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In vitro Activity and Heteroresistance of Omadacycline Against Clinical Staphylococcus aureus Isolates From China Reveal the Impact of Omadacycline Susceptibility by Branched-Chain Amino Acid Transport System II Carrier Protein, Na/Pi Cotransporter Family Protein, and Fibronectin-Binding Protein.

ABSTRACT: Omadacycline (Omad), a new tetracycline (Tet)-class broad-spectrum aminomethylcycline, has been reported to exhibit excellent potency against Gram-positive bacteria, including Staphylococcus aureus and Enterococci. The aim of this study was to evaluate the in vitro activity and heteroresistance characteristics of Omad in clinical S. aureus isolates from China and investigate Omad resistance mechanisms. A sample of 263 non-duplicate clinical S. aureus isolates [127 methicillin-resistant (MRSA) and 136 methicillin-sensitive (MSSA)] were collected retrospectively. Our data indicated that Omad exhibited excellent in vitro activity against both MRSA and MSSA. Omad heteroresistance frequencies were 3.17% (4/126) in MRSA and 12.78% (17/133) in MSSA. No mutations in Tet target sites, (five 16SrRNA copies and 30S ribosomal protein S10) were present in heteroresistance-derived clones, whereas Tet target site mutations contribute to induced Omad resistance in S. aureus in vitro. RNA sequencing (RNA-Seq) revealed that overexpression of branched-chain amino acid transport system II carrier protein and Na/Pi cotransporter family protein contributes to Omad heteroresistance emergence. Whole-genome sequencing demonstrated that the genetic mutation of fibronectin-binding protein (FnBP) could increase the Omad MIC. In conclusion, Omad heteroresistance risk should be considered in clinical isolates with MICs ? 0.5 mg/L and Omad susceptibility in S. aureus may be affected by efflux pump proteins (i.e., a branched-chain amino acid transport system II carrier protein and an Na/Pi cotransporter family protein), and FnBP.

SUBMITTER: Bai B

PROVIDER: S-EPMC6856048 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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In vitro Activity and Heteroresistance of Omadacycline Against Clinical Staphylococcus aureus Isolates From China Reveal the Impact of Omadacycline Susceptibility by Branched-Chain Amino Acid Transport System II Carrier Protein, Na/Pi Cotransporter Family Protein, and Fibronectin-Binding Protein.

Bai Bing B Lin Zhiwei Z Pu Zhangya Z Xu Guangjian G Zhang Fan F Chen Zhong Z Sun Xiang X Zheng Jinxin J Li Peiyu P Deng Qiwen Q Yu Zhijian Z

Frontiers in microbiology 20191108

Omadacycline (Omad), a new tetracycline (Tet)-class broad-spectrum aminomethylcycline, has been reported to exhibit excellent potency against Gram-positive bacteria, including Staphylococcus aureus and Enterococci. The aim of this study was to evaluate the in vitro activity and heteroresistance characteristics of Omad in clinical S. aureus isolates from China and investigate Omad resistance mechanisms. A sample of 263 non-duplicate clinical S. aureus isolates [127 met ...[more]

PMID: 31787948

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Project description:Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637-651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (DeltaF), which suggested that this site lies in an environment that is affected by conformational change in the protein. DeltaF was substrate dependent (no DeltaF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1-18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.

Project description:BACKGROUND: Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S. aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotypes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA. RESULTS: Seven different fnbB allelic variants were identified. Some strains that cluster by phylogenetic analysis contain different fnbB variants, whereas more divergent strains contain the same fnbB variant. The phylogeny of fnbB alleles does not match the phylogeny of fnbA alleles. Some FnBPA and FnBPB isotypes that are specified by human S. aureus strains are also found in bovine strains. The seven fnbB allelic variants encode seven distinct isotypes of the FnBPB A domain that are 61 to 85% identical in amino acid sequence. Variant amino acid residues were mapped on a three-dimensional model of the FnBPB A domain and were predicted to be surface-exposed. They are responsible for the antigenic diversity detected with polyclonal antibody and a monoclonal antibody raised against isotype I. Ligand binding by recombinant FnBPB N23 isotypes was compared by ELISA-based solid phase assays and surface plasmon resonance. Each bound to immobilized fibrinogen, elastin and fibronectin dose-dependently and saturably with similar affinities. Binding to fibronectin was surprising because the A domains do not contain any known motifs that mediate binding to fibronectin. This raises the possibility that the A domain of FnBPB contains a novel fibronectin binding motif that binds fibronectin by a novel mechanism. CONCLUSIONS: Seven different isoforms of FnBPB A domain retain ligand-binding functions but are antigenically distinct. The variation in FnBPA and FnBPB occurs in human and bovine S. aureus strains and may act as an immune evasion mechanism. All seven isotypes of FnBPB are capable of binding fibronectin though none contain any known fibronectin-binding motifs. These results have implications for the development of vaccines or immunotherapeutics that target FnBPB.

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In vitro Activity and Heteroresistance of Omadacycline Against Clinical Staphylococcus aureus Isolates From China Reveal the Impact of Omadacycline Susceptibility by Branched-Chain Amino Acid Transport System II Carrier Protein, Na/Pi Cotransporter Family Protein, and Fibronectin-Binding Protein.

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