Antimicrobial activity of vaginal isolates of Corynebacterium amycolatum against Acinetobacter baumannii

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Introduction

Acinetobacter baumannii is a Gram-negative aerobic opportunistic pathogen included in the ESKAPE group of pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, Enterobacter spp.), which causes nosocomial infections in immunocompromised individuals and is multidrug resistant [1]. A. baumannii is the causative agent of various infections, including skin and soft tissue infections, wound infections, bacteremia, endocarditis, urinary tract infections, meningitis and pneumonia [2]. One of the mechanisms of nosocomial infection development is the ability of A. baumannii to form biofilms on biotic (host mucous membranes) and abiotic surfaces (e.g., catheters), which give the pathogen an advantage for long-term persistence and resistance to antibacterial drugs [3]. Cells enclosed in biofilms have limited metabolic activity and are protected by an extracellular matrix, which makes them resistant to antibiotics and the innate immune factors of the host [4, 5].

The World Health Organization recognizes A. baumannii as a serious threat to human health, listing it as a priority pathogen for which novel control strategies are urgently needed as an alternative to antibiotics [6].

Among these strategies, researchers are focusing on antibacterial compounds produced by microorganisms. These compounds, often released into the extracellular environment, can act as mediators of cell-cell communication, allowing microbial communities to colonize a selected ecological niche [7].

Recently, microorganisms of the genus Corynebacterium spp. have attracted research interest. The important role of certain corynebacterial species in microbiome-mediated protection against pathogens has been described. For example, Corynebacterium xerosis exhibits both inhibitory and destructive activity against biofilm formation by S. aureus, Streptococcus mutans, Escherichia coli, and P. aeruginosa [8]. Additionally, Corynebacterium pseudodiphtheriticum and Corynebacterium accolens have been shown to suppress nasal colonization by S. aureus [9, 10].

Our previous studies have shown the ability of cell-free supernatants (CFS) of vaginal isolates of C. amycolatum to suppress the growth properties (planktonic culture growth and biofilm formation) of S. aureus, P. aeruginosa and K. pneumoniae, as well as to destroy the pre-formed biofilms of these pathogens [11, 12].

However, to date, no studies have evaluated the antimicrobial activity of Corynebacterium spp. against A. baumannii.

The aim of this study was to investigate the antimicrobial activity of C. amycolatum CFS against clinical isolates of A. baumannii.

Materials and methods

Bacterial strains and growth conditions

Six clinical isolates of A. baumannii (1107, 1124, 1209, 1221, 1287, 1301) with multidrug resistance were kindly provided by the Research Center for Microbiology of Orenburg State Medical University (Russia). For the experiment, A. baumannii strains were cultured in tryptic soy broth (TSB; Research Center for Pharmacotherapy, Russia) at 37°C. Four strains of C. amycolatum (ICIS 9, ICIS 53, ICIS 82 and ICIS 99) were initially isolated from vaginal swabs of healthy women of reproductive age and deposited in the Network Collection of Symbiotic Microorganisms and Their Consortia of the Institute of Cellular and Intracellular Symbiosis, Ural Branch of the Russian Academy of Sciences. Previously, C. amycolatum strains were identified using whole-genome sequencing. Annotated genomic sequences were deposited in the GenBank database, bioproject PRJNA339674, with accession numbers MTPT00000000, MIFV00000000, JAXKQN000000000 and JAIUSU000000000. The Corynebacterium strains were stored in brain heart infusion broth (BHI; HiMedia Laboratories Pvt. Ltd.) containing 20% (v/v) glycerol at −80°C.

Preparation of C. amycolatum CFS

For the experiment, C. amycolatum was grown overnight, inoculated into BHI, and cultured for 24 hours at 37°C. Bacterial cells were precipitated by centrifugation (9000g at 4°C for 15 min). The CFS of each strain was sterilized by filtration through a 0.22 μm membrane filter (Millipore, Nihon) and immediately used in experiments in undiluted form.

Determination of the influence of C. amycolatum CFS on the growth of A. baumannii planktonic culture

The effect of C. amycolatum CFS on the growth of planktonic cultures of test strains was studied in sterile 96-well polystyrene plates (Sigma-Aldrich Chemie) according to the method described by M. Scillato et al. [13] with minor modifications. Aliquots of 100 μl of daily agar cultures of test strains (3 × 10⁵ CFU/mL) prepared on TSB were transferred to each well of a 96-well plate and 100 μL of C. amycolatum CFS was added. BHI broth was used as a control instead of CFS. Each sample was tested in parallel in 4 wells. The plates were incubated aerobically for 24 hours at 37°C. The optical density of the culture was measured every hour at a wavelength of 630 nm using a microplate spectrophotometer (Molecular Devices, LLC).

Determination of the influence of C. amycolatum CFS on biofilm formation by A. baumannii

The formation of biofilms by A. baumannii test strains in the presence of C. amycolatum CFS was evaluated in 96-well polystyrene plates using the method described by A. Algburi et al. [14] with minor modifications. Overnight cultures of A. baumannii strains were suspended in TSB until a final concentration of approximately 10⁵ CFU/mL was reached. Then, 100 µL of the test strain suspension and 100 µL of CFS were added to each well of the plate. BHI was added to the control wells. The plates were incubated at 37°C for 24 hours. The effect of CFS on the formation of the A. baumannii biofilm was determined by the change in spectrophotometric absorption in the experimental wells compared to the control wells. Absorption was measured at 540 nm using a microplate spectrophotometer (Molecular Devices, LLC).

Determination of the influence of C. amycolatum CFS on the production of exopolysaccharides by A. baumannii

The effect of C. amycolatum CFS on the production of exopolysaccharides (EPS) by A. baumannii was evaluated according to the method described by A. Chiba et al. [15], with minor modifications. Clinical isolates of A. baumannii were grown overnight in TSB medium and resuspended in the same medium to approximately 10⁷ CFU/ml. Then, 2 mL of one of the C. amycolatum CFS was added to 2 mL of the bacterial suspension and incubated for 24 hours at 37°C. The control was treated with BHI. After incubation, the cell suspensions were centrifuged (9000g for 10 min at 25°C) and 1.5 M NaCl (1 mL) was added. The cell suspension was then centrifuged again (5000g for 10 min at 25°C), the supernatant (40 µL) was mixed with 5% phenol (40 µL), sulfuric acid (2 mL) was added, and the mixture was incubated at 30°C for 10 min. The effect of CFS on the production of EPS by A. baumannii was determined by the change in spectrophotometric absorption in the experimental wells compared to the control wells. Absorption was measured at 490 nm using a microplate spectrophotometer (Molecular Devices, LLC).

Scanning electron and luminescent microscopy

The effect of C. amycolatum CFS on biofilm formation in test strains of A. baumannii was studied using scanning electron microscopy (SEM) and luminescence microscopy at the Yuri Gagarin Center for the Identification and Support of Gifted Children (Orenburg, Russia). A. baumannii biofilms were visualized using SEM on a TESCAN MIRA 3 microscope according to the method [12]. For fluorescence microscopy, samples were prepared according to the method of O.Contreras-Martínez et al. [16] with minor modifications. 10 × 10 mm cover glasses (Medpolymer) were placed in sterile 12-well polystyrene plates, 100 μL of C. amycolatum CFS was added to each well containing 100 μL of A. baumannii suspension, and the plate was incubated for 24 hours at 37°C. The wells of the plate were washed twice with phosphate-buffered solution (pH 7.2), the cover glass was removed, dried, and placed on a microscope slide. Then, acridine orange (5 μL, 100 mg/L) and ethidium bromide (5 μL, 100 mg/L) were mixed, applied to the glass surface, covered with a larger cover glass (24.0 × 24.0 mm), and stained for 5 min at 25°C in the dark. The slides were viewed under an Axio Scope. A1 microscope (Carl Zeiss) with a luminescent module at 100× magnification in an immersion system and photographed with an Axiocam 208 color digital camera (Carl Zeiss).

Identification of compounds in C. amycolatum ICIS 99 CFS using gas chromatography-mass spectrometry

The C. amycolatum ICIS 99 strain was cultivated in a 750 mL Erlenmeyer flask containing 100 mL of BHI. The culture was incubated with orbital shaking at 150 rpm, 37°C for 48 hours. The inoculum volume was 10%. After cultivation, the culture fluid was poured from the flasks into test tubes, centrifuged at 9000 rpm at 4°C, and filtered through sterile Millipore (Merck) membrane filters with a pore diameter of 0.22 μm. The resulting filtrate was extracted with a mixture of organic solvents (chloroform, butyl acetate, methanol, and diethyl ether) at a ratio of extractant to culture fluid of 1:1 for 6 hours at 25°C and constant stirring at a speed of 220 rpm. The extracts obtained were evaporated to dryness on a UL-1100 (Ulab) rotary evaporator under vacuum at a temperature below 40°C. After filtration and evaporation, the final product was obtained in the form of a dry extract. The extract obtained was analyzed using a Shimadzu GCMS-QP2010 Ultra (Shimadzu) mass spectrometer according to the method described above¹. Spectral peaks were identified using the NIST mass spectral library (The NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library Version 2.0 g build May 19 2011).

Statistical analysis was performed using Statistica v. 6.0 software (StatSoft, Inc.). The results are presented as mean values and standard errors (M ± m) obtained in at least three independent experiments. Data visualization and statistical processing were performed using GraphPad Prism v. 9.4.1 software (GraphPad).

Results

The CFS of vaginal isolates of C. amycolatum were tested for antimicrobial activity against test strains of A. baumannii isolated from various clinical samples. It was found that the CFS of C. amycolatum inhibited the growth of planktonic cultures of all test strains (Fig. 1). The CFS of strain ICIS 99 had the greatest effect. The CFS of strain ICIS 9 was less effective against clinical isolates of A. baumannii, and had a stimulating effect on A. baumannii 1107. It is important to note that in preliminary studies using the delayed antagonism method on a solid nutrient medium, C. amycolatum CFS showed no antimicrobial activity against all test strains of A. baumannii (data not shown).

 

Fig. 1. Dynamics of bacterial growth of clinical isolates of A. baumannii in the presence of C. amycolatum CFS.

The ordinate axes show the optical density of the culture at a wavelength of 630 nm, the abscissa axes show time, h.

 

When assessing the effect of C. amycolatum CFS on biofilm formation by A. baumannii, a multidirectional effect was observed, which depended on both the CFS itself and the test strain. C. amycolatum CFS caused a decrease in biofilm formation in strains 1107, 1209, 1221 and 1287 (p < 0.0005 and p < 0.001) (Fig. 2). With regard to A. baumannii 1301, CFS mainly exhibited an indifferent effect. It is important to note that of all vaginal isolates of C. amycolatum, only ICIS 99 suppressed biofilm formation of all test strains of A. baumannii.

 

Fig. 2. The effect of CFS of vaginal isolates of C. amycolatum on biofilm formation by A. baumannii.

Error bars represent standard errors of the mean calculated using data from at least 3 independent experiments. *p < 0.05, **p < 0.001, ***p < 0.0005 compared to control.

 

An increase in the EPS production capacity of A. baumannii was observed in all clinical isolates after co-cultivation with C. amycolatum (Table 1).

 

Table 1. Production of EPS by clinical isolates of A. baumannii after co-cultivation with C. amycolatum CFS (М ± m)

Strain	Initial EPS production	C. amycolatum ICIS 9	C. amycolatum ICIS 53	C. amycolatum ICIS 82	C. amycolatum ICIS 99
A. baumannii 1107	0,171 ± 0,003	0,198 ± 0,001*	0,187 ± 0,001	0,195 ± 0,001*	0,188 ± 0,001*
A. baumannii 1124	0,190 ± 0,001	0,250 ± 0,001*	0,219 ± 0,001*	0,234 ± 0,001*	0,243 ± 0,005*
A. baumannii 1209	0,137 ± 0,001	0,143 ± 0,001	0,148 ± 0,001	0,141 ± 0,001	0,161 ± 0,001*
A. baumannii 1221	0,153 ± 0,001	0,168 ± 0,001*	0,175 ± 0,001*	0,214 ± 0,003*	0,171 ± 0,001*
A. baumannii 1287	0,140 ± 0,001	0,180 ± 0,001*	0,152 ± 0,001	0,175 ± 0,001*	0,146 ± 0,002
A. baumannii 1301	0,172 ± 0,002	0,207 ± 0,002*	0,179 ± 0,002	0,213 ± 0,001*	0,183 ± 0,001

Note. *p < 0.05 compared to the original EPS production.

 

The morphology of A. baumannii cells and biofilms before and after co-cultivation with C. amycolatum CFS was further investigated using electron and fluorescence microscopy. For this purpose, A. baumannii strain 1124 was examined after interaction with the CFS of C. amycolatum ICIS 99. The biofilms of A. baumannii 1124 had a dense, lumpy structure with fibers and adhesions between cells; the cells were even and smooth, and the membrane was not damaged (Fig. 3, a–c). Co-cultivation of A. baumannii 1124 with CFS of ICIS 99 resulted in the absence of dense cell clusters. The cells were scattered across the entire surface of the glass, with no fibers or adhesions between cells. The cells were flat, smooth and the cell membrane was not damaged (Fig. 3, d–e).

 

Fig. 3. SEM images showing the effect of C. amycolatum ICIS 99 CFS on biofilm formation by A. baumannii 1124.

a–c — control; d–e — biofilm formation in the presence of C. amycolatum ICIS 99 CFS.

 

Images taken using fluorescence microscopy are shown in Fig. 4. Acridine orange diffuses through intact cytoplasmic membranes in living cells, where it interacts with DNA, emitting bright green fluorescence. In contrast, bromide etidium penetrates only cells with damaged membranes and cell walls in dead cells, interacts with DNA and produces orange-red fluorescence [16]. As shown in Fig. 4, a, b, untreated A. baumannii 1124 (control) grew well after 24 hours and exhibited completely green fluorescence, while A. baumannii 1124 cells after co-cultivation with CFS of strain ICIS 99 mainly exhibited orange-red fluorescence, which characterized the presence of dead cells.

 

Fig. 4. Images obtained using fluorescence microscopy showing the effect of CFS of C. amycolatum ICIS 99 on biofilm formation by A. baumannii 1124.

a–c — control; b–d — biofilm formation in the presence of CFS of C. amycolatum ICIS 99. Live cells appear green, while dead cells appear red and orange.

 

Gas chromatography-mass spectrometry (GC-MS) was used to study the composition of C. amycolatum ICIS 99 CFS. The names of the compounds, retention times, and degree of similarity (%) are given in Table 2.

 

Table 2. Identification of compounds present in CFS of C. amycolatum ICIS 99 using GC-MS/MS

Compound	Retention time, min	%
1,4-Diazabicyclo[4.3.0]nonane-2,5-dioxo-3-isobutyl	13.889	4.20
1,2-Benzoldicarboxylic acid, dibutyl ester	13.961	4.54
Ergotamine-3',6',18-trione, 12'-hydroxy-2'-methyl-5'-(phenylmethyl)-, (5'alpha)-	16.515	3.52
Pyrrolo[1, 2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-	16.760	3.77

 

GC-MS analysis of the extract obtained from CFS of C. amycolatum ICIS 99 showed the presence of four volatile compounds eluting between 13.130 and 27.238 min (Fig. 5). The identified compounds are shown in Fig. 6.

 

Fig. 5. Chromatographic mass spectrum of CFS extract of C. amycolatum ICIS 99.

 

Fig. 6. Spectra and chemical structure of identified compounds present in CFS of C. amycolatum ICIS 99.

a — 1,4-diaza-2,5-dioxo-3-isobutylbicyclo[4.3.0]nonane; b — 1,2-benzenedicarboxylic acid, dibutyl ester; c — ergothione-3',6',18-trione, 12'-hydroxy-2'-methyl-5'-(phenylmethyl)-, (5'alpha)-; d — pyrrolo[1, 2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-.

 

Discussion

For a long time, microorganisms of the genus Corynebacterium spp. were considered exclusively as pathogens causing serious diseases in humans, including zoonotic infections [17]. A few studies published in the mid-20th century on the antimicrobial and immunomodulatory activity of certain Corynebacterium spp. strains were not followed up [18]. Over the past decade, the paradigm regarding this group of microorganisms has shifted dramatically. The important role of Corynebacterium spp. in maintaining the colonization resistance of various biotopes of the human body is being studied [10, 19]. C. pseudodiphtheriticum, C. accolens and C. amycolatum are increasingly recognized as potential probiotic bacteria [20–23].

It has previously been shown that vaginal isolates of C. amycolatum had antimicrobial activity against multidrug-resistant bacterial pathogens, including S. aureus, P. aeruginosa and K. pneumoniae [11, 12]. Antimicrobial activity is recognized as one of the important features of probiotics, as it promotes competition with pathogenic bacteria [24, 25].

This study is the first to demonstrate the antimicrobial activity of CFS against A. baumannii, which is a priority pathogen due to its growing resistance to antibiotics [6]. A distinctive feature of this pathogen is the high heterogeneity observed among isolates due to its highly dynamic genome [26]. The high prevalence of A. baumannii strains in clinical settings is explained by their ability to form antibiotic-resistant biofilms that protect bacteria from external stress factors [3].

The results of the study demonstrate that C. amycolatum CFS significantly suppressed the growth of planktonic culture and biofilm formation of test strains of A. baumannii. The greatest effect was exerted by CFS of strain ICIS 99. In a single case, the authors observed stimulation of the growth of the planktonic culture of test strain A. baumannii 1107 after co-cultivation with CFS of ICIS 9. The authors regarded this effect as a strain-specific interaction. It is possible that ICIS 9 secretes metabolic products or signaling molecules released into the CFS that may have a stimulating effect on the growth of the A. baumannii 1107 strain. However, analysis of the literature did not confirm these results.

The in vitro studies were supported by SEM and fluorescence microscopy data. SEM results revealed that after co-cultivation with C. amycolatum CFS, the pathogen cells were scattered across the glass surface and their cell membranes were intact. Fluorescence microscopy revealed a lack of metabolic activity in most cells, indicating their death.

Capsular EPS plays an important role in biofilm formation in A. baumannii, acting as a structural component of the matrix, facilitating cell attachment and aggregation, and forming a protective barrier with viscoelastic, cohesive, and hydrating properties, allowing A. baumannii biofilms to withstand environmental stress and innate factors of the host immune system [27]. It should be noted that during co-cultivation of A. baumannii test strains with C. amycolatum CFS, an increase in EPS production was observed. The authors interpreted the finding as a protective response of the pathogen to stress. According to the literature, Gram-negative bacteria produce EPS, including capsular EPS, which help them cope with and adapt to various environmental stressors [27].

GC-MS analysis of the CFS extract obtained from C. amycolatum ICIS 99 revealed the presence of four volatile compounds, each of which has a diverse spectrum of biological activity. The 1,4-diaza-2,5-dioxo-3-isobutylbicyclo[4.3.0]nonane compound, first identified in the endophytic actinomycete Nocardiopsis sp. GRG 2, exhibits antibacterial, anti-biofilm, and antioxidant activity [28, 29]. Ergotaman-3',6',18-trione,12'-hydroxy-2'-methyl-5'-(phenylmethyl)-,(5'alpha)- has antimicrobial and anti-inflammatory properties [30]. Pyrrolo-pyrazine derivatives (pyrrolo[1, 2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-) exhibit antimicrobial, antifungal, antioxidant, and antitumor activity [31, 32]. Pyrrolizine and pyrazole derivatives may be active against A. baumannii [33, 34].

It is possible that all identified compounds in the CFS extract of C. amycolatum ICIS 99 exhibit antimicrobial activity against clinical isolates of A. baumannii. On the other hand, the observed reduction in the growth of planktonic culture and biofilm formation of A. baumannii may be due to the synergistic action of these compounds.

The data obtained on the antimicrobial and antibiofilm activity of C. amycolatum CFS against A. baumannii are another argument pointing to the probiotic properties of C. amycolatum, and the chemical compounds identified in CFS open up prospects for further research aimed at developing new strategies to combat infections caused by A. baumannii.

Conclusion

This study demonstrates that CFS from vaginal isolates of C. amycolatum exhibit antimicrobial and antibacterial activity against A. baumannii. GC-MS analysis of the CFS extract of C. amycolatum ICIS 99 revealed the presence of four volatile compounds, which, according to the literature, have a wide range of biological activity. The results of this study open up opportunities for studying microorganisms of the genus Corynebacterium spp. as a source of unique biologically active compounds with antibacterial activity, as well as for further in-depth research on the separation, purification, determination of structural and functional characteristics, and evaluation of the mechanism of action of the identified compounds.

 

¹ Stroganova E.A., Gladysheva I.V., Cherkasov S.V. Patent 2802776. Russian Federation. IPC C12N 1/20, C12P 13/00, C12R 1/15 Agent for producing organic compounds with antibacterial and antioxidant activity. Applicant and patent holder: Federal State Budgetary Institution OFIT UrO RAS. No. 2023101178; filed 20.01.2023; published 01.09.2023. Bulletin No. 25.

About the authors

Irina V. Gladysheva

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Author for correspondence.
Email: gladishevaiv@yandex.ru
ORCID iD: 0000-0001-6231-7028

Cand. Sci. (Med.), senior researcher, Laboratory of microbial ecology and dysbiosis, Institute of Cellular and Intracellular Symbiosis, Ural Branch of the Russian Academy of Sciences

Russian Federation, Orenburg

Elena V. Ivaschenko

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: ivaschenkoev@yandex.ru
ORCID iD: 0009-0007-9152-3032

laboratory assistant-researcher, Laboratory of biomedical technologies, Institute of Cellular and Intracellular Symbiosis of the Ural branch of the Russian Academy of Sciences

Russian Federation, Orenburg

Ekaterina S. Filonchikova

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: filonchikova@inbox.ru
ORCID iD: 0000-0001-9644-2978

researcher, Laboratory of biomedical technologies, Institute of Cellular and Intracellular Symbiosis, Ural branch of the Russian Academy of Sciences

Russian Federation, Orenburg

Elena A. Shchuplova

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: khanina83@yandex.ru
ORCID iD: 0000-0002-2546-5416

Cand. Sci. (Biol.), senior researcher, Laboratory of microbial ecology and dysbiosis, Institute of Cellular and Intracellular Symbiosis, Ural Branch of the Russian Academy of Sciences

Russian Federation, Orenburg

Sergey V. Cherkasov

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: cherkasovsv@yandex.ru
ORCID iD: 0000-0002-0707-2977

Dr. Sci. (Med.), Academician of the Russian Academy of Sciences, chief researcher, Laboratory of biomedical technologies, Institute of Cellular and Intracellular Symbiosis of the Ural Branch of the Russian Academy of Sciences

Russian Federation, Orenburg

References

Supplementary files