In Vitro Activity of Sodium Bituminosulfonate: Susceptibility Data for the Revival of an Old Antimicrobial

Abstract

Revival of old antibiotic compounds is a promising strategy to strengthen the antimicrobial armamentarium in the era of increasing resistance and limited development pipelines. To exploit their full potential, their reinvestigation using current standards is needed. We aimed to investigate the in vitro activity of the old antimicrobial agent sodium bituminosulfonate in accordance with the current recommendations for antimicrobial susceptibility testing (AST) and to generate susceptibility data reflecting the current epidemiological situation. The in vitro activity of sodium bituminosulfonate was tested on consecutive clinical isolates, including 12 methicillin-resistant Staphylococcus aureus (MRSA) and 12 methicillin-susceptible S. aureus (MSSA), 24 coagulase-negative staphylococci (CoNS), 60 streptococci, 12 Enterococcus faecalis, 12 Enterococcus faecium (including two vancomycin-resistant strains), 12 Enterobacterales, 12 nonfermenting Gram-negative bacilli, and 12 Cutibacterium [Propionibacterium] acnes. AST of sodium bituminosulfonate was performed using broth microdilution method for Gram-positive cocci and Gram-negative rods and by agar dilution method for C. acnes. Sodium bituminosulfonate demonstrated activity against Gram-positive pathogens with minimal inhibitory concentration 90 (MIC₉₀) values (g/L) for MRSA 0.25, MSSA 1, CoNS 16, Streptococcus pyogenes 0.03, Streptococcus agalactiae 0.125, Streptococcus dysgalactiae ≤0.015, Streptococcus pneumoniae ≤0.015, viridans streptococci 0.03, E. faecalis 0.25, E. faecium 0.5, and C. acnes 0.03 (without blood supplement). MIC values for Gram-negative bacteria were considerably higher. Blood-supplemented media proved to be unsuitable for activity testing of this agent. Sodium bituminosulfonate may represent an alternative to classical antibiotics for topical use. Although it has been clinically used for many decades, well-designed randomized trials are needed for the effective revival of this old antimicrobial.

Background

The infections with antibiotic-resistant bacteria are increasing worldwide, leading to an enormous clinical and public health burden.¹ However, the current development pipeline does not meet the needs of the community for effective antimicrobials. Recent analysis demonstrated that the clinical development is dominated by derivatives of established antibiotic classes and most candidates display limited innovation.² It has been emphasized that antibiotics without cross-resistance with existent classes are urgently needed.²

In a situation wherein new antimicrobial agents are scarce, revisiting old antibiotics is a promising strategy.³ However, often there is a need to exploit their full potential, that is, reinvestigation using modern methodological and regulatory standards.⁴ Generating present-day susceptibility data that reflect current epidemiological situation is an indispensable part of the revival process.⁵

Sodium bituminosulfonate is an old antimicrobial agent that is an active ingredient derived from the sulfur-rich oil shale. Dark and pale derivatives of sulfonated shale oil have been used for more than a century for various indications,^6,7 with the most common applications being in the area of dermatology including skin infections.^8,9 Data on the antimicrobial activity of sodium bituminosulfonate are limited to a few publications.^10–13 This study aimed to investigate in vitro activity of sodium bituminosulfonate in accordance with the current recommendations for antimicrobial susceptibility testing (AST).^14–16 AST was performed for a broad spectrum of bacterial species comprising Gram-positive cocci including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), Gram-negative rods, and the anaerobic bacterium Cutibacterium [Propionibacterium] acnes.

Methods

Bacterial strains

The in vitro activity of sodium bituminosulfonate was tested on clinical isolates collected during the routine diagnostics at the Institute of Medical Microbiology, University Hospital Münster. For each species, consecutive isolates of that particular species were included without selection. Only one isolate per patient was eligible. Tested staphylococci comprised 24 S. aureus isolates, including 12 MRSA and 12 methicillin-susceptible S. aureus (MSSA) isolates, as well as 24 coagulase-negative staphylococci (CoNS), including 12 S. epidermidis, 2 S. hominis, 2 S. haemolyticus, 2 S. capitis, 2 S. saprophyticus, 2 S. auricularis, and 2 S. lugdunensis isolates. Streptococcal isolates comprised Streptococcus pyogenes (n = 12), Streptococcus agalactiae (n = 12), Streptococcus dysgalactiae (n = 12), Streptococcus pneumoniae (n = 12), as well as 12 viridans streptococci including 2 Streptococcus anginosus, 2 Streptococcus intermedius, 2 Streptococcus constellatus, 2 Streptococcus mitis, 2 Streptococcus salivarius, and 2 Streptococcus sanguinis isolates. Enterococci included 12 Enterococcus faecalis and 12 Enterococcus faecium isolates. In addition, Gram-negative bacteria from the Enterobacterales order (Enterobacteriaceae, Morganellaceae, and Yersiniaceae) and from the group of nonfermenting Gram-negative bacilli were included. For Enterobacterales (n = 12), one isolate for each of the following species was tested: Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Citrobacter koseri, Proteus mirabilis, Proteus vulgaris, Serratia marcescens, Morganella morganii, and Providencia rettgeri. For nonfermenting Gram-negative bacilli (n = 12), two isolates for each of the following species were tested: Pseudomonas aeruginosa, Acinetobacter baumannii, Acinetobacter lwoffii, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Burkholderia cepacia. Furthermore, 12 C. acnes strains isolated from positive blood cultures were part of the tested collection.

Antimicrobial susceptibility testing

Sodium bituminosulfonate substance (Ichthyol^® light) was provided by the manufacturer Ichthyol-Gesellschaft (Hamburg, Germany). Sodium bituminosulfonate was tested in double dilution concentration steps ranging from 0.015 to 256 g/L.

AST of Gram-positive cocci and Gram-negative rods was performed using the broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) and the International Organization for Standardization (ISO).^14,16 In brief, bacterial suspensions with the turbidity of a 0.5 McFarland standard were prepared from overnight agar cultures and diluted in cation-adjusted Mueller–Hinton broth (CA-MHB, BD Diagnostics, Heidelberg, Germany) to achieve a final inoculum in test of ∼5 × 10⁵ cfu/mL. Real bacterial concentration was confirmed by the colony counting of serial dilutions. The microtiter plates were incubated at 36°C and read visually after 18 ± 2 hr. As reference strains, S. aureus ATCC 29213, S. pneumoniae ATCC 49619, E. faecalis ATCC 29212, and E. coli ATCC 25922 were included.

Since preliminary experiments showed unfeasibility of testing sodium bituminosulfonate by broth microdilution with the blood-supplemented medium, brain–heart-infusion (BHI; Merck, Darmstadt, Germany) broth was used for streptococci instead of CA-MHB with 2.5–5% lysed horse blood (LHB) recommended by CLSI for this group of microorganisms.¹⁷ To compare the test performance for streptococci with BHI broth or CA-MHB with LHB, vancomycin minimal inhibitory concentrations (MICs) were generated for the reference strain S. pneumoniae ATCC 49619 using these two nutrient media.

AST of C. acnes was performed by the agar dilution method in accordance with the CLSI recommendations for AST of anaerobic bacteria.¹⁵ In brief, bacterial suspensions with the turbidity of a 0.5 McFarland standard were prepared from cultures incubated in anaerobic atmosphere on supplemented Brucella agar (BD Diagnostics) for 48 hr. Then, 1.5 μL of these bacterial suspensions were inoculated onto the surface of agar plates containing two-fold dilutions of sodium bituminosulfonate. The medium used for anaerobic AST was Brucella agar (BD Diagnostics, Heidelberg, Germany), supplemented with 5 μg/mL hemin (Sigma-Aldrich, Saint Louis, MO), 1 μg/mL vitamin K₁ (Sigma-Aldrich), and 5% laked sheep blood (Thermo Fisher Scientific, Wesel, Germany), as recommended by CLSI.¹⁵

Since preliminary experiments showed technical interference while testing sodium bituminosulfonate with the blood-supplemented medium by broth microdilution, an alternative medium without blood supplement was also used for anaerobic AST by agar dilution. As alternative medium, the same Brucella agar (BD Diagnostics) has been used, supplemented with 5 μg/mL hemin (Sigma-Aldrich), 1 μg/mL vitamin K₁ (Sigma-Aldrich), but not containing blood. To assess the test performance for C. acnes with or without blood supplement, vancomycin MICs were generated for the reference strains Clostridioides [Clostridium] difficile ATCC 700057 and C. acnes ATCC 6919 using these two media. Growth control without antimicrobial was included to confirm sufficient growth and anaerobiosis. Final bacterial concentration in test was confirmed by plating serial dilutions onto supplemented Brucella agar and colony counting after 48 hr of incubation in an anaerobic atmosphere. Inoculated agar plates were incubated at 36 ± 1°C in an anaerobic jar and read visually after 48 hr.

All MIC determinations were performed in triplicate and median value was calculated for analysis.

Results

Preliminary experiments demonstrated unfeasibility of testing sodium bituminosulfonate with blood-supplemented medium due to the occurrence of an unspecific turbidity, particularly at concentrations of 0.25 to 8 g/L sodium bituminosulfonate. During preliminary comparative experiments, no difference was detected in vancomycin MIC results, generated for the reference strain S. pneumoniae ATCC 49619 with BHI broth or CA-MHB with LHB (Supplementary Table S1).¹⁷ The vancomycin MIC for this reference strain was in quality control (QC) range with both broths (Supplementary Table S1). This proves that MICs generated for streptococci in this report are true values although nutrient medium has to be modified for this group of organisms. Similarly, there was no difference between vancomycin MIC results, generated for the reference strains C. difficile ATCC 700057 and C. acnes ATCC 6919 using Brucella agar with or without blood supplement (Supplementary Table S2). The vancomycin MIC for the QC strain C. difficile ATCC 700057 was within the recommended QC range with both agars (Supplementary Table S2).

MIC₅₀, MIC₉₀, and MIC ranges of sodium bituminosulfonate for clinical strains of Gram-positive cocci and Gram-negative rods are demonstrated in Table 1. MIC for QC reference strain S. aureus ATCC 29213 was 0.06 or 0.125 g/L throughout the testing. MIC for QC reference strains S. pneumoniae ATCC 49619 and E. faecalis ATCC 29212 was ≤0.015 and 0.25 g/L, respectively. These results may be used for establishing of tentative QC ranges for AST of sodium bituminosulfonate. MIC for reference strain E. coli ATCC 25922 was 256 g/L throughout the testing.

Table 1.

Antimicrobial Activity of Sodium Bituminosulfonate

Microorganisms	MIC₅₀, g/L	MIC₉₀, g/L	MIC range, g/L
Staphylococci
MRSA, n = 12	0.25	0.25	0.125 to 0.25
MSSA, n = 12	0.25	1	0.06 to 2
CoNS, n = 24	1	16	0.03 to 32
Streptococci
Streptococcus pyogenes, n = 12	≤0.015	0.03	≤0.015 to 0.03
Streptococcus agalactiae, n = 12	0.06	0.125	≤0.015 to 0.125
Streptococcus dysgalactiae, n = 12	≤0.015	≤0.015	≤0.015 to ≤0.015
Streptococcus pneumoniae, n = 12	≤0.015	≤0.015	≤0.015 to ≤0.015
Viridans streptococci, n = 12	≤0.015	0.03	≤0.015 to 0.03
Enterococci
Enterococcus faecalis, n = 12	0.125	0.25	0.125 to 0.25
Enterococcus faecium^a, n = 12	0.25	0.5	0.125 to 0.5
Enterobacterales, n = 12	128	256	64 to >256
Nonfermenting Gram-negative bacilli, n = 12	64	>256	16 to >256

Including one vanA isolate of VRE and one vanB VRE isolate.

CoNS, coagulase-negative staphylococci; MIC, minimal inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible S. aureus; VRE, vancomycin-resistant enterococci.

Table 2 gives MIC₅₀, MIC₉₀, and MIC ranges of sodium bituminosulfonate for clinical strains of C. acnes. We used agar dilution instead of broth microdilution for C. acnes, because the latter method is currently not recommended by CLSI for anaerobic bacteria except Bacteroides spp. and Parabacteroides spp.¹⁵ For the reference strain C. difficile ATCC 700057, MIC values of vancomycin were equal when tested on agar supplemented or not supplemented with blood, with vancomycin MIC being within the recommended QC range (Supplementary Table S2). Similarly, reference strain C. acnes ATCC 6919 showed the same vancomycin MIC on agar with and without blood supplement (Supplementary Table S2). However, MIC values of sodium bituminosulfonate were considerably higher when tested with blood supplement (Table 2). Hence, blood supplement seems to disturb the in vitro activity of sodium bituminosulfonate. The impact of blood additive on the activity of sodium bituminosulfonate could only be determined by the agar dilution method. It is difficult to prove whether blood supplement disturbs the activity of sodium bituminosulfonate also in the broth microdilution setting, because the MICs cannot be properly read due to unspecific broth turbidity with blood supplement. We, therefore, assume that blood-supplemented nutrient media are generally not appropriate for AST of sodium bituminosulfonate, and respective deviation from the guideline has to be made for this antimicrobial agent.

Table 2.

Antimicrobial Activity of Sodium Bituminosulfonate Against Cutibacterium acnes Clinical Strains (n = 12)

Medium	MIC₅₀, g/L	MIC₉₀, g/L	MIC range, g/L
Brucella agar supplemented with 5 μg/mL hemin, 1 μg/mL vitamin K1 (without blood supplement)	0.03^a	0.03	0.015–0.03
Brucella agar supplemented with 5 μg/mL hemin, 1 μg/mL vitamin K1, and 5% laked sheep blood^b	4	8	1–8

Median MIC for the reference strain C. acnes ATCC 6919 was 0.03 and 8 g/L without blood supplement and with blood supplement, respectively.

Medium recommended by the CLSI for antimicrobial susceptibility testing of anaerobic bacteria.

CLSI, Clinical and Laboratory Standards Institute.

Discussion

The early in vitro data on antimicrobial activity of sodium bituminosulfonate and its derivatives have been reported already in the 19th century.¹⁰ Only few reports of antimicrobial activity were published afterward,^11–13 so that the available data are very scarce. Obviously, the methodology of AST in these old reports^10–13 did not meet the requirements of current AST standards. Furthermore, the susceptibility data obtained decades ago on reference strains and a limited number of clinical isolates do not reflect the current epidemiological situation. In addition, different oil shale derivatives and different formulations were tested throughout reports,^10–13 whereas now focusing on most promising candidates is important. Overall, data from previous publications showed much higher activity of sodium bituminosulfonate against Gram-positive bacteria than against Gram-negative bacteria. This is in agreement with our study (Table 1).

MIC values of sodium bituminosulfonate for the most Gram-positive bacteria tested were much higher than common MIC values of classical antibiotics. It should, however, be considered that sodium bituminosulfonate is applied topically and, theoretically, a topical concentration up to 1,200 g/L can be achieved when 100% substance (liquid with a relative density of 1.2) is applied. Some of the commercially available topical formulations contain 20% sodium bituminosulfonate. The sodium bituminosulfonate concentration of 240 g/L in such a topical product greatly exceeds the MICs that were determined for Gram-positive bacteria in our study. Indeed, the pharmacokinetic–pharmacodynamic concept of clinical efficacy of antimicrobial drugs takes into account the index of achievable concentration at the action site and the microorganism's MIC.⁵ Interestingly, even with Gram-negative bacteria, MIC was only seldom off-scale (>256 g/L) and was achieved with concentrations of 256 g/L or lower for most isolates (Table 1).

C. acnes strains showed low MICs of sodium bituminosulfonate when tested without blood supplement (Table 2). Blood additive was demonstrated in this study to adversely affect in vitro activity of sodium bituminosulfonate. We, therefore, assume that MIC values determined without blood supplement better reflect real activity of sodium bituminosulfonate. This study included C. acnes strains isolated from blood, which typically represent skin contaminants. C. acnes has been associated with pathogenesis of acne vulgaris,¹⁸ and sodium bituminosulfonate has been applied for this indication for about a century.^6,7 The in vitro activity data from our study generally support this treatment concept as well as treatment of other skin infections with sodium bituminosulfonate.

In conclusion, sodium bituminosulfonate possesses in vitro activity in particular against Gram-positive pathogens and may represent an alternative to classical antibiotics for topical use. This topical antimicrobial agent could reduce the selection pressure and resistance development toward common antibiotics usually applied for decolonization purposes (e.g., mupirocin for nasal MRSA eradication) and, if unavoidable, for topical treatment of skin infections (e.g., fusidic acid).^19,20 Its activity includes MRSA and VRE, which are high priority pathogens according to the WHO priority list for research and development of new antibiotics for antibiotic-resistant bacteria. In contrast, MIC values for Gram-negative bacteria were much higher so that low in vitro activity of sodium bituminosulfonate against these pathogens can be assumed. Although sodium bituminosulfonate formulations have been used for many decades and reports and studies of clinical success are available,^6,21–23 well-designed randomized controlled trials complying with the standards of modern evidence-based medicine are necessary for the effective revival of this old antimicrobial.

Footnotes

Acknowledgments

We are thankful to Damayanti Kaiser and Barbara Grünastel for excellent technical assistance.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by a research grant from Ichthyol-Gesellschaft Cordes, Hermanni & Co (AF730384 to K.B.).

Supplementary Material

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Supplementary Material

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