Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis

ABSTRACT Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA ). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

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Total Variation in the penA Gene of Neisseria meningitidis: Correlation between Susceptibility to β-Lactam Antibiotics and penA Gene Heterogeneity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00353-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3317-3324 ◽

Cited By ~ 26

Author(s):

Sara Thulin ◽

Per Olcén ◽

Hans Fredlund ◽

Magnus Unemo

Keyword(s):

Neisseria Meningitidis ◽

Penicillin G ◽

Penicillin Binding Protein ◽

Wild Type ◽

Gene Encoding ◽

Variable Regions ◽

Resistance To Penicillin ◽

The Relationship ◽

Reference Strains

ABSTRACT In recent decades, the prevalence of Neisseria meningitidis isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Peni) for most strains is due mainly to mosaic structures in the penA gene, encoding penicillin-binding protein 2. In this study, susceptibility to β-lactam antibiotics was determined for 60 Swedish clinical N. meningitidis isolates and 19 reference strains. The penA gene was sequenced and compared to 237 penA sequences from GenBank in order to explore the total identified variation of penA. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type penA gene. By studying the final 1,143 to 1,149 bp of penA in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of penA mosaic structures was found especially for penicillin G and ampicillin. The Peni isolates comprised an MIC of >0.094 μg/ml for penicillin G and an MIC of >0.064 μg/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pens or Peni. In comparison with the wild-type penA sequence, two specific Peni sites were altered in all except two mosaic penA sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between penA sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the penA gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.7.2208-2218.2002 ◽

2002 ◽

Vol 46 (7) ◽

pp. 2208-2218 ◽

Cited By ~ 135

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.

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Cloning and Sequencing of the Gene Encoding Toho-2, a Class A β-Lactamase Preferentially Inhibited by Tazobactam

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.5.1181 ◽

1998 ◽

Vol 42 (5) ◽

pp. 1181-1186 ◽

Cited By ~ 67

Author(s):

Ling Ma ◽

Yoshikazu Ishii ◽

Masaji Ishiguro ◽

Hiroshi Matsuzawa ◽

Keizo Yamaguchi

Keyword(s):

Amino Acid ◽

Acid Resistance ◽

Penicillin G ◽

Amino Acid Residues ◽

Incompatibility Group ◽

Gene Encoding ◽

Reading Frame ◽

E Coli ◽

Class A ◽

Hydrolysis Rates

ABSTRACT Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with β-lactam antibiotics. The β-lactamase (Toho-2) purified from the bacteria hydrolyzed β-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum β-lactamases, Toho-2 was inhibited 16-fold better by the β-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to β-lactams was transferred by conjugation from E. coliTUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. D89862) designated asbla toho was found to have 76.3% identity to class A β-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 β-lactamase and 47.5% identity to TEM-1 β-lactamase. Therefore, the newly isolated β-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group β-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other β-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.

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Nucleotide sequence of the murE gene of Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/m89-175 ◽

1989 ◽

Vol 35 (11) ◽

pp. 1051-1054 ◽

Cited By ~ 8

Author(s):

Jing-Song Tao ◽

Edward E. Ishiguro

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Nucleotide Sequence ◽

Diaminopimelic Acid ◽

Amino Acid Residues ◽

Penicillin Binding Protein ◽

Coding Region ◽

Base Pairs ◽

Gene Encoding ◽

Peptidoglycan Synthesis

The nucleotide sequence of the murE gene encoding the diaminopimelic acid adding enzyme of Escherichia coli is reported. The coding region consisted of 1413 base pairs and was separated from the ftsI (penicillin-binding protein 3) gene by 61 base pairs. The deduced primary structure of MurE comprised 471 amino acid residues with a molecular mass of 50.6 kilodaltons.Key words: Escherichia coli, murE, peptidoglycan synthesis, diaminopimelic acid adding enzyme.

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Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

Journal of Bacteriology ◽

10.1128/jb.186.15.4885-4893.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4885-4893 ◽

Cited By ~ 154

Author(s):

Takane Katayama ◽

Akiko Sakuma ◽

Takatoshi Kimura ◽

Yutaka Makimura ◽

Jun Hiratake ◽

...

Keyword(s):

Amino Acid ◽

Genomic Library ◽

Bifidobacterium Bifidum ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Amino Acid Residues ◽

Gene Encoding ◽

Sugar Chain ◽

Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

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Determination of D- and L-amino acid residues in peptides with fluorescent chiral tagging reagents by high-performance liquid chromatography

Chromatographia ◽

10.1007/bf02315131 ◽

1995 ◽

Vol 40 (11-12) ◽

pp. 645-651 ◽

Cited By ~ 19

Author(s):

T. Toyo'oka ◽

Y. -M. Liu

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Amino Acid ◽

High Performance ◽

Amino Acid Residues

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Mosaic-Like Structure of Penicillin-Binding Protein 2 Gene (penA) in Clinical Isolates of Neisseria gonorrhoeae with Reduced Susceptibility to Cefixime

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.12.3744-3749.2002 ◽

2002 ◽

Vol 46 (12) ◽

pp. 3744-3749 ◽

Cited By ~ 132

Author(s):

Satoshi Ameyama ◽

Shoichi Onodera ◽

Masahiro Takahata ◽

Shinzaburo Minami ◽

Nobuko Maki ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Neisseria Gonorrhoeae ◽

Neisseria Meningitidis ◽

Binding Protein ◽

Clinical Isolates ◽

Penicillin Binding Protein ◽

Gene Sequences ◽

Neisseria Flavescens ◽

Neisseria Perflava

ABSTRACT Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 μg/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 μg/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.

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RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4314 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4314-4326 ◽

Cited By ~ 21

Author(s):

C Mann ◽

J Y Micouin ◽

N Chiannilkulchai ◽

I Treich ◽

J M Buhler ◽

...

Keyword(s):

Cell Division ◽

Amino Acid ◽

Rna Polymerase ◽

Essential Gene ◽

Temperature Sensitive ◽

G1 Arrest ◽

Restrictive Temperature ◽

Amino Acid Residues ◽

Gene Encoding ◽

Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

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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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