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Molecular Characterization of Fluoroquinolone-Resistant Moraxella catarrhalis Variants Generated In Vitro by Stepwise Selection.

ABSTRACT: Moraxella catarrhalis causes respiratory infections. In this study, fluoroquinolone-resistant strains were selected in vitro to evaluate the mechanism of fluoroquinolone resistance. Strains with reduced fluoroquinolone susceptibility were obtained by stepwise selection in levofloxacin, and fluoroquinolone targets gyr and par were sequenced. Six novel mutations in GyrA (D84Y, T594dup, and A722dup), GyrB (E479K and D439N), and ParE (Q395R) involved in M. catarrhalis resistance to fluoroquinolones were revealed.

SUBMITTER: Yamada K

PROVIDER: S-EPMC5610493 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Molecular Characterization of Fluoroquinolone-Resistant Moraxella catarrhalis Variants Generated In Vitro by Stepwise Selection.

Yamada Kageto K Saito Ryoichi R Muto Saori S Kashiwa Machiko M Tamamori Yoshiko Y Fujisaki Shingo S

Antimicrobial agents and chemotherapy 20170922 10

Moraxella catarrhalis causes respiratory infections. In this study, fluoroquinolone-resistant strains were selected in vitro to evaluate the mechanism of fluoroquinolone resistance. Strains with reduced fluoroquinolone susceptibility were obtained by stepwise selection in levofloxacin, and fluoroquinolone targets gyr and par were sequenced. Six novel mutations in GyrA (D84Y, T594dup, and A722dup), GyrB (E479K and D439N), and ParE (Q395R) involved in M. catarrhalis< ...[more]

PMID: 28760904

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Alternations in DNA gyrase genes in low-level fluoroquinolone-resistant Moraxella catarrhalis strains isolated in Poland.

Project description:PurposeThe purpose of this study was to investigate the molecular mechanisms of fluoroquinolone resistance in Moraxella catarrhalis clinical strains isolated in Lublin, Poland.Materials and methodsA total of 150 non-duplicate clinical strains of M. catarrhalis were obtained from individuals with signs of upper respiratory tract infection. Bacterial identification was corroborated on the basis of phenotypic and biochemical characteristics as well as with the use of molecular tests. The antimicrobial susceptibility of M. catarrhalis isolates was determined using the disk diffusion method and Etest. Mutations in the gyrase (gyrA and gyrB) and topoisomerase (parC and parE) genes were determined by polymerase chain reaction and sequencing.ResultsIt was observed that 16.7% of the studied isolates were drug resistant. Resistance to tetracycline was detected for 12% of the strains. Resistance to nalidixic acid, moxifloxacin, and levofloxacin was exhibited by 2.7% of the strains; 1.3% of the strains were resistant to trimethoprim/sulfamethoxazole and 0.7% to erythromycin. Minimum inhibitory concentration values of the four strains demonstrating fluoroquinolone resistance were: 6-12 mg/L for nalidixic acid, 1-1.5 mg/L for levofloxacin, 1 mg/L for moxifloxacin, and 0.25-0.5 mg/L for ciprofloxacin. The research resulted in the detection of mutations in 4 strains, in gyrase gyrA and gyrB genes. In gyrA gene, there occurred mutation G412C as well as four silent transition mutations. Within gyrB gene, there occurred mutation, substitution A1481G, as well as two identical silent mutations.ConclusionOur findings reveal that resistance to fluoroquinolones in M. catarrhalis is connected with amino acid substitutions in gyrA and gyrB genes. To our knowledge, this work is the first description of fluoroquinolone-resistant clinical strains of M. catarrhalis with described mutations in gyrA and gyrB genes isolated in Poland and in Europe.

| S-EPMC6084068 | biostudies-literature

Characterization of a trifunctional glucosyltransferase essential for Moraxella catarrhalis lipooligosaccharide assembly.

Project description:The human respiratory tract pathogen Moraxella catarrhalis expresses lipooligosaccharides (LOS), glycolipid surface moieties that are associated with enhanced colonization and virulence. Recent studies have delineated the major steps required for the biosynthesis and assembly of the M. catarrhalis LOS molecule. We previously demonstrated that the glucosyltransferase enzyme Lgt3 is responsible for the addition of at least one glucose (Glc) molecule, at the ?-(1-4) position, to the inner core of the LOS molecule. Our data further suggested a potential multifunctional role for Lgt3 in LOS biosynthesis. The studies reported here demonstrate that the Lgt3 enzyme possesses two glycosyltransferase domains (A1 and A2) similar to that of other bifunctional glycosyltransferase enzymes involved in surface polysaccharide biosynthesis in Escherichia coli, Pasteurella multocida and Streptococcus pyogenes. Each Lgt3 domain contains a conserved DXD motif, shown to be involved in the catalytic activity of other glycosyltransferases. To determine the function of each domain, A1 (N-terminal), A2 (C-terminal) and double A1A2 site-directed DAD to AAA mutants were constructed and the resulting LOS phenotypes of these modified strains were analyzed. Our studies indicate that the Lgt3 N-terminal A1 catalytic domain is responsible for the addition of the first ?-(1-3) Glc to the first Glc on the inner core. The C-terminal catalytic domain A2 then adds the ?-(1-4) Glc and the ?-(1-6) Glc, confirming the bifunctional nature of this domain. The results from these experiments demonstrate that Lgt3 is a novel, multifunctional transferase responsible for the addition of three Glcs with differing linkages onto the inner core of M. catarrhalis LOS.

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Modular arrangement of allelic variants explains the divergence in Moraxella catarrhalis UspA protein function.

Project description:Ubiquitous surface protein A molecules (UspAs) of Moraxella catarrhalis are large, nonfimbrial, autotransporter proteins that can be visualized as a "fuzzy" layer on the bacterial surface by transmission electron microscopy. Previous studies attributed a wide array of functions and binding activities to the closely related UspA1, UspA2, and/or UspA2H protein, yet the molecular and phylogenetic relationships among these activities remain largely unexplored. To address this issue, we determined the nucleotide sequence of the uspA1 genes from a variety of independent M. catarrhalis isolates and compared the deduced amino acid sequences to those of the previously characterized UspA1, UspA2, and UspA2H proteins. Rather than being conserved proteins, we observed a striking divergence of individual UspA1, UspA2, and UspA2H proteins resulting from the modular assortment of unrelated "cassettes" of peptide sequence. The exchange of certain variant cassettes correlates with strain-specific differences in UspA protein function and confers differing phenotypes upon these mucosal surface pathogens.

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Characterization of the Moraxella catarrhalis uspA1 and uspA2 genes and their encoded products.

Project description:The uspA1 and uspA2 genes of M. catarrhalis O35E encode two different surface-exposed proteins which were previously shown to share a 140-amino-acid region with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997). The N-terminal amino acid sequences of the mature forms of both UspA1 and UspA2 from strain O35E were determined after enzymatic treatment to remove the N-terminal pyroglutamyl residue that had blocked Edman degradation. Mass spectrometric analysis indicated that the molecular mass of UspA1 from M. catarrhalis O35E was 83,500 +/- 116 Da. Nucleotide sequence analysis of the uspA1 and uspA2 genes from three other M. catarrhalis strains (TTA24, ATCC 25238, and V1171) revealed that the encoded protein products were very similar to those from strain O35E. Western blot analysis was used to confirm that each of these three strains of M. catarrhalis expressed both UspA1 and UspA2 proteins. Several different and repetitive amino acid motifs were present in both UspA1 and UspA2 from these four strains, and some of these were predicted to form coiled coils. Linear DNA templates were used in an in vitro transcription-translation system to determine the sizes of the monomeric forms of the UspA1 and UspA2 proteins from strains O35E and TTA24.

| S-EPMC93893 | biostudies-literature

Moraxella catarrhalis expresses an unusual Hfq protein.

Project description:The Hfq protein is recognized as a global regulatory molecule that facilitates certain RNA-RNA interactions in bacteria. BLAST analysis identified a 630-nucleotide open reading frame in the genome of Moraxella catarrhalis ATCC 43617 that was highly conserved among M. catarrhalis strains and which encoded a predicted protein with significant homology to the Hfq protein of Escherichia coli. This protein, containing 210 amino acids, was more than twice as large as the Hfq proteins previously described for other bacteria. The C-terminal half of the M. catarrhalis Hfq protein was very hydrophilic and contained two different types of amino acid repeats. A mutation in the M. catarrhalis hfq gene affected both the growth rate of this organism and its sensitivity to at least two different types of stress in vitro. Provision of the wild-type M. catarrhalis hfq gene in trans eliminated these phenotypic differences in the hfq mutant. This M. catarrhalis hfq mutant exhibited altered expression of some cell envelope proteins relative to the wild-type parent strain and also had a growth advantage in a continuous flow biofilm system. The presence of the wild-type M. catarrhalis hfq gene in trans in an E. coli hfq mutant fully reversed the modest growth deficiency of this E. coli mutant and partially reversed the stress sensitivity of this E. coli mutant to methyl viologen. The use of an electrophoretic mobility shift assay showed that this M. catarrhalis Hfq protein could bind RNA derived from a gene whose expression was altered in the M. catarrhalis hfq mutant.

| S-EPMC2423088 | biostudies-literature

Molecular characterization of the BRO beta-lactamase of Moraxella (Branhamella) catarrhalis.

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