Dataset Information

Mutations of the Transporter Proteins GlpT and UhpT Confer Fosfomycin Resistance in Staphylococcus aureus.

ABSTRACT: With the increasing spread of methicillin-resistant Staphylococcus aureus worldwide, fosfomycin has begun to be used more often, either alone or in combination with other antibiotics, for treating methicillin-resistant S. aureus infections, resulting in the emergence of fosfomycin-resistant strains. Fosfomycin resistance is reported to be mediated by fosfomycin-modifying enzymes (FosA, FosB, FosC, and FosX) and mutations of the target enzyme MurA or the membrane transporter proteins UhpT and GlpT. Our previous studies indicated that the fos genes might not the major fosfomycin resistance mechanism in S. aureus, whereas mutations of glpT and uhpT seemed to be more related to fosfomycin resistance. However, the precise role of these two genes in S. aureus fosfomycin resistance remains unclear. The aim of the present study was to investigate the role of glpT and uhpT in S. aureus fosfomycin resistance. Homologous recombination was used to knockout the uhpT and glpT genes in S. aureus Newman. Gene complementation was generated by the plasmid pRB473 carrying these two genes. The fosfomycin minimal inhibitory concentration (MIC) of the strains was measured by the E-test to observe the influence of gene deletion on antibiotic susceptibility. In addition, growth curves were constructed to determine whether the mutations have a significant influence on bacterial growth. Deletion of uhpT, glpT, and both of them led to increased fosfomycin MIC 0.5 ?g/ml to 32 ?g/ml, 4 ?g/ml, and >1024 ?g/ml, respectively. By complementing uhpT and glpT into the deletion mutants, the fosfomycin MIC decreased from 32 to 0.5 ?g/ml and from 4 to 0.25 ?g/ml, respectively. Moreover, the transporter gene-deleted strains showed no obvious difference in growth curves compared to the parental strain. In summary, our study strongly suggests that mutations of uhpT and glpT lead to fosfomycin resistance in S. aureus, and that uhpT mutation may play a more important role. The high resistance and low biological fitness cost resulting from uhpT and glpT deletion suggest that these strains might have an evolutionary advantage in a fosfomycin-rich clinical situation, which should be closely monitored.

SUBMITTER: Xu S

PROVIDER: S-EPMC5437707 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Mutations of the Transporter Proteins GlpT and UhpT Confer Fosfomycin Resistance in Staphylococcus aureus.

Xu Su S Fu Zhuyingjie Z Zhou Ying Y Liu Yang Y Xu Xiaogang X Wang Minggui M

Frontiers in microbiology 20170519

With the increasing spread of methicillin-resistant Staphylococcus aureus worldwide, fosfomycin has begun to be used more often, either alone or in combination with other antibiotics, for treating methicillin-resistant S. aureus infections, resulting in the emergence of fosfomycin-resistant strains. Fosfomycin resistance is reported to be mediated by fosfomycin-modifying enzymes (FosA, FosB, FosC, and FosX) and mutations of the target enzyme MurA or the membrane transporter protein ...[more]

PMID: 28579984

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Project description:In China, fosfomycin alone or in combination with other antibiotics is commonly used in the treatment of infections caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Although fosfomycin-resistant S. aureus strains have continued to emerge and increase, the research on them is rare. In order to determine the prevalence and mechanisms of fosfomycin resistance in MRSA clinical isolates, a total of 96 non-duplicate MRSA isolates were collected from blood and cerebrospinal fluid samples at Huashan Hospital in Shanghai, China between 2004 and 2014. Antimicrobial susceptibility testing was performed by agar dilution. Meanwhile, the fosfomycin-resistance-related genes, fosB, murA, glpT, and uhpT, were amplified by PCR and subjected to sequencing analysis. Multilocus sequence typing (MLST) was conducted to assess strain types. The minimum inhibitory concentration (MIC) of fosfomycin for the 96 MRSA strains ranged from 1.0 to >1,024 mg/L, and approximately 70% (67/96) of the isolates were resistant to fosfomycin (MIC ≥ 64.0 mg/L). Nine isolates with MICs ≥ 128 mg/L carried fosB gene. Twenty-five distinct mutations were detected in the murA (7), glpT (10), and uhpT (8) genes. While five of the murA mutations and five of the glpT mutations were observed only in fosfomycin-sensitive isolates and one of the murA mutation was found both in fosfomycin-resistant and fosfomycin-sensitive isolates, the remaining 14 mutations (1 murA, 5 glpT, and all uhpT mutations) were present only in fosfomycin-resistant isolates. MLST analysis demonstrated that the majority (46/67) of the glpT and/or uhpT mutants belong to ST5, the predominant sequence type among the fosfomycin-resistant MRSA isolates. In conclusion, there is a high rate of fosfomycin resistance in MRSA strains. The mutations in the murA, glpT, and uhpT genes are common in fosfomycin-resistant MRSA strains, and may play a greater role in the development of fosfomycin resistance than the presence of the fosB gene in these organisms.

Project description:BackgroundFosfomycin exhibits excellent in vitro activity against multidrug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Increasing fosfomycin resistance among clinical MRSA isolates was reported previously, but little is known about the relative abundance of Fosfomycin resistance genes in MRSA isolates circulating in Taiwan.MethodsAll MRSA isolates, collected in 2002 and 2012 by the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program, were used in this study. Susceptibility to various antimicrobial agents, including fosfomycin, was determined by broth microdilution. Genetic determinants of fosfomycin resistance, including fosB carriage and murA, glpT and uhpT mutations, were investigated using PCR and sequencing of amplicons. Staphylococcal protein A (spa) typing was also performed to determine the genetic relatedness of MRSA isolates.ResultsA total of 969 MRSA strains, 495 in the year 2002 and 474 in the year 2012, were analyzed. The overall in vitro susceptibility was 8.2% to erythromycin, 18.0% to clindamycin, 29.0% to tetracycline, 44.6% to ciprofloxacin, 57.5% to trimethoprim/sulfamethoxazole, 86.9% to rifampicin, 92.9% to fosfomycin and 100% to linezolid and vancomycin. A significant increase in the fosfomycin resistance rate was observed from 3.4% in 2002 to 11.0% in 2012. Of 68 fosfomycin-resistant MRSA isolates, several genetic backgrounds probably contributing to fosfomycin resistance were identified. Twelve isolates harbored the fosB gene, and various mutations in murA, uhpT, and glpT genes were noted in 11, 59, and 66 isolates, respectively. The most prevalent gene mutations were found in the combination of uhpT and glpT genes (58 isolates). The vast majority of the fosfomycin-resistant MRSA isolates belonged to spa type t002.ConclusionsAn increased fosfomycin resistance rate of MRSA isolates was observed in our present study, mostly due to mutations in the glpT and uhpT genes. Clonal spread probably contributed to the increased fosfomycin resistance.

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Mutations of the Transporter Proteins GlpT and UhpT Confer Fosfomycin Resistance in Staphylococcus aureus.

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Mutations of the Transporter Proteins GlpT and UhpT Confer Fosfomycin Resistance in <i>Staphylococcus aureus</i>.

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