Impaired Alanine Transport or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to β-lactam Antibiotics

Abstract

Prolonging the clinical effectiveness of β-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between β-lactams and DCS. DCS resensitized MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics.

Whereas many bacteria can exhibit resistance to select antimicrobials, isolates of the human pathogen Staphylococcus aureus can express resistance to all licensed antistaphylococcal drugs. This results in significant morbidity and mortality rates, with up to 20% of patients with systemic methicillin-resistant S. aureus (MRSA) infections dying, despite receiving treatment with antistaphylococcal drugs [1]. As part of our efforts to identify improved therapeutic approaches for MRSA infections, Waters et al [2] recently described the novel use of β-lactam antibiotics to attenuate the virulence of MRSA-induced invasive pneumonia and sepsis. They demonstrated that oxacillin-induced repression of the Agr quorum-sensing system and altered cell wall architecture resulted in down-regulated toxin production and increased MRSA killing by phagocytic cells, respectively [2]. Supporting these in vitro data, a randomized controlled trial involving 60 patients showed that the β-lactam antibiotic flucloxacillin in combination with vancomycin shortened the duration of MRSA bacteremia from 3 to 1.9 days [3, 4].

Because expression of methicillin resistance in S. aureus affects fitness and virulence and is a regulated phenotype, further therapeutic interventions may also be possible. The complexity of the methicillin resistance phenotype is evident among clinical isolates of MRSA, which express either low-level, heterogeneous methicillin resistance or homogeneous, high-level resistance (HoR) [5–7]. Exposure of isolates with heterogeneous resistance to β-lactam antibiotics induces expression of mecA, which encodes the alternative penicillin-binding protein 2a (PBP2a) and can select for mutations in accessory genes resulting in a HoR phenotype, including mutations that affect the stringent response and c-di-AMP signaling [8–12]. Because accessory genes can influence the expression of methicillin resistance in MRSA, targeting the pathways associated with such genes may identify new ways to increase the susceptibility of MRSA to β-lactams.

To pursue this possibility, we performed a forward genetic screen to identify loci that affect the expression of resistance to β-lactam antibiotics in MRSA. Using the Nebraska Transposon Mutant Library (NTML), which comprises 1952 sequence-defined transposon insertion mutants [13], we found that inactivation of a putative amino acid permease gene, cycA, reduced resistance to cefoxitin, the β-lactam drug recommended by the Clinical and Laboratory Standards Institute for measuring mecA-mediated methicillin resistance in MRSA isolates. Amino acid transport and susceptibility to oxacillin and d-cycloserine (DCS) were compared in the wild-type and cycA mutant grown in chemically defined media (CDM), CDM supplemented with glucose (CDMG) and other complex media. The activity against MRSA of DCS and β-lactams, alone and in combination, was measured in vitro and in a mouse model of bacteremia. Peptidoglycan (PG) analysis was performed to compare the impact of the cycA mutation or exposure to DCS on cell wall structure and cross-linking. The results of our experiments suggest that therapeutic strategies targeting alanine transport, required for resistance to β-lactams and DCS, and a reevaluation of DCS may be important in efforts to restore the efficacy of β-lactam antibiotics against MRSA.

MATERIALS AND METHODS

Bacterial Strains, Growth Conditions, and Antimicrobial Susceptibility Testing

Bacterial strains (Supplementary Table 2) were grown in Luria-Bertani medium (Sigma), brain-heart infusion (BHI), trypic soya broth (TSB), Mueller-Hinton (MH), nutrient, sheep blood BHI, chemically defined medium (CDM), or CDM with 14 mmol/L glucose (CDMG) [14].

The minimum inhibitory concentrations (MICs) were determined in accordance with Clinical and Laboratory Standards Institute guidelines, using plate and broth dilution assays in MH, or MH with 2% sodium chloride (NaCl) for oxacillin and nafcillin. Oxacillin MICs were also measured using E-tests (Oxoid) on MH with 2% NaCl. Quality control strains American Type Culture Collection (ATCC) 29213 and ATCC 25923 were used for oxacillin and cefoxitin MIC assays, respectively.

Identification of Cefoxitin-Susceptible MRSA Mutant NE810

Cefoxitin disks (30 μg; Oxoid) were used to measure susceptibility of NTML mutants. The cefoxitin zone diameter for JE2 was 18 mm compared to >35 mm for NE1868 (mecA::Em^r) and 22 mm for NE810. The cycA transposon insertion in NE810 was verified by means of polymerase chain reaction using the primers NE810_Fwd and NE810_Rev (Supplementary Table 3). Phage 80α was used to transduce the NE810 cycA allele into JE2 and other strains. Genome sequencing was performed by MicrobesNG using the USA300_FPR3757 genome as a reference. To complement NE810, cycA was amplified from JE2 on a 1608–base pair fragment using primers NE810F1_Fwd and NE810F1_Rev (Supplementary Table 3) and cloned into pLI50 using the Clontech In-Fusion kit.

mecA Transcription Analysis

Quantitative reverse-transcription polymerase chain reaction was performed on a Roche LightCycler with primers mecA1_Fwd and mecA1_Rev for mecA and gyrB_Fwd and gyrB_Rev for gyrB (internal standard) (Supplementary Table 3), as described elsewhere [2]. Data presented are the average of 3 experiments with standard errors.

Amino Acid Transport Studies

Amino acid analysis in spent media from cultures grown in CDM or CDMG was performed as described elsewhere [15].

Analysis of PG Composition in NE810 and JE2 Treated With DCS

Independent quadruplicate 50-mL cultures were grown to an optical density at A₆₀₀ nm of 0.5, dosed with DCS at 0, 8, 20, or 32 μg/mL for 100 minutes, then harvested and resuspended in 5 mL of phosphate-buffered saline (PBS) (Supplementary Figure 3) before PG was extracted as described previously [16]. Mass spectrometry was performed with a Waters XevoG2-XS QTof mass spectrometer. Structural characterization of muropeptides was determined based on their mass spectrometric data and tandem mass spectrometric fragmentation pattern, matched with PG composition and structure reported elsewhere [17–20].

Antibiotic Synergy Analysis Using Microdilution Checkerboard Assay

Antibiotic synergism was measured using the checkerboard microdilution method in 96-well plates inoculated with 5 × 10⁵ colony-forming units (CFUs)/mL, and growth or no growth was assessed after 24 hours at 37°C. The fractional inhibitory concentration (FIC) index (ΣFIC) was calculated for each drug combination in triplicate experiments, with an ΣFIC of ≤0.5 considered synergistic.

Kill Curve Assays

Overnight cultures adjusted to 10⁷ CFUs/mL were exposed to 0.125×, 0.25×, and 0.5× MIC of oxacillin, cefoxitin, and DCS alone or in combination, and the CFUs per milliliter were enumerated at 0, 2, 4, 8, and 24 hours. Data are presented at the antibiotic concentrations where synergy was measured (ie, 0.5× MIC for JE2, USA300, DAR173, DAR22, DAR113, BH1CC, and RP62A, and 0.25× MIC for DAR169). Synergism was defined as a ≥2 log¹⁰ decrease in the CFU count (CFUs per milliliter) in cell suspensions exposed to DCS/β-lactam combinations compared with the most effective individual drug after 8 hours.

Mouse Infection Experiments

Age-matched 6- to 8-week-old, outbred CD1 female mice (Charles River) were used in a nonlethal model of bacteremia. JE2 and NE810 cultures were grown to an optical density at A₆₀₀ nm of 0.5 in BHI, washed in PBS, and adjusted to 1 × 10⁸ CFUs/mL. Mice were infected intravenously (via the tail vein) with 5 × 10⁶ CFUs (n  =  10 mice per group). The infections were left untreated (PBS control) or treated with either oxacillin (75 mg/kg/12 h), DCS (30 mg/kg/12 h) or a combination of both (first antibiotic dose administered 16 hours after infection), before being euthanized after 5 days. Bacteria present in homogenized spleens and kidneys recovered from the mice were enumerated on blood agar.

Ethics Statement

Mouse experiments were approved by the UK Home Office (license no. 40/3602) and the University of Liverpool Animal Welfare and Ethics Committee. This study was carried out in strict accordance with the UK Animals (Scientific Procedures) Act 1986. All efforts were made to minimize suffering.

Statistical Analysis

Two-tailed Student t tests and 1-way analysis of variance with Kruskal-Wallis test followed by Dunn multiple-comparisons test in the GraphPad Prism software 8.1.2 application (for the mouse infection experiments) were used to determine statistically significant differences in assays performed during this study. Differences were considered at P < .05.

RESULTS

Mutation of cycA Increases the Susceptibility of MRSA to β-Lactam Antibiotics and d-cycloserine

To identify new ways of controlling expression of methicillin resistance, we sought to identify novel mutations involved in this phenotype. An unbiased screen of the NTML to identify mutants with increased susceptibility to cefoxitin identified NE810 (SAUSA300_1642) (Supplementary Figure 1A), which also exhibited a >128-fold increase in susceptibility to oxacillin (Supplementary Figure 1B). NE810 was previously identified among several NTML mutants reported to be more susceptible to amoxicillin [21] but was not investigated further. Expression of mecA was not affected in NE810 (Supplementary Figure 1C), and genome sequence analysis revealed an intact staphylococcal cassette chromosome mec (SCCmec) element and the absence of any other mutations. NE810 was successfully complemented (Supplementary Figure 1B), and transduction of the SAUSA300_1642 allele into several MRSA strains from a number of clonal complexes and with different SCCmec types was also accompanied by increased cefoxitin and oxacillin susceptibility (Table 1).

Table 1.

Antibacterial and Drug Synergy of d-Cycloserine and β-Lactam Antibiotics, Alone and in β-Lactam Combinations, Against 14 Staphylococcus aureus strains and Staphylococcus epidermidis RP62A^a

	Antibiotic MIC, μg/mLb
Strain	OX	NAF	DCS	FOX	DCS/FOX (ΣFIC)	CEC	DCS/CEC (ΣFIC)	CTX	DCS/CTX (ΣFIC)	TZP	DCS/TZP (ΣFIC)	IPM	DCS/IPM (ΣFIC)
JE2	64	32	32	64	8/8 (0.37)	64	8/2 (0.28)	64	8/4 (0.31)	32	8/0.5 (0.26)	1	NDc
NE810 (cycA)	0.25	0.5	2	8	ND	4	ND	8	ND	2	ND	0.125	ND
USA300	64	32	32	64	8/8 (0.37)	64	8/8 (0.37)	64	8/8 (0.37)	64	8/2 (0.28)	1	ND
USA300 cycA	0.25	1	2	8	ND	4	ND	8	ND	4	ND	0.125	ND
DAR173	128	128	32	256	4/32 (0.25)	128	8/4 (0.28)	512	8/4 (0.25)	128	4/4 (0.15)	64	4/2 (0.15)
DAR173 cycA	0.5	8	4	32	ND	16	ND	16	ND	4	ND	1	ND
DAR22	128	128	32	128	8/8 (0.31)	128	8/8 (0.31)	512	8/4 (0.25)	128	4/4 (0.15)	128	4/1 (0.13)
DAR22 cycA	0.5	8	0.5	16	ND	16	ND	16	ND	4	ND	0.5	ND
DAR169	32	32	32	32	8/2 (0.31)	128	4/32 (0.37)	64	4/8 (0.25)	8	4/1 (0.25)	2	ND
DAR169 cycA	16	4	0.5	16	ND	64	ND	16	ND	2	ND	0.5	ND
COL	512	256	64	512	8/128 (0.37)	128	16/32 (0.5)	>2048	ND	128	32/0.5 (0.5)	256	8/64 (0.37)
DAR113	128	64	32	128	8/8 (0.3)	64	8/4 (0.3)	256	8/8 (0.2)	128	8/4 (0.2)	16	8/0.5 (0.2)
BH1CC	256	512	32	256	8/32 (0.3)	128	8/8 (0.3)	1028	8/16 (0.2)	256	8/4 (0.2)	64	8/1 (0.2)
BH14B(04)	128	64	16	128	4/32 (0.5)	128	4/32 (0.5)	512	4/32 (0.31)	64	4/1 (0.26)	64	2/8 (0.25)
BH8(03)	128	128	32	128	8/32 (0.5)	256	8/64 (0.5)	128	8/16 (0.25)	128	8/8 (0.3)	64	8/0.5 (0.1)
BH6	128	128	32	128	8/16 (0.37)	256	8/64 (0.5)	512	8/32 (0.31)	128	8/4 (0.28)	32	8/0.5 (0.26)
DAR202	64	64	32	128	4/32 (0.37)	128	4/32 (0.37)	64	8/16 (0.5)	64	8/2 (0.28)	4	8/1 (0.5)
DAR45	2	0.5	32	4	ND	32	8/2 (0.31)	4	ND	2	ND	0.5	ND
DAR13	128	32	32	128	4/32 (0.37)	128	8/4 (0.28)	128	4/32 (0.37)	32	8/1 (0.28)	8	4/2 (0.37)
RP62A	128	2	32	64	6/16 (0.5)	64	6/16 (0.5)	32	8/8 (0.5)	8	8/2 (0.5)	32	8/1 (0.2)

Abbreviations: CEC, cefaclor; CTX, cefotaxime; DCS, d-cycloserine; ΣFIC, fractional inhibitory concentration index; FOX, cefoxitin; IPM, imipenem; MIC, minimum inhibitory concentration; NAF, nafcillin; ND, not determined; OX, oxacillin; TZP, tazobactin.

^aThe antibiotics differ in penicillin-binding protein (PBP) specificity: oxacillin, PBP1, PBP2, and PBP3; nafcillin, PBP1; cefoxitin, PBP4; cefaclor, PBP3; cefotaxime, PBP2; piperacillin-tazobactin, PBP3/β-lactamase inhibitor; and imipenem, PBP1.

^bMIC values are given in micrograms per millilter for individual antibiotics, and as fractional inhibitory concentrations (FICs) (with ΣFIC in parentheses) for antibiotics in combination. ΣFIC = FIC A + FIC B, where FIC A is the MIC of DCS in combination with the β-lactam/MIC of DCS alone, and FIC B is the MIC of the β-lactam in combination with DCS/MIC of the β-lactam alone. The combination is considered synergistic when the ΣFIC is ≤0.5 and indifferent when it is >0.5 to <2.

^cND if strain is susceptible (or hyperresistant) to β-lactam antibiotic or for cycA mutants with reduced DCS and β-lactam MICs.

SAUSA300_1642 is annotated as a serine/alanine/glycine transporter with homology to CycA in Mycobacterium tuberculosis [22, 23], required for DCS resistance in mycobacteria [22, 23]. In contrast to the observations in Mycobacteria, our data showed that NE810 and several unrelated MRSA strains carrying the cycA mutation were significantly more susceptible to DCS than the wild-type JE2 (Supplementary Figure 1D and Table 1). The cycA mutation also reduced the DCS MIC of the methicillin-susceptible S. aureus strains 8325-4 and ATCC 29213 from 32 to 4 μg/mL. DCS inhibits alanine racemase (Alr), which converts l-alanine to d-alanine and the Ddl d-alanyl-d-alanine (d-ala-d-ala) ligase [24]. A mutant in the putative ddl SAUSA300_2039 gene is not available in the NTML, suggesting that it may be essential. However, the alr mutant NE1713 was significantly more susceptible to cefoxitin (Supplementary Figure 2A) (MIC, 16 μg/mL) and DCS (Supplementary Figure 2B) (MIC, <0.25 μg/mL), consistent with an important role for d-alanine in resistance to both antibiotics.

CycA is Required for Alanine Transport and d-ala-d-ala Incorporation Into the Peptidoglycan Stem Peptide

To investigate the role of CycA as a potential permease, JE2 and NE810 were grown for 8 hours in CDM containing 14 mmol/L glucose (CDMG) and amino acid consumption in spent media was measured. Although no growth rate or yield difference was noted between JE2 and NE810 in CDMG (Figure 1A), alanine uptake by NE810 was significantly impaired compared with JE2 (Figure 1B). Use of other amino acids, including serine and glycine, was similar NE810 and JE2 (Supplementary Figure 3). Impaired alanine transport in the cycA mutant grown in CDMG was correlated with increased susceptibility to oxacillin (1 μg/mL) (Figure 1C) and DCS (1 μg/mL) (Figure 1D). These data demonstrate for the first time that CycA in S. aureus is required for alanine transport.

Figure 1.

Mutation of cycA impairs alanine uptake. A, Growth of JE2 and NE810 in chemically defined medium supplemented with glucose (CDMG). B, Alanine consumption by JE2 and NE810 grown aerobically in CDMG. Residual amino acid was measured in spent media after 2, 4, 6 and 8 hours of growth. C, D, Growth of JE2 and NE810 cultures for 12 hours in CDMG supplemented with 1 μg/mL oxacillin (C) or 1 μg/mL d-cycloserine (DCS) (D). Cell density was measured as optical density at A600 nm (OD₆₀₀).

Quantitative PG compositional analysis was performed using ultraperformance liquid chromatographic analysis of muramidase-digested muropeptide fragments extracted from exponential phase cultures of JE2 and NE810 grown for 220 minutes in TSB media (Supplementary Figure 4). The PG profile of the cycA mutant revealed a significant accumulation of tripeptides compared with wild-type JE2 (Figure 2A and 2B), which was associated with a significant reduction in cross-linking (Figure 2C). In NE810, the dimer, trimer, and tetramer fractions were decreased, which was accompanied by a concomitant increase in the monomer fraction (Figure 2D).

Figure 2.

Mutation of cycA or d-cycloserine (DCS) treatment impacts peptidoglycan peptide stem length and reduces cell wall crosslinking. A, Representative UV chromatograms of peptidoglycan from wild-type JE2, NE810 and JE2 treated with increasing concentrations of DCS (8, 20 and 32 μg/mL). Muropeptides with tripeptide stems are numbered 1–3. The Proposed structures of the 3 muropeptides with tripeptide stems identified in NE810 and DCS-treated JE2 cells. The theoretical and observed neutral masses determined by mass spectrometry (MS) are indicated. B, Relative abundance of muropeptides with tripeptides in the stem. C, Relative cross-linking efficiency. D, Relative proportions of cell wall muropeptide fractions based on oligomerization. All errors bars represent 95% confidence interval (n = 4). Significant differences determined using Students t-test are denoted using asterisks (*P < .05; **P < .01; ***P < .001). Abbreviations: d-Gln, d-glutamine; d-Glu, d-glutamic acid; l-Ala, L352 alanine; l-Lys, l-lysine; NAG, n-acetylglucosamine; NAM, n-acetylmuramic acid.

Consistent with these data, exposure of JE2 to DCS 8 μg/mL was also associated with a similar accumulation in muropeptides with tripeptide stems (Figure 2B), reduced cross-linking (Figure 2C), increased muropeptide monomers, and reduced dimers, trimers, and tetramers (Figure 2D). DCS had a strong dose-dependent effect on the accumulation of muropeptides with tripeptide stems, reduced cross-linking, and accumulation of monomers (Figure 2A–2D). Subinhibitory (0.25× MIC) and 4× MIC DCS concentrations, were previously shown to be associated with incorporation of an incomplete stem peptide (tripeptide) [24] and reduced d-ala-d-ala levels [25], respectively. These data show that impaired d-ala incorporation in the cycA mutant or following exposure to DCS is accompanied by reduced PG cross-linking and increased β-lactam susceptibility.

Mutation of cycA or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to β-Lactam Antibiotics

Previously reported synergy between DCS and β-lactam antibiotics [24, 26] suggests that impaired alanine uptake in the cycA mutant may have the same impact on cell wall biosynthesis as DCS-mediated inhibition of Alr and Ddl. To further investigate this, we compared the activity of DCS and β-lactam antibiotics, alone and in combination, against JE2 and NE810. Checkerboard microdilution assay ΣFICs (ΣFIC ≤0.5) revealed synergy between DCS and several licensed β-lactam antibiotics with different PBP selectivity against JE2 and USA300 FPR3757 (Table 1). Oxacillin and nafcillin were not included in checkerboard assays because measurement of their MICs involves supplementing the media with 2% NaCl, which distorts the MIC of DCS (data not shown).

Using the MRSA strains JE2, USA300, DAR173, DAR22, DAR169, and their corresponding cycA mutants, the kinetics of killing by DCS, oxacillin, and cefoxitin, alone and in combination, was measured over 24 hours using antibiotic concentrations corresponding to 0.125×, 0.25×, and 0.5× fold MICs. Recovery of growth in media supplemented with oxacillin or cefoxitin alone was evident after 8 hours (Figure 3), reflecting the selection and expansion of HoR mutants as described elsewhere [2, 25, 27]. Recovery of growth in cultures exposed to DCS alone was also evident (Figure 3), which may correlate with our observation that mutants resistant to DCS (on BHI agar supplemented with 128 μg/mL DCS) arise at a rate of approximately 5.5 × 10^–8 per cell per generation.

Figure 3.

In vitro kill curves for d-cycloserine (DCS), oxacillin, and cefoxitin with JE2, USA300 FPR3757, DAR173, DAR22, DAR169, and their isogenic cycA mutants. Antibiotics at the concentrations indicated (in micrograms per milliliter) were added to suspensions of overnight bacterial cultures adjusted to 10⁷ CFUs/mL in brain-heart infusion (optical density at A600 nm, 0.05), incubated at 37^oC and the CFU count (CFUs/mL) measured at 0, 2, 4, 8 and 24 hours. The data presented are the means of 3 independent experiments, with standard errors of the mean. Antibiotic synergism was defined as a ≥2 log¹⁰ decrease in CFU count in cell suspensions exposed to DCS/β-lactam combinations, compared with the most effective individual antibiotic alone. Abbreviation: SCCmec, staphylococcal cassette chromosome mec.

Using combinations of DCS and oxacillin or cefoxitin at 0.125× MIC did not achieve a ≥2 log¹⁰ reduction in the number of CFUs per milliliter (data not shown). However, at 0.5× MIC for strains JE2, USA300, DAR173, and DAR22, DCS (16 μg/mL)/oxacillin (32 μg/mL) and DCS (16 μg/mL)/cefoxitin (32 μg/mL) combinations achieved a ≥5 log¹⁰ reduction in CFUs/ compared with oxacillin, cefoxitin, or DCS alone (Figure 3). For strain DAR169, DCS/β-lactam combinations at 0.25-fold MIC was sufficient to achieve a ≥5 log¹⁰ reduction in CFUs recovered, compared with the individual antibiotics (Figure 3). DCS/β-lactam combinations at 0.5× MIC were also able to achieve ≥5 log¹⁰ reduction in the number of CFUs per millilter against the methicillin-resistant Staphylococcus epidermidis strain RP62A [28], compared with either antibiotic alone (Supplementary Figure 5). Checkerboard experiments with 14 MRSA strains and MRSE strain RP62A further revealed synergy (ΣFIC ≤0.5) between DCS and a range of β-lactam antibiotics with different PBP specificities, namely cefoxitin (PBP4), cefaclor (PBP3), cefotaxime (PBP2), piperacillin-tazobactin (PBP3/ β-lactamase inhibitor), and imipenem (PBP1) (Table 1).

This synergy seems to be specific to β-lactams, and no synergy (ΣFIC >0.5) was measured between DCS and several antibiotics that are used topically or systemically to decolonize or treat patients colonized or infected with S. aureus or MRSA (clindamycin, trimethoprim, mupirocin, and ciprofloxacin), or several antibiotics to which S. aureus isolates commonly exhibit resistance (tobramycin, kanamycin, and spectinomycin) (Supplementary Table 1). Furthermore, the cycA mutation had no impact on susceptibility to any of these non–β-lactam antibiotics (apart from ermB-encoded clindamycin resistance on the transposon).

Combination Therapy with DCS and Oxacillin Significantly Reduces the Bacterial Burden in the Kidneys and Spleen of Mice Infected with MRSA

The virulence of the NE810 mutant and the therapeutic potential of oxacillin in combination with DCS in the treatment of MRSA infections were assessed in mice. Treatment with oxacillin or DCS alone significantly reduced the number of CFUs recovered from the kidneys of mice infected with JE2 (Figure 4). Furthermore, the oxacillin/DCS combination was significantly more effective than either antibiotic alone, and the combination was equally effective in reducing the bacterial burden in the kidneys of animals infected with JE2 or NE810 when compared with no treatment (P ≤ .001) (Figure 4) demonstrating the capacity of DCS to significantly potentiate the activity of β-lactam antibiotics against MRSA under in vivo conditions. Unexpectedly, oxacillin- or DCS-mediated eradication of NE810 infections in the kidneys was similar to findings with JE2 (Figure 4). In the spleen, only oxacillin/DCS combination treatment was associated with a significant reduction in the number of CFUs recovered from mice infected with JE2 or NE810 (Supplementary Figure 6).

Figure 4.

Combination therapy with d-cycloserine and oxacillin significantly reduces the bacterial burden in the kidneys of mice infected with MRSA. The number of colony-forming units (CFU) recovered from the kidneys of mice infected by tail vein injection with 5 × 106 JE2 or NE810 (CycA) and left untreated or treated with 75mg of oxacillin (Ox)/kg, 30mg of DCS/kg or a combination of both Ox and DCS delivered subcutaneously every 12 hours for 5 days. The first antibiotic dose was given 16 hours after infection. Significant differences determined using one-way ANOVA with Kruskal-Wallis test followed by Dunn’s multiple comparisons test are denoted using asterisks (*P ≤ .05, **P ≤ .01, ****P ≤ .0001). The limit of detection (50 colonies) is indicated with a hashed red line.

Alanine Transport and Resistance to Oxacillin and DCS in Chemically Defined Medium are not Dependent on cycA

The failure of oxacillin or DCS treatment to enhance the eradication of NE810 infections in the mouse bacteremia model prompted us to further characterize the growth conditions used for the in vitro antibiotic susceptibility assays. Specifically, we investigated the role of glucose, which was previously reported to increase the growth requirement for amino acids [15], and which we reasoned may be important for CycA-dependent alanine transport. Growth of JE2 and NE810 were similar in CDM lacking glucose (Figure 5A), and uptake of alanine (Figure 5B) and other amino acids (Supplementary Figure 7) was unchanged in NE810 compared with JE2. Furthermore, NE810 and JE2 grew equally well in CDM supplemented with oxacillin and DSC (Figure 5C and 5D). These data explain in part why the cycA mutant does not exhibit increased β-lactam and DCS susceptibility in the mouse bacteremia model and further reveal the strong correlation between alanine transport and susceptibility to oxacillin and DCS.

Figure 5.

Alanine transport and resistance to oxacillin and d-cycloserine (DCS) in chemically defined medium (CDM) are cycA independent. A, Growth of JE2 and NE810 in CDM lacking glucose. Cell density was measured as optical density at A600 nm (OD₆₀₀). B, Alanine consumption by JE2 and NE810 grown aerobically in CDM. Residual amino acid was measured in spent media after 2, 4, 6, and 8 hours growth. C, D, Growth (A₆₀₀) of JE2 and NE810 cultures for 12 hours in CDM supplemented with 1 μg/mL oxacillin (C) or 1 μg/mL DCS (D).

DISCUSSION

The exploitation of antibiotic repurposing as part of concerted efforts to address the antimicrobial resistance crisis has been hampered by a lack of mechanistic data to explain demonstrated therapeutic potential and the perception that studies attempting to identify new uses for existing drugs are not hypothesis driven. In the current study, we revealed that CycA was required for full expression of resistance to β-lactam antibiotics and DCS. Loss of function of this putative alanine transporter significantly increased the susceptibility of MRSA to β-lactam antibiotics, an outcome that could be reproduced through exposure to DCS, which targets the Alr and Ddl enzymes in the early steps of cell wall biosynthesis.

The potential of β-lactam/DCS combinations for treatment of MRSA infections follows a recent report that DCS can also potentiate the activity of vancomycin against a laboratory-generated vancomycin-resistant highly-resistant S. aureus strain in vitro and in a silkworm infection model [29]. The excellent safety profile of β-lactam antibiotics makes these drugs particularly attractive as components of combination antimicrobial therapies. When used in the treatment of tuberculosis, DCS (trade name Seromycin; The Chao Centre) is typically administered orally in 250-mg tablets twice daily for up to 2 years. At this dosage, the DCS concentration in blood serum is generally 25–30 μg/mL, similar to the concentrations used in our in vitro and in vivo experiments. The known neurological side effects associated with DCS therapy [30, 31] mean that this antibiotic is unlikely to be considered for the treatment of MRSA infections unless alternative therapeutic approaches have been exhausted. Combination therapy with DCS and oxacillin was significantly more effective than either drug alone over a 5-day therapeutic window, suggesting that further studies on using DCS to augment β-lactams to treat recalcitrant staphylococcal infections are merited.

Mutation of cycA increases the susceptibility of MRSA to β-lactam antibiotics and results in hypersusceptibility to DCS, whereas a cycA point mutation in Mycobacterium bovis contributes, in part, to increased DCS resistance, presumably by interfering with transport into the cell [23]. In Escherichia coli, cycA mutations can also result in increased resistance or have no effect on DCS susceptibility depending on the growth media [32–36], suggesting that CycA is primarily important for DCS resistance under conditions when its contribution to amino acid transport is also important. Our data showing that mutation of cycA was not associated with increased DCS resistance strongly suggest that CycA has no role in uptake of this antibiotic in S. aureus.

Under growth conditions where CycA is required for alanine transport (in nutrient/glucose-replete media), mutation of cycA and DCS exposure have similar effects on the structure of S. aureus PG (Figure 6). Consistent with previous studies in S. aureus [24] and in M. tuberculosis [37], our studies showed a dose-dependent accumulation of muropeptides with a tripeptide stem in MRSA exposed to DCS. The cycA mutation was also associated with the increased accumulation of muropeptides with a tripeptide stem. These data indicate that a reduced intracellular alanine pool or inhibition of Alr and Ddl is associated with reduced d-ala-d-ala incorporation into the PG stem peptide. The increased accumulation of tripeptides in turn interferes with normal PBP transpeptidase activity and offers a plausible explanation for increased susceptibility to β-lactam antibiotics. The importance of the terminal stem peptide d-ala-d-ala for β-lactam resistance has previously been reported. Mutation of the murF-encoded ligase, which catalyses of the d-ala-d-ala into the stem peptide also increased β-lactam (but not DCS) susceptibility [38, 39]. Similarly, growth of a HoR MRSA strain in media supplemented with high concentrations of glycine was accompanied by replacement of the d-ala-d-ala with d-ala-gly and decreased methicillin resistance [17].

Figure 6.

Proposed model depicting how impaired alanine transport associated with mutation of CycA or exposure to d-cycloserine (DCS) can inhibit the d-alanine pathway for peptidoglycan biosynthesis, leading to increased susceptibility to β-lactam antibiotics. Abbreviations: Alr, alanine racemase; CDM, chemically defined medium; CDMG, CDM supplemented with glucose; d-ala, d-alanine; d-ala-d-ala, d-alanyl-d-alanine; Ddl, d-ala-d-ala ligase; d-glx, d-glutamine; l-ala, l-alanine; NAG, n-acetylglucosamine; NAM, n-acetylmuramic acid; PG, peptidoglycan.

Impaired uptake of alanine in CDMG was correlated with increased susceptibility to oxacillin and DCS, suggesting that alanine utilisation via CycA is important to make d-alanine available for cell wall biosynthesis and consequently resistance to β-lactams. Consistent with this, NE810 also exhibited increased oxacillin susceptibility in BHI, TSB, and MH media. However, no change in alanine transport or susceptibility to oxacillin and DCS was measured in CDM lacking glucose, which may explain the failure of oxacillin and DCS to more efficiently eradicate NE810 infections in the mouse bacteremia model. The availability of nutrients such as glucose and amino acids varies in different niches colonized by S. aureus during infection, ranging from glucose rich in organs such as the liver [40] to glucose depleted in established abscesses [41].

In turn, this affects the role of amino acids as carbon sources [15, 42], and potentially the activity of CycA in alanine transport and β-lactam susceptibility. Furthermore, normal alanine transport in the cycA mutant grown in CDM indicates that an alternative alanine transport mechanism(s) may be active under these growth conditions (Figure 6). Identification of this alternative alanine permease may be important in the development of therapeutic strategies targeting alanine transport to increase β-lactam susceptibility in MRSA, and elucidation of the role of glucose in the control of alanine transport should provide new insights into β-lactam resistance.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Acknowledgments. We thank Craig Winstanley and Kate Reddington for generously providing bacterial strains.

Disclaimer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Financial support. This study was supported by the Irish Health Research Board (grants HRA_POR/2012/51 and HRA-POR-2015-1158 to J.P.O'G.), Science Foundation Ireland (grant 16/TIDA/4137 to J.P.O'G.), the UK Medical Research Council (grant MR/M020045/1 to A. K.), the National Institutes of Health (grant AI083211 to P. D. F), and Svenska Forskningsrådet Formas (F. C).

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.