation of amoxicillin-susceptible strain 26695 with the penicillin binding protein 1 gene (pbp1) from B258 increased the amoxicillin resistance of 26695 to equal that of B258, while studies using biotinylated amoxicillin showed a decrease in the binding of amoxicillin to the PBP1 of B258. Transformation with 4pbp1fragments, each encompassing several amino acid substitutions, combined with site-directed mutagenesis studies, identified 3 amino acid substitutions in PBP1 of B258 which affected amoxicillin susceptibility (Val 469 Met, Phe 473 Leu, and Ser 543 Arg). Homology modeling showed the spatial orientation of these specific amino acid changes in PBP1 from 26695 and B258. The results of these studies demonstrate that amoxicillin resistance in the clinical U.S. isolate B258 is due solely to an altered PBP1 protein with a lower binding affinity for amoxicillin. Homology modeling analyses using previously identified amino acid substitutions of amoxicillin-resistant PBP1s demonstrate the importance of specific amino acid substitutions in PBP1 that affect the binding of amoxicillin in the putative binding cleft, defining those substitutions deemed most important in amoxicillin resistance.

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Molecular characteristics of penicillin-binding protein 2b, 2x, and 1a sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates in Shenyang, China

Canadian Journal of Microbiology ◽

10.1139/w08-030 ◽

2008 ◽

Vol 54 (6) ◽

pp. 489-494 ◽

Cited By ~ 2

Author(s):

Su Fei Tian ◽

Yun Zhuo Chu ◽

Bai Yi Chen

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Sequence Analysis ◽

Dna Sequences ◽

Multilocus Sequence Typing ◽

Molecular Characteristics ◽

Penicillin Binding Protein ◽

Genetic Changes ◽

Penicillin Binding Proteins ◽

Amino Acid Profiles

The aim of this sudy was to investigate the nature of the amino acid motifs found in penicillin-binding proteins (PBP) 2b, 2x, and 1a of penicillin-nonsusceptible Streptococcus pneumoniae isolates from Shenyang, China, and to obtain information regarding the prevalence of alterations within the motifs or in positions flanking the motifs. For 18 clinical isolates comprising 4 penicillin-susceptible S. pneumoniae, 5 penicillin-intermediate S. pneumoniae, and 9 penicillin-resistant S. pneumoniae. the DNA sequences of PBP2b, PBP2x, and PBP1a transpeptidase domains were determined and then genotyped by multilocus sequence typing. Sequence analysis revealed that most penicillin-nonsusceptible S. pneumoniae isolates (penicillin MIC ≥ 1.5 μg/mL and cefotaxime MIC ≥ 2 μg/mL) shared identical PBP2b, PBP2x, and PBP1a amino acid profiles. Most penicillin-resistant S. pneumoniae isolates were ST320 (4-16-19-15-6-20-1), the double-locus variant of the Taiwan19F-14 clone. This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of β-lactam resistance in Shenyang, China.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01316-05 ◽

2006 ◽

Vol 50 (7) ◽

pp. 2487-2492 ◽

Cited By ~ 50

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.

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