Bacteriophage-Antibiotic Combination Strategy: an Alternative against Methicillin-Resistant Phenotypes of Staphylococcus aureus

Comparative time-kill experiments with Staphylococcus aureus bacteriophage (phage) Sb-1 alone and phage-antibiotic combinations (PACs) against two methicillin-resistant S. aureus (MRSA) strains have shown synergy with both daptomycin-phage and vancomycin-phage combinations. PACs prevented development of phage resistance and demonstrated bactericidal activity for all triple combinations. In addition, the extracellular membrane vesicle (MV) formation and the potential impact of phage on MV suppression were examined.

ABSTRACT

Comparative time-kill experiments with Staphylococcus aureus bacteriophage (phage) Sb-1 alone and phage-antibiotic combinations (PACs) against two methicillin-resistant S. aureus (MRSA) strains have shown synergy with both daptomycin-phage and vancomycin-phage combinations. PACs prevented development of phage resistance and demonstrated bactericidal activity for all triple combinations. In addition, the extracellular membrane vesicle (MV) formation and the potential impact of phage on MV suppression were examined. Our results demonstrate the potential of PAC for combating MRSA infections.

INTRODUCTION

Staphylococcus aureus is the leading cause of bacteremia and infective endocarditis that contribute significantly to morbidity and mortality (1–6). For decades, the antimicrobial treatment of choice for invasive methicillin-resistant S. aureus (MRSA) infections has been vancomycin (VAN); however, treatment failures and development of heterogeneous VAN intermediate-resistant S. aureus (hVISA), and VAN intermediate-resistant S. aureus (VISA) strains have led to the use of daptomycin (DAP) and ceftaroline (CPT), with bactericidal activity against MRSA (7–10). Despite the use of these alternatives, persistent bacteremia, development of DAP-nonsusceptible (DNS) strains, and mortality are still common in severe infections caused by S. aureus (11, 12).

Bacteriophages (phages), as natural predators of bacteria (13, 14), can evade the resistance developed to antibiotics through their different mechanisms of action (15–19). The hypothesis behind testing phage-antibiotic combinations (PACs) is that two distinctive selective pressures on bacterial growth can be more impactful than either alone (14, 20). Interestingly, Gram-positive bacteria, including S. aureus, naturally produce extracellular membrane vesicles (MVs) into their extracellular environment (21). These MVs may play a critical role in host immune evasion and transfer of virulence factors through the agr regulatory system and phenol-soluble modulins (22–29). The objective of this work was to investigate PAC therapy particularly in the context of existing antimicrobial resistance to individual agents (30–37) and the impact of PAC on MV formation with a bacteriophage used in Georgian hospitals (38).

S. aureus strain selection and in vitro susceptibility.

Eight well-characterized MRSA strains were screened for Sb-1 and antibiotic susceptibility (Table 1). Purified Sb-1 bacteriophage (a myophage categorized as Herelleviridae; GenBank accession no. HQ163896) was purchased commercially (38–40). Phage-mediated lysis and release of phage progeny particles were confirmed by formation of individual plaques on selected S. aureus strains (41). Sb-1 efficiency of plating (EOP) for strains D712 (DNS VISA, agr2) and MW2 (MRSA, agr2,3) (42) was equal to 1, 10⁻³, respectively. The two strains were used for 24-h time-kill experiments and MV studies. Bactericidal activity was defined as a >3-log₁₀-CFU/ml reduction from baseline. Synergy between two agents was defined as a >2-log₁₀-CFU/ml reduction compared with the most potent agent. Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple-comparison test (with P < 0.05 considered significant).

TABLE 1.

MIC values and Sb-1 sensitivity screening tests

Isolate	Phenotype	Strain type	Sb-1 sensitivitya	MIC (mg/liter) ofb :
				VAN	DAP	CPT	CFZ
COL	MRSA	USA100/ST250	C	2	0.25	0.25	32
D712	DNS VISA	USA100/ST5	C	4	4	0.5	>64
JH1	MRSA	USA100/ST5	C	1	0.25	0.25	16
494	MRSA	USA100/ST5	C	1	0.5	0.5	16
N315	MRSA	USA100/ST5	T	0.5	<0.13	0.25	64
MW2	MRSA	USA400/ST1	T	0.5	0.25	0.25	>64
D592	hVISA	USA100/ST5	T	2	0.5	2	>64
JO3	DNS	USA300/ST8	R	1	4	0.25	8

^aSb-1 produced clear or turbid spots or was resistant to phage infection. C, clear or high sensitivity to Sb-1 bacteriophage with distinct plaque formation (EOP of 0.1 to 1 compared with reference strain); T, turbid or medium sensitivity to Sb-1 with distinct plaque formation (EOP of 0.0001 to 0.01 compared with reference strain); R, resistant or no disturbance of bacterial lawn and no plaque formation.

^bVAN, vancomycin; DAP, daptomycin; CPT, ceftaroline; CFZ, cefazolin.

Time-kill analyses for assessing the PAC efficacy.

All triple combinations against D712 reached below the detection limit in 24 h (Fig. 1). Even though D712 is a DNS VISA strain, combinations of phage-DAP or phage-VAN were synergistic with significant reduction in log₁₀ CFU/ml versus the single antibiotics DAP and VAN, respectively (P < 0.0001). Our hypothesis regarding this observation is that the complementary action of PACs reduces colony counts while inhibiting the developed mechanisms for phage resistance, because PAC is not impacted by the antibiotic resistance status of the bacterial cell (14, 43, 44). Phage monotherapy revealed static activity with a 1.45-log₁₀-CFU/ml reduction from the initial inoculum, which was greater than those of any of the other individual agents. Other dual therapies, i.e., phage-cefazolin (CFZ) and phage-CPT, were as effective as phage alone (P > 0.05).

FIG 1.

(A, B) Time-kill experiments versus MRSA strain MW2 and DNS VISA strain D712. Triple combinations are highlighted as they demonstrated bactericidal activity compared with single antibiotics at the end of 24 h exposure. (C, D) Relative fluorescence units as identifiers of MV formation versus various treatments. Comparisons are versus growth control for each strain. VAN, vancomycin; DAP, D, daptomycin; CPT, ceftaroline; CFZ, cefazolin; Phage, P, bacteriophage Sb-1; ns, not significant.

Regarding the MW2 strain, phage monotherapy caused a 0.4-log₁₀-CFU/ml reduction from the initial inoculum. This observation correlates with our EOP assays categorizing MW2 phage susceptibility as intermediate. The phage-DAP and phage-VAN regimens were synergistic, causing significant reduction in log₁₀ CFU/ml versus the single antibiotics DAP and VAN, respectively (P < 0.0001). Phage-CPT was as effective as phage alone (P > 0.05), whereas the phage-CFZ regimen caused a 1.4-log₁₀-CFU/ml reduction compared with single-phage therapy (P = 0.005).

Resistance tests.

Resistance screening was performed using the double-drop method (41, 45) (Table 2). No evidence of bacterial resistance to Sb-1 was observed at the end of 24 h in time-kill samples with PAC, whereas phage-alone regimens developed resistance. Antibiotic resistance tests were performed as described previously against DAP, VAN, CPT, and CFZ (46, 47), and no resistance/elevated MICs were observed in any of the regimens against strain D712 at the end of 24 h.

TABLE 2.

Resistance check for bacteriophage and antibiotics at the end of 24 h exposure

Regimena	D712		MW2
	Phage resistanceb	Antibiotic resistance/elevated MIC (μg/ml)	Phage resistanceb	Antibiotic resistance/elevated MIC (μg/ml)
P	R	–c	R	–c
P-VAN	S		S
VAN	NA		NA
P-VAN-CFZ	S		S
P-VAN-CPT	S		S
P-DAP	S		S
DAP	NA		NA
P-DAP-CFZ	S		S
P-DAP-CPT	S		S
P-CFZ	S		S
P-CPT	S		S
GC	S	–c	S	–c

^aVAN, vancomycin; DAP, daptomycin; CPT, ceftaroline; CFZ, cefazolin; P, bacteriophage Sb-1; GC, growth control.

^bBacterial isolates were susceptible at the end of 24-h time-kill experiment. S, clear spot in double-drop method; R, resistant (phage activity was not observed within bacterial spot). Regimens not including bacteriophage were not tested for bacteriophage resistance. NA, not applicable.

^c–, antibiotic resistance/elevated MIC values were not detected. The resistance/elevated MIC test was performed against DAP, VAN, and CPT. D712 already has MIC values of >64 against CFZ.

Membrane vesicle formation measurements.

Our hypothesis regarding MV experiments was that phages may stimulate vesicle shedding through AgrA-mediated quorum sensing (24) (Fig. 1). To test this hypothesis, we measured membrane vesicle formation in all regimens/treatments, including growth control, using modified Bolte method (48). Although MVs activate the immune response, which is critical in cases like bacteremia, they can also worsen the disease state in circumstances such as infective endocarditis due to high production of specific proinflammatory cytokines (49, 50). Moreover, reduction in MV formation might improve overall antimicrobial efficacy if MVs act as “sinks” or decoys capable of binding to phages or antibacterials and effectively reducing the local concentration of active drug (51).

Strain D712 demonstrated significantly higher MV formation with β-lactam antibiotics than with either β-lactam-phage combination (P < 0.0001). A similar trend was observed with strain MW2, in that CFZ, VAN, or DAP regimens formed significantly greater amounts of MVs than corresponding PAC regimens (P < 0.0001), whereas CPT did not follow this trend in strain MW2. Interestingly, the MW2 strain exhibited 6-fold-higher MVs for growth control than the D712 strain. Therefore, suppression of MV formation was more pronounced with MW2 than with D712, and suppression of MVs in PACs versus D712 was not significant (P > 0.5).

Phage quantification and the impact of antibiotics on phage replication.

Phage quantification was assessed using the modified small-drop agar overlay method (41). Phage populations at the end of the 24-h time-kill analysis ranged between 10⁴ and 10⁶ PFU/ml for all treatments and bacterial strains. While the phage population (attached plus unattached) at the start of the experiment was 6 × 10⁵ PFU/ml (bacteriophage-to-bacterial ratio, 0.6), the number of unattached bacteriophages increased up to 10⁶ during the experiment. In general, mean phage counts for MW2 were less than D712 because of lower sensitivity. However, no specific pattern of phage increase/decrease was observed with addition of antibiotic(s).

The limitations of this study include the limited number of tested organisms, inclusion of only one bacteriophage, and lack of dose escalation/de-escalation experiments. The findings of this study introduce PAC as a promising alternative for combating multidrug-resistant infections. In addition, PAC may allow for a reduction in required antibiotic exposures, minimizing antibiotic adverse events and strengthening antibiotic stewardship efforts.