Staphylococcus aureus is one of the most common bacterial pathogens worldwide in both community and hospital settings. Methicillin-resistant Staphylococcus aureus (MRSA) is an important multi-resistant pathogen. To date, vancomycin has remained the cornerstone drug in the management of invasive MRSA infections. However, the continuous rise in the vancomycin minimum inhibitory concentration (MIC), known as the “vancomycin MIC creep” phenomenon, poses a significant challenge to MRSA therapy; therefore, fosfomycin has recently been used alone or in combination with other antibiotics in treating MRSA infections (del Rio et al., 2016; VanEperen and Segreti, 2016). Nevertheless, this situation has inevitably led to the emergence of fosfomycin-resistant MRSA strains. In a recent review, the susceptibility of S. aureus to fosfomycin ranged between 33.2 and 100% in the nine available studies (frequency = 91.7%, 95% confidence interval 88.7–94.9%); in seven of the studies susceptibility rate was >90% (Vardakas et al., 2016). According to the CHINET surveillance program in China in 2010, 29.5% of the MRSA clinical isolates were resistant to fosfomycin (Guo et al., 2013). And Yu et al. (2010) reported a fosfomycin susceptible rate of 33.2%.

The mechanisms of action and resistance of fosfomycin have been studied for decades. Fosfomycin was first discovered in 1969 as an effective bactericidal agent against Gram-positive and Gram-negative organisms. The mechanism of action of fosfomycin differs from that of most commonly used antimicrobials. In general, fosfomycin is transported across the bacterial wall primarily with the help of the glycerol-3-phosphate (G-3-P) transport (GlpT) system. In the presence of glucose-6-phosphate (G-6-P), the hexose phosphate uptake transport (UhpT) system is induced, and provides an alternative route to the GlpT system. UhpT are important membrane transporter proteins for small molecules, including fosfomycin (Castaneda-Garcia et al., 2009). When transported into the cytosol of a bacterium, fosfomycin deactivates the target protein UDP-N-acetylglucosamine-3-enolpyruvyltransferase (MurA, encoded by the murA gene), thereby preventing the formation of N-acetylmuramic acid from N-acetylglucosamine and phosphoenolpyruvate, which is the initial step in peptidoglycan chain formation of the bacterial wall (Kahan et al., 1974). The key resistance mechanisms to fosfomycin include the loss or reduced production of transporters, reduced affinity to MurA, and production of fosfomycin-modifying enzymes (Sastry and Doi, 2016).

However, to date, the mechanisms contributing to fosfomycin resistance have been mostly studied in Gram-negative bacteria, with few related studies on Gram-positive bacteria. We have conducted several studies to investigate the fosfomycin resistance mechanisms in Gram-positive cocci, including Enterococcus faecium and S. aureus (Xu et al., 2013; Chen et al., 2014; Fu et al., 2016a,b). These previous studies indicated that the fos gene was not the major mechanism of fosfomycin resistance in MRSA isolates from our hospital, whereas mutations of glpT and uhpT seemed to be more closely related to fosfomycin resistance. However, the exact roles of these two genes in S. aureus fosfomycin resistance remain unclear. Thus, we designed the present study to investigate the roles of glpT and uhpT in S. aureus fosfomycin resistance.

The deletion mutants showed considerably increased MIC values to fosfomycin compared to that of the parental strain. The Newman-ΔuhpT&glpT strain, in which both transporter genes were knocked out, showed high-level resistance (MIC > 1024 μg/ml) to fosfomycin, as determined by the E-test (Table 3). When uhpT or glpT was knocked out from S. aureus Newman, the fosfomycin MICs increased from 0.5 to 32 μg/ml or 4 μg/ml, respectively.

Complementing uhpT and glpT led to a reduced fosfomycin MIC in the deletion mutants and the clinical fosfomycin-resistant S. aureus strains with defects at both sites. By complementing plasmid pRB473-uhpT into Newman-ΔuhpT, the strain’s fosfomycin MIC decreased from 32 to 0.5 μg/ml. Similarly, by complementing glpT into Newman-ΔglpT, strain’s fosfomycin MIC decreased from 4 to 0.25 μg/ml. S. aureus SA2, SA94, and SA30 were clinical fosfomycin-resistant strains, with mutations of both uhpT and glpT, uhpT only, and glpT only, respectively. When complemented with the functional transporter genes, the fosfomycin MICs decreased considerably, as shown in Table 3.

In vitro bacterial growth curves of the wild-type strain S. aureus Newman and the deletion mutants were compared to evaluate the potential fitness cost of these resistant-conferring mutations. As shown in Figure 1, no significant depression in growth was observed in Newman-ΔuhpT and Newman-ΔglpT compared to the wild-type strain. However, the strain Newman-ΔuhpT&glpT presented slight growth inhibition compared to the wild-type.

Phenotype Microarray analysis was performed using carbon utilization panels, PM1 and PM2, in 190 carbon substrates. The changes in carbon metabolism were listed in Figure 2. S. aureus Newman showed metabolic advantage over Newman-ΔuhpT and Newman-ΔuhpT&glpT in wells containing G-6-P (Figure 2, PM1, well C1). G-3-P was not included in the substrate list, and there was no obvious change found in Newman-ΔglpT.

Intravenous fosfomycin is broadly used in the treatment of multidrug-resistant pathogens in Europe and Asia owing to its unique antibiotic mechanism, high permeability, and high susceptibility rate (Falagas et al., 2009). Falagas et al. (2010) performed fosfomycin susceptibility testing in non-urinary MRSA isolates, among which 99.2% (129/130) were found to be susceptible. The same group reviewed the susceptibility data of Gram-positive cocci, and reported a cumulative susceptibility rate of 87.9% (4240/4892) in S. aureus (Falagas et al., 2009).

Fosfomycin-resistance mechanism has been well described for Gram-negative bacteria such as E. coli (Kim et al., 1996; Horii et al., 1999; Huang et al., 2003; Takahata et al., 2010). In E. coli, GlpT and UhpT are responsible for fosfomycin uptake. Mutations or insertional inactivation in the glpT and/or uhpT genes or their regulatory genes lead to the loss of function of the transporters and fosfomycin resistance. The inactivation of either uhpT or glpT conferred a moderate fosfomycin resistance, (MICs increased from 2 to 8 μg/ml and 32 μg/ml, respectively, compared to the wild type) (Takahata et al., 2010). In P. aeruginosa, the inactivation of glpT produced significant decrease in fosfomycin MIC, from 8 to 1024 μg/ml (Castaneda-Garcia et al., 2009; Takahata et al., 2010). Modification or overexpression of murA, production of fosfomycin-modifying enzymes, are also associated with fosfomycin resistance (Garcia et al., 1995; Kim et al., 1996; Bernat et al., 1997; Horii et al., 1999; Fillgrove et al., 2003; Roberts et al., 2013).

There is less known of fosfomycin resistance mechanism in Gram-positive cocci. In previous works, we collected MRSA clinical strains, and found that only the minority of the fosfomycin-resistant MRSA strains carried the fos gene or murA mutation, while glpT and uhpT mutations were common (82.1%, 55/67, vs. 77.6%, 52/67, respectively) (Fu et al., 2016a,b). This fact indicated that, fosB or murA mutation is not the major contributor to fosfomycin resistance in MRSA, while mutations within the glpT and/or uhpT genes might play an important role in S. aureus fosfomycin resistance. In the present study, we established uhpT and/or glpT deletion mutants. Knocking out both genes resulted in high-level fosfomycin resistance (MIC > 1024 μg/ml). Complementing either of the two genes into the deletion mutants and clinical mutated strains resulted in a decreased fosfomycin MIC. Direct comparison of uhpT and glpT according to the level of increase of the MIC suggested that uhpT has a greater effect on the strain’s MIC to fosfomycin than glpT.

To evaluate the possible, the in vitro fitness cost of the transporter gene mutation, we compared growth curves between the fosfomycin-sensitive wild-type strain, laboratory deletion mutant strain, and clinical strains with defects on uhpT and/or glpT. Previous reports have shown that mutations of uhpT and glpT can compromise the growth of strains of E. coli, Klebsiella pneumoniae, and Proteus mirabilis (Li Pira et al., 1987; Marchese et al., 2003). A probable mechanism might be that mutations of the glpT and/or uhpT transporting systems prevent carbon source getting into the cytoplasm, and therefore disturb cell metabolism (Venkateswaran and Wu, 1972; Nilsson et al., 2003). But in P. aeruginosa, the glpT mutation was found to lead to fosfomycin resistance with no obvious fitness cost (Castaneda-Garcia et al., 2009). In the present study, there was only a slight reduction of growth observed in the strain Newman-ΔuhpT&glpT compared to the wild type, and no significant growth suppression was observed either in the laboratory deletion mutant strains or in clinical strains with defects on uhpT and/or glpT, which is similar as observed in P. aeruginosa. So S. aureus might also compensate the disadvantage in energy obtainment caused by uhpT and/or glpT mutation through other transporting systems. But further study is still in need for verification.

We observed that G-6-P utilization was defected in both Newman-ΔuhpT and Newman-ΔuhpT&glpT. UhpT is the membrane transporter of this substrate, deletion mutants of uhpT showed defects in G-6-P metabolism is as expected. The G-3-P metabolism in S. aureus seems to be more complicated. G-3-P seems to be an intermediate product in carbon/phosphorus metabolism pathway. As another low G+C Gram-positive bacteria, B. subtilis shares similar carbon metabolism pattern as S. aureus. In B. subtilis, G-3-P is produced from glycerol with glycerol kinase. And G-3-P dehydrogenase can oxidize G-3-P to dihydroxyacetone phosphate, an intermediated in glycolysis (Holmberg et al., 1990). We have not observed significant change in metabolism. This may be because that G-3-P utilization defect is easily compensated by other pathways.

In summary, the results of our study strongly suggest that mutations of uhpT and glpT lead to fosfomycin resistance in S. aureus, and that the uhpT mutation may play a more important role. The high resistance and low fitness cost resulting from uhpT and glpT mutations suggest that these mutated strains might have an evolutionary advantage in a fosfomycin-rich clinical situation. The widely observed uhpT or glpT mutation in S. aureus might be a threat in hospital settings. Further studies are needed to evaluate the frequency of S. aureus fosfomycin mutants, and virulence of these mutants.

Designed and conceived the experiments: YL, XX, and MW. Performed the experiments: SX, ZF, and YZ. Analyzed the data: SX, ZF, and YZ. Wrote and reviewed the manuscript: SX, ZF, XX, and YL.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.