Sub-lethal doses of surotomycin and vancomycin have similar effects on Clostridium difficile virulence factor production in vitro

Abstract

Purpose

Clostridium difficile is an anaerobic spore-forming bacterial pathogen that causes a spectrum of illness severity ranging from mild diarrhoea to severe life-threatening pseudomembranous colitis. C. difficile infection (CDI) is antibiotic-associated and primarily mediated by two exotoxins, Toxins A and B. We and others have shown that some antibiotics stimulate Toxin A and B production by C. difficile in a strain-specific manner. Still, the effects of newer anti-C. difficile antibiotics on this process and spore formation remain to be investigated.

Methodology

Surotomycin (formally CB-183,315) is a novel, minimally absorbed, narrow-spectrum antibiotic. We determined the effects of surotomycin on C. difficile growth, toxin production and sporulation in historical and BI/NAP1/027 epidemic strains of C. difficile.

Results/Key findings

While antibiotic free controls showed toxin production during the stationary phase growth, all strains exposed to sub-inhibitory concentrations of surotomycin and vancomycin demonstrated increased TcdA and TcdB production during early (log phase) growth by all strains. However, this effect was not observed at 24 or 48 h post-treatment by any of the C. difficile strains exposed to either antibiotic. Additionally, all doses of surotomycin and vancomycin suppressed spore formation in all tested strains.

Conclusion

In summary, these findings demonstrate that surotomycin and vancomycin have similar effects on exotoxin production and sporulation by C. difficile in vitro. Furthermore, since spores contribute to recurrent infection, the ability of surotomycin to suppress spore formation may explain its ability to disrupt the reinfection cycle in the clinical setting.

Introduction

Clostridium difficile is a notorious nosocomial pathogen that causes antibiotic-associated diarrhoea in hospitals and healthcare facilities worldwide. In the United States, C. difficile-associated deaths have increased 400 % over the last decade, resulting in greater than 20 000 deaths per year [1]. Similar trends in C. difficile infections (CDI) have also been reported in Canada and Europe [2, 3]. Recent increases in CDI severity and mortality are largely attributed to the rapid dissemination of epidemic strains and increased resistance to conventional antimicrobial therapies [4, 5]. Recurrent C. difficile infections are also of major clinical concern, as approximately 25 % of all CDI patients will develop a second infection following standard antibiotic treatment. Of these, nearly 40 % will experience additional recurrent CDI infections [6].

Effective therapies for treating patients with severe and recurrent CDI remain limited. Oral administration of vancomycin in combination with intravenous metronidazole is the recommended treatment for an initial episode of fulminant CDI [7]. However, examples of treatment failure and disease recurrence for these antibiotics are now being reported [8]. In 2011 fidaxomicin was approved by the U.S. Food and Drug Administration (FDA) to clinically treat CDI [9], representing the first new CDI-approved antibiotic treatment in over 20 years. More recently, surotomycin (CB-183,315), a narrow-spectrum cyclic lipopeptide antibiotic, was developed as an alternative therapy for treating severe cases of CDI and for the prevention of recurrent disease. In a Phase 2 controlled trial, patients receiving surotomycin displayed significantly lower CDI recurrence rates when compared to those treated with vancomycin [10]. Subsequent Phase 3 clinical trials later showed that surotomycin had a cure rate and sustained clinical response that was neither superior nor inferior to that of vancomycin [11, 12].

In vitro, surotomycin displays strong activity against C. difficile [8] and is capable of killing C. difficile organisms during exponential- and stationary-phase growth as well as during the spore germination phase [13]. At inhibitory concentrations, surotomycin suppresses Toxin A (TcdA) and Toxin B (TcdB) production. Specifically, Bouillaut et al. [13] and Endres et al. [14] both demonstrated that surotomycin at supra-inhibitory concentrations [4x and 40x, and 8x and 80x the minimum inhibitory concentrations (MIC), respectively] did not enhance TcdA and TcdB production in stationary phase organisms. In addition, Endres et al. reported that no differences in toxin production were observed when sub-inhibitory concentrations of surotomycin (0.5x MIC) were added to stationary-phase cultures [14]. Work done by Chilton et al. further showed that in a human gut model of CDI, high concentrations of surotomycin rapidly reduced toxin production and resolved simulated C. difficile infection to the same extent as vancomycin [15].

Previous work by our laboratory and others has demonstrated that some antibiotics at sub-MIC doses stimulate early and prolonged TcdA and TcdB production by rapidly growing C. difficile organisms [16–18]. Based on these findings, this study was designed to determine the effects of sub-inhibitory concentrations of surotomycin on soluble TcdA and TcdB protein production and sporulation by multiple strains of C. difficile during exponential and early-stationary-phase growth. Better understanding of the effects of surotomycin on exotoxin production and sporulation may be important when considering surotomycin as an alternative antimicrobial therapy for patients with severe or recurrent C. difficile infection.

Methods

C. difficile strains and growth conditions

Four strains of C. difficile were studied. American Type Culture Collection (ATCC) strain 9689 is a strain determined as toxinotype 0 by pulsed-field gel electrophoresis. Hines VA strain 5325 is a historical BI/NAP1/027 clinical isolate collected in 1993 (specific REA type BI 1 and toxinotype III, a kind gift from Dr Stuart Johnson; Hines VA Medical Center, Hines, IL). Strain 2989, a recent epidemic BI/NAP1/027 clinical isolate provided by Cubist Pharmaceuticals, was collected during the LCD-DR-09-03 Phase 2 study. All growth experiments were performed in a Bactron 300 anaerobic chamber in an atmosphere containing 5 % carbon dioxide, 5 % hydrogen and 90 % nitrogen. C. difficile strains used in this study were grown in brain–heart infusion (BHI) medium. All cultures grown in the presence of surotomycin were supplemented with additional Ca²⁺ at a final concentration of 50 mg l⁻¹. Prior to the initiation of the proposed studies, the inherent level of Ca²⁺ in BHI liquid medium was first determined by Laboratory Specialists, Inc. (Westlake, OH). CaCl₂·2H₂O was then added to the BHI medium as recommended by Laboratory Specialists, Inc. to achieve a final concentration of 50 mg l⁻¹.

Determination of minimum inhibitory concentrations (MIC)

Analytical grade stock samples of surotomycin were provided by Cubist Pharmaceuticals. The minimum inhibitory concentrations (MICs) of surotomycin and vancomycin were determined for all C. difficile strains by microbroth dilution assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for such testing in anaerobes [19], and as we have previously described [16]. In brief, 200 µl of a stationary overnight culture was used to inoculate 20 ml of pre-reduced BHI broth. Cultures were grown anaerobically for approximately 2 h at 37 °C until a turbidity equal to the 0.5 McFarland standard (OD₆₃₀ of 0.08–0.1; ~1×10⁶ c.f.u. ml⁻¹) was achieved. The prepared C. difficile (50 µl) was then added to duplicate wells of a 96-well plate containing 50 µl of 2-fold serially diluted (0.25–32 µg ml⁻¹) surotomycin. Plates were incubated anaerobically at 37 °C for 48 h and growth (turbidity) was assessed by a microplate reader (OD₆₃₀). MICs were defined as the lowest antibiotic concentration that inhibited measurable bacterial growth (i.e. OD630 equal to the negative control). MICs were performed five times in triplicate.

Growth curves

C. difficile isolates were cultured anaerobically overnight in 10 ml BHI, whereupon bacteria were collected by centrifugation and washed once with reduced 0.9 % saline. The pellet was resuspended to the original volume (10 ml) in fresh (reduced and warmed to 37 °C) BHI then 1 ml of washed bacteria was added to 100 ml of fresh, pre-equilibrated BHI for a 1 % inoculum. This was allowed to grow ~1 h to an OD₆₃₀ of 0.08–0.1. Ten millilitres of this culture was then added to 990 ml of fresh, pre-reduced BHI and grown anaerobically for ~2 h to an OD₆₃₀ of 0.08–0.1 (10 % inoculum). Aliquots (99 mLs) of this 10 % culture were divided among five individual sterile 250 ml flasks. Surotomycin was prepared in calcium-supplemented BHI (see Methods above) as a 100× stock solution with reference to the highest concentration required. Vancomycin was prepared in 0.9 % saline as a 100x stock solution with reference to the highest concentration required. Two-fold serial dilutions were made from this stock in BHI and 0.9 % saline, respectively, and 1 ml of each appropriate stock was added to 99 ml of C. difficile culture to give final concentrations of 2×, 1×, 1/4× and 1/8× MIC (as recommended by the sponsor), respectively. At times 0 h (antibiotics added) and 6, 12, 24 and 48 h after antibiotic addition, 10 ml samples were removed from each C. difficile culture and a small aliquot (10 µl) was used to determine viable c.f.u. ml⁻¹. Resultant supernatants were filter sterilized through 0.22 µm syringe filters and frozen at −70 °C for soluble TcdA/B production by ELISA.

Toxin production

Soluble TcdA and TcdB protein levels were measured (in combination) in collected culture supernatant samples using the Wampole Tox A/B II kit (TechLabs, Blacksburg, VA) according to the manufacturer’s recommendations. TcdB purified from a NAP1 isolate (a kind gift from Dr Jimmy Ballard, University of Oklahoma Health Sciences Center; stock concentration 300 µg ml⁻¹) was used to construct a standard curve [16, 20]. Samples were diluted when necessary to obtain readings within the linear range of the standard (500–7 ng ml⁻¹). All samples were tested in duplicate.

Sporulation

To determine the number of spores produced by C. difficile strains over the growth cycle, 0.5 ml aliquots were collected from antibiotic- and control-treated cultures at 24 and 48 h post-treatment. Collected samples were mixed with 1 ml of 100 % ethanol for one hour with rotation at room temperature to kill vegetative organisms [21]. Samples were then pelleted by centrifugation (13 000 g for 10 min) and washed twice in dPBS. Following the last wash, pellets were resuspended in 0.5 ml dPBS. Spores were enumerated by serially diluting the samples in 0.9 % saline and plating onto BHI agar plates supplemented with 0.1 % sodium taurocholate. Plates were incubated anaerobically at 37 °C for 24 h and resultant colony-forming units/ml calculated. Approximately 0.1 % of spores estimated by visual inspection using light microscopic analysis germinate and grow as colony-forming units on BHI agar plates (unpublished data). These values are in agreement with germination rates and outgrowth efficiencies reported by Lawley et al. [21].

Results

Minimum inhibitory concentrations of historical and NAP1 strains

The minimum inhibitory concentrations (MICs) for surotomycin and vancomycin were determined for the 9689 strain and the 5325 and 2989 historic and epidemic BI/NAP1/027 isolates, respectively. According to the CLSI and National Committee for Clinical Laboratory Standards (NCCLS) interpretative categories for resistance, sensitive organisms have an MIC<8.0 µg ml⁻¹. All strains tested displayed sensitivity to surotomycin. The 5325 and 2989 strains had MIC susceptibility values of 2.0 µg ml⁻¹ and the 9689 strain an MIC of 1.0 µg ml⁻¹ (Table 1). Similar susceptibilities were also observed for vancomycin, with MIC values of 2.0 µg ml⁻¹ for the 9689 and 5325 strains and 4.0 µg ml⁻¹ for the 2989 strain (Table 1). These MIC values are similar to those previously reported by others [8, 13].

Table 1. Minimum inhibitory concentrations (MICs) for surotomycin and vancomycin were determined for American Type Culture Collection (ATCC) strain 9689 (toxinotype 0), a historical BI/NAP1/027 clinical isolate (Hines VA strain 5325) and a recent epidemic BI/NAP1/027 clinical isolate (strain 2989 provided by Cubist Pharmaceuticals).

Strain	Vancomycin MIC (µg ml−1)	Surotomycin MIC (µg ml−1)
9689	2	1
5325	2	2
2989	4	2

Growth of C. difficile in the presence of surotomycin and vancomycin

There were minimal variations among the growth rates of all strains of C. difficile in antibiotic-free media, and all three peaked at ~10⁸ c.f.u. ml⁻¹ by 6 h and were in the stationary phase by 12 h (Fig. 1a). Minor effects were observed on growth following the addition of sub-inhibitory concentrations of surotomycin (1/8X and 1/4X MIC; Fig. 1b, c). Specifically, all three cultures reached stationary phase growth at 6–12 h, though the viability of strains 535 and 2989, and not 9689, decreased by ~1 log at 48 h in 1/4X MIC surotomycin (Fig. 1b, c). In addition, surotomycin at 1X MIC suppressed growth in all C. difficile strains at 6–12 h; however, their viability recovered to levels similar to antibiotic-free cultures by 24 h (Fig. 1d). Exposure to 2× MIC surotomycin resulted in a rapid decline in growth by all strains, and by 12 h all 2× MIC surotomycin cultures displayed minimal viability and were terminated (Fig. 1e).

Fig. 1.

Effects of surotomycin on growth of C. difficile historical 9689 and epidemic 9689 and 5325 strains. (a) Antibiotic-free cultures; (b) 1/8× MIC; (c) 1/4× MIC; (d) 1× MIC; and (e) 2× MIC. Surotomycin was added, at the final concentrations indicated, during early log phase growth (designated Time 0). Samples were collected in duplicate over 48 h for quantification of viable C. difficile.

Analogous growth trends were observed in cultures exposed to antibiotic-free and sub-MIC doses of vancomycin (Fig. 2a–c), as growth peaked at 6–12 h and reached a maximum of ~10⁸–10⁹ c.f.u. ml⁻¹ (Fig. 2a–c) in all treatments. In contrast to 1× MIC surotomycin, all organisms exposed to 1× MIC vancomycin did not enter exponential growth until 24 h (Fig. 2d), and the maximum growth was ~2 logs lower at 48 h for all strains except 2989 (Fig. 2d). Similar to surotomycin, all 2× MIC vancomycin cultures were terminated at 12 h due to a considerable decline in viability (Fig. 2e).

Fig. 2.

Effects of vancomycin ongrowth of C. difficile historical 9689 and epidemic 9689 and 5325 strains. (a) Antibiotic-free cultures; (b) 1/8× MIC; (c) 1/4× MIC; and (d) 1× MIC; and (e) 2× MIC. Vancomycin was added, at the final concentrations indicated, during early log phase growth (designated Time 0). Samples were collected in duplicate over 48 h for quantification of viable C. difficile.

The effects of surotomycin and vancomycin on TcdA and TcdB exotoxin production

In antibiotic-free media, toxin production started at 6 h for all strains and continued throughout the 48 h experiment, albeit at decidedly different levels (data not shown). As organisms entered the exponential phase growth (6 h), sub-inhibitory concentrations of surotomycin (1/4× and 1/8×) induced approximately 3- to 4-fold more TcdA/B production by all strains when compared to antibiotic-free cultures (Fig. 3a). Similar increases in toxin production were also observed by the 9689 strain during early stationary phase growth (12 h: Fig. 3a). Furthermore, 1× MIC surotomycin also stimulated TcdA/B production by the historical 9689 C. difficile strain at 6–12 h of growth (Fig. 3a). However, by mid- to late-stationary phase growth (24 and 48 h), TcdA/B levels in each surotomycin-treated culture were similar to those of the antibiotic-free controls (Fig. 3a).

Fig. 3.

Comparison of soluble TcdA/TcdB production following exposure to sub-inhibitory and inhibitory concentrations of surotomycin and vancomycin. Supernatant samples were collected at 6, 12, 24 and 48 h from C. difficile ATCC 9689, BI 5325 and 2989 strain cultures containing either nothing, 1/8×, 1/4× or 1× MIC doses of (a) surotomycin or (b) vancomycin. Collected samples were screened for detection of TcdA/TcdB by commercial ELISA and are given as relative values compared to the drug-free culture. Data shown are the means of 3 replicates from 2 experiments.

The effects of vancomycin on TcdA/B production by C. difficile were comparable to those of surotomycin. Specifically, increased TcdA/B was measured during log phase (6 h) growth by all three strains (Fig. 3b), including an approximate 7-fold increase in the 9689 1/8× MIC vancomycin culture (Fig. 3b). Similarly, sub-inhibitory concentrations of vancomycin enhanced TcdA/B levels during early-stationary phase growth, most notably in the 9689 and 5325 isolates (~6 fold and ~4 fold, respectively; Fig. 3b). Greater toxin production was also observed by the 9689 and 5325 stains at 12 h following 1× vancomycin exposure (Fig. 3b). Like surotomycin, soluble TcdA/B concentrations in all vancomycin-treated cultures were similar to those of the antibiotic-free controls during mid- to late-stationary phase growth at 24 and 48 h (Fig. 3b).

Surotomycin and vancomycin decrease sporulation by C. difficile

Little to no formation of viable spores was observed prior to 24 h growth by any of the tested strains under any culture condition (data not shown). However, by 24 h pronounced sporulation was observed in all antibiotic-free cultures, and by 48 h the number of isolated spores increased in all strains by ~2 log (Fig. 4a, b). As expected, a greater number of spores were produced by the BI/NAP1/027 5325 and 2989 isolates when compared to the 9689 strain (Fig. 4a–d) [22]. At both 24 and 48 h, surotomycin dose dependently reduced the production of viable spores in all strains of C. difficile, including a ~4 log decrease in all 1× MIC cultures at 48 h when compared to the drug-free control (Fig. 4a, b).

Fig. 4.

Comparison of spore production at 24 and 48 h following exposure to sub-inhibitory and inhibitory concentrations of antibiotics. Strains 9689, 5325 (Historical BI) and 2989 (Epidemic BI) strains of C. difficile were exposed to either nothing, surotomycin (a and b) or vancomycin (c and d) at 24 and 48 h, respectively, at the final concentrations indicated. Samples were collected and spores isolated by mixing an equal volume of culture supernatant with 100 % ethanol followed by centrifugation. Harvested spores were enumerated by serially diluting in PBS and plating onto BHI agar plates. Plates were incubated anaerobically at 37 °C for 72 h.

Similar responses were also observed by all C. difficile strains following exposure to vancomycin. As shown in Fig. 4(c, d), vancomycin dose dependently affected spore formation during the stationary phase by the same strains of C. difficile. These sporulation levels were comparable to those observed for surotomycin and parallel those previously reported by our group [23].

Discussion

C. difficile infections are becoming more common, more lethal and more resistant to standard antimicrobial therapies, yet the options for managing these infections remain limited. While several antibiotics, including metronidazole, tigecycline, rifaximin, nitazoxanide and ramoplanin, have shown success in treating CDI, the only FDA-approved drugs for treating C. difficile disease are vancomycin and fidaxomicin [24]. Given the burden of CDI and its increasing rate of recurrence, surotomycin was recently developed to offer clinicians an alternative option for treating severe and relapse infections. Surotomycin has a narrow spectrum of activity that compromises the cell wall integrity of C. difficile organisms while minimally targeting Gram-negative species like Bacteroides fragilis. Following reports that surotomycin did not meet key efficacy endpoints of non-inferiority when compared to vancomycin, enthusiasm for utilizing the drug to treat severe CDI diminished [11, 12]. Still, surotomycin displays strong activity against C. difficile, including strains that are vancomycin-resistant or have reduced susceptibility to metronidazole [8].

The primary risk factor for developing C. difficile infection is antibiotic use, and due to their impact on the indigenous colonic microflora, all antibiotics carry the potential for triggering CDI. Interestingly, a newer concept concerning the role of antibiotics in C. difficile pathogenesis has emerged, and several studies by ourselves and others suggest that, in addition to their role in promoting C. difficile colonization, certain antibiotics may directly influence the pathogenicity of C. difficile organisms [16–18, 25, 26] by enhancing virulence factor expression. Specifically, at sub-inhibitory concentrations, some antibiotics induced TcdA and TcdB production as well as sporulation by several strains of C. difficile. In most cases, this effect was observed during the early (non-stationary) stages of growth when toxin production by C. difficile is typically not detected. The clinical significance of this in vitro observation is unclear, but stimulating toxin production during infection could exacerbate, and not improve, the clinical symptoms associated with CDI. Furthermore, nearly all authors have concluded that this phenomenon between antibiotic exposure and increased toxin production by C. difficile is inconsistent and reflects both strain- and antibiotic-specific responses.

In agreement with previous reports [16–18], data collected in our current study showed that sub-inhibitory concentrations of surotomycin also initiated early toxin production in both toxinotype III (BI/NAP1/027) strains and the toxinotype 0 strain (9689) of C. difficile when compared to the antibiotic-free medium. Specifically, surotomycin at 1/4× and 1/8× MIC up-regulated TcdA/B protein production during the log (6 h) and early stationary (12 h) phases of growth. Comparable increases in TcdA/B levels were also observed in cultures containing 1/4× and 1/8× MIC vancomycin. In contrast to the early timepoints, all sub-inhibitory surotomycin and vancomycin cultures at the mid- and late-stationary growth phases (i.e. 24 and 48 h) had overall toxin levels similar to or below those of the drug-free controls. These later results are similar to those of Endres et al., who demonstrated that sub-MIC concentrations of surotomycin (1/2× MIC) did not enhance TcdA or TcdB production by an epidemic BI/NAP1/027 strain at 24 h in vitro [14]. Additionally, inhibitory concentrations of surotomycin clearly restricted toxin production and growth of C. difficile at the 24 and 48 h time points, as TcdA and TcdB levels in cultures exposed to 1× MIC surotomycin were ~60–90 % lower than that of the no-treatment controls. This is encouraging from a clinical standpoint considering that surotomycin is found at ~200–400 µg ml⁻¹ in the gut during treatment [15]. Similar to surotomycin, TcdA/B levels were considerably reduced in cultures containing inhibitory concentrations of vancomycin.

Our analysis also showed that surotomycin dose-dependently reduced sporulation in both the historical and epidemic BI/NAP1/027 strains and the toxinotype 0 of C. difficile. In fact, a ~4 log decrease in the 1× MIC cultures was observed in the high-spore-forming 5325 and 2989 epidemic strains at 48 h. Spores serve as a major risk factor for new and recurrent infections in healthcare environments, and thus treatments that inhibit spore formation in patients suffering from CDI are critical to reducing the transmission of disease. The ability of surotomycin, even at sub-MIC concentrations, to suppress spore formation could theoretically explain the low recurrence rate of CDI in patients receiving surotomycin therapy during the Phase 2 trial [10, 27, 28]. Other factors, however, may be more important in determining the clinical outcomes as noted in the Phase 3 trials of surotomycin [11, 12].

In conclusion, C. difficile is a serious nosocomial pathogen that has reached epidemic levels across the globe. Because of this, there is a pressing need for the development of newer treatments to manage severe and recurrent cases of CDI. Based on the current study, very low concentrations of surotomycin (~1–2 µg ml⁻¹) inhibited toxin production and sporulation by C. difficile, which is theoretically advantageous when considering drug development for CDI. Other factors, however, may be important in reducing clinical CDI recurrence. In addition, the data collected from this study further contribute to the growing body of knowledge regarding the influence of antibiotics on the virulence of C. difficile.