An in vitro study of interactions of Candida albicans with Klebsiella pneumoniae and Enterococcus faecalis isolated from intestinal microbiome of HIV infected patients

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INTRODUCTION

The intestinal microbiome is an integrated system of interacting microorganisms; this system is capable of self-regulating by forming different types of microbial relationships [1][2]. Microbial antagonism is one of the factors contributing to development of microbiocenosis [3]. Symbiont antagonism is characterized by production of antimicrobial substances [3], lytic enzymes (peptidase, amylase) destroying structures of microorganisms or molecules secreted by them [4][5]. Some bacteria produce low molecular weight substances, which change growth properties and bacteria persistence by affecting their genetic program [6][7], inhibiting antioxidant systems of competitors [8][9], activating metabolic shunts they need in their competition over food and iron sources [10].

Interactions between bacterial and fungal microbiomes in the intestinal biotope play a pivotal role in creation and maintenance of symbiosis and are associated with the risk of development of candidamycosis [3]. Bifidobacteria and lactobacilli create antagonistic relationships with fungi, which are aimed to prevent excessive fungal colonization of different biotopes [4][11]. Some studies have demonstrated the mutual effect of virulence factors in opportunistic pathogenic bacteria and fungi on the macroorganism, including development of pathological processes [12]. In their competition over receptors present in the mucus membrane, micromycetes and opportunistic pathogenic bacteria enter into antagonistic relationships with the indigenous microbiota. However, when the population of opportunistic pathogenic bacteria reaches high levels of density, they become antagonistic toward fungi of the genus Candida [13].

Antagonism factors and targeted effect of opportunistic pathogenic bacteria from different taxonomic groups on fungi as well as the conditions required for implementation of antagonistic relationships and the factors of antagonism regulation in opportunistic pathogenic symbionts need further study. The mechanism of survival of fungi amidst “dual” antagonism (of indigenous and facultative bacteria) of intestinal symbionts remains unclear. Identification of bio-communicative mechanisms involved in prevention of development of endogenous candida infection is critically important for HIV infected patients.

The aim of the study was to identify in vitro targets for klebsiellas and enterococci antagonism factors in Candida albicans isolated from the intestinal microbiome of HIV infected patients.

MATERIALS AND METHODS

A total of 89 children diagnosed with HIV infection took part in the study; all of them were hospitalized to the respiratory infection department at the Kemerovo Regional Clinical Hospital for Infectious Diseases in 2019–2021; 10 (11%) children were hospitalized because of secondary bacterial diseases (pneumonia, tonsillitis) and 79 (89%) children were hospitalized with acute respiratory viral infections. The mean age of the patients was 24 ± 2 months; there were 49 (55%) boys and 40 (45%) girls. Most of the children (76.3%) had the 2nd stage of HIV infection (2A — 4.5%; 2B — 56.1%; 2C — 15.7%); 14.6% had the 3rd stage; 8.9% had the 4th stage. The stages of HIV infection are consistent with Pokrovsky’s classification (2001), including the amendments adopted in 2006.¹

The study was approved by the Ethics Committee of the Kemerovo State Medical University (minutes No. 5 of 31/1/2019). All the patients included in the study had their informed consent signed so that results of the study could be used for scientific purposes.

The tests were performed on 38 C. albicans strains, 28 Klebsiella pneumoniae strains, and 30 Enterococcus faecalis strains isolated from the intestinal biotope. The isolation of microorganisms was performed using selective and differential media. To extract K. pneumoniae, we used HiChrome Klebsiella Selective Agar Base (HIMEDIA); the specimens were cultured at 37ºC for 24 hours. The purple-magenta-colored colonies were reseeded on Kligler’s medium (State Scientific Center of Applied Microbiology and Biotechnology (SSC AMB)) to accumulate pure culture and to do preliminary analysis of the biochemical properties. To isolate E. faecalis, we used the Enterococcus Agar medium (SSC AMB) for seeding, selected typical colonies in 24 hours, analyzed the morphology, and accumulated pure cultures. C. albicans fungi were isolated using the HiChrome Candida Agar (HIMEDIA); then we selected colonies corresponding to C. albicans by color using the differential scale in the manufacturer’s instruction. To eliminate the risk of a false-negative result in the tests involving inhibition of fungal morphogenesis, all the strains were assessed for their ability to form germ tubes in the horse serum three hours after the culturing at 37ºC [14]. The final species-level identification of all microorganisms was performed using the VITEK 2 Compact analyser (BioMerieux). The tests were performed on E. faecalis–C. albicans pairs; each symbiont in the pair was obtained from the same patient to prevent the risk of the outsider phenomenon [15]. As a result, there were 26 pairs of symbionts. The tests were performed twice in 3 replications.

The effect of K. pneumoniae on the morphological transformation of C. albicans was assessed [14]. At first, K. pneumoniae cultures were grown in Mueller– Hinton broth (SSC AMB) for 18 hours at 37ºC; C. albicans fungi were grown on Sabouraud agar (SSC AMB) for 24 hours to match the completion of the exponential growth stage [16]. The C. albicans suspension was prepared in the sterile 0.9% NaCl solution with turbidity of 0.5 McFarland units, being equal to 1–5 × 10⁶ CFU/ml) [17]. The Klebsiellas suspension was diluted 100 times to have the similar turbidity. The final concentration of Klebsiellas was 1 × 10⁶ CFU/ml. 100 μl of K. pneumoniae and C. albicans suspension was placed into a tube with 0.5 ml of horse serum (Microgen NPO). Microorganisms were incubated at 37ºC. Three hours after, “crushed drop” smears were prepared and 100 cells of C. albicans fungi examined under a Carl Zeiss Primostar microscope; the percentage of cells forming germ tubes was recorded. Untreated C. albicans cultures were used as control cultures, which were also assessed for their ability to form germ tubes in the protein-based medium.

The effect of E. faecalis exometabolites on the C. albicans catalase was assessed using the methods [9] including modifications, i.e. using stable ammonium molybdate instead of unstable potassium iodide. The two-day broth culture of E. faecalis was used to produce the supernatant by centrifuging the culture two times at 3000 rpm for 15 min. The supernatant liquid was separated from bacterial cells using membrane filters. C. albicans cultures were used to prepare suspension in the sterile 0.9% saline solution with turbidity of 0.5 McFarland units. Test samples were prepared by mixing 0.1 ml of C. albicans suspension, 2.6 ml of Sabouraud broth, and 0.3 ml of supernatant from broth cultures of E. faecalis. The values of catalase activity of fungal broth cultures not exposed to exometabolites of enterococci (0.1 ml of fungal suspension and 2.9 ml of broth) were used as reference variables. To measure the catalase activity, we added 1 ml of 0.0125 M solution of H₂О₂ to the test samples and to the reference samples of 0.2 ml; 10 minutes after, the reaction was discontinued by adding 1 ml of 4% ammonium molybdate solution. The non-inactivated H₂О₂ reacted with ammonium molybdate to produce colored complexes, optical density (OD) of which was measured with the SF 2000 spectrophotometer (OKB Spectr) at λ = 550 nm compared to the medium. The catalase activity was calculated using the formula [9]. The obtained results were compared with the catalase activity of C. albicans cultures, which were not treated with enterococcus supernatants.

To perform the statistical analysis, we used the IBM SPSS Statistics/PS IMAGO software package («IBM/Predictive Solutions Sp z.o.o.»). The normality of data distribution was verified with the Shapiro–Wilk test. The comparative analysis was performed using non-parametric methods for assessment of statistical significance (the χ2 and Mann–Whitney tests) [18]. The experimental data are presented as average values and standard deviation, the median, and interquartile range [the 25th and 75th percentiles]. In statistical hypothesis testing, the significance level was equal to or less than 0.05 [18].

RESULTS

The tests showed that K. pneumoniae inhibited the ability of fungi to form germ tubes. When cocultured with Klebsiellas, on average, 12.3 [ 6.33; 15]% of yeast cells produced germ tubes, while the fungal monoculture contained 29.8 [ 25; 36, 7]% of cells with blastospore transformation. Thus, the inhibition of morphological transformation of C. albicans in associations with K. pneumoniae accounted for 58.72% (p < 0.01).

Enterococcus supernatants affected C. albicans in different ways (Table 1). In 65.4% of cases, enterococci inhibited the catalase of micromycetes; in 19.2% of cases, the catalase activity in fungi did not demonstrate any changes after treatment with E. faecalis supernatants; only in 15.4% of cases, the catalase production increased. The initial catalase level in untreated C. albicans cultures averaged 1.02 [ 0.87; 1.13] µmol/min OD; after the treatment with E. faecalis exometabolites, it decreased to 0.55 [0.36; 0.73] µmol/min OD (p < 0.05).

 

The influence of E. faecalis exometabolites on the catalase activity of C. albicans (M ± SD)

 

On average, the catalase activity of C. albicans was inhibited by 46.1% (p < 0.05).

The obtained results demonstrate that antagonism of Klebsiellas and enterococci toward C. albicans has different target points, different intensity and is a product of competition in the multicomponent microbial community.

DISCUSSION

As the number of HIV infected people is increasing, candidiasis has become a common concomitant condition; therefore, a special emphasis is placed on new approaches in prevention of the process progression and timely diagnosis of opportunistic mycosis prior to the onset of symptoms [19]. Using of biocoenotic relationships and factors making it possible to regulate biological properties of C. albicans in microbiocenoses and counteract the implementation of their pathogenic potential offers promising prospects [20][21]. Regardless of the biotope, resident microbiota regulates the virulence of C. albicans. Lactic acid and bacteriocins produced by Lactobacillus spp. play an essential role in inhibiting the activity of proliferation genes, impeding the growth and production of hyphae in C. albicans, and decreasing the expression of hyphal wall proteins 1 (Als3 and Hwp1) [11][12]. The anti-biofilm effect toward fungi is produced by oleic and pentadecanoic acids resulting from the metabolism of fatty acids of anaerobic bacteria [22]. As for E. faecalis, their antagonism is strongly associated with the secretion of bacteriocin (ENTV) inhibiting the development of hyphae and affecting the formation of C. albicans biofilms. Microorganisms of the genus Bacteroides, Prevotella, Bifidobacterium decrease the anti-complementary activity of fungi [23].

When describing the interaction between fungi and the opportunistic pathogenic microbiota, most studies focus on mutual enhancement of antagonism toward indigenous bacteria [23]. In bacterial and fungal associations, fungi tend to act as “helpers” of facultative microorganisms. Candida metabolites enhance the anti-lysozyme activity of Staphylococcus aureus, Klebsiella spp., E. coli lac–/hly+, and have a direct inhibitory effect on the anti-lysozyme factor of bifidobacteria [24]. The component of the cell wall — C. albicans b-1,3-glucan increases the antibiotic resistance of St. aureus [25]. In the meantime, there are data demonstrating that similar to indigenous microorganisms, opportunistic pathogenic bacteria display antagonism toward fungi in high-density populations [22][26].

Some molecular mechanisms of intermicrobial interactions of C. albicans with K. pneumoniae and E. faecalis isolated from HIV infected patients have been studied. By and large, the studied bacterial species demonstrate antagonism toward C. albicans, showing the consistency with the data provided by researchers from other countries [13].

It has been found that K. pneumoniae have an effective biopotential for regulation of the population size of C. albicans. The morphological transformation of C. albicans is the target for K. pneumoniae. Fungal morphogenesis into the hyphal form is seen as the pathogenicity factor for micromycetes, as they have a wider range of adhesins for mucosal surfaces, increased propagation in tissues, and the increased number of phospholipases, which concentrate on the termini of hyphal elements [27].

Antagonism toward fungi has been demonstrated not only by K. pneumoniae, but also by E. faecalis. E. faecalis inhibited catalase, which is a powerful antioxidant enzyme in microorganisms with aerobic respiration [28][29]. Changes in the activity or inhibition of enzymes in antioxidant systems of microorganisms can result in accumulation of toxic forms of oxygen, thus affecting the permeability of a membrane, intake rate of nutrients and, eventually, the proliferation rate of microorganisms [30][31].

CONCLUSION

The obtained results support the data on the role of facultative bacteria in functioning of the intestinal microbiome and demonstrate their regulating effect on C. albicans. The antagonism of facultative bacteria toward C. albicans is based on the inhibition of morphological transformation and catalase production, opening up promising opportunities for methods aimed at prevention of candidiasis and involving enhanced antagonism of not only resident, but also transient microbiota. The results of tests and approaches to exploration of bacterial and fungal relationships have significant research and practical potential, making it possible to perform in vitro modeling of the process aimed at control of biological properties of Candida fungi by using the antagonism factors in the intestinal bacterial microbiota.

 

1. Decree No. 166 of the Ministry of Health and Social Development of the Russian Federation, adopted on 17/3/2006, The Instruction for Completing of Annual Form No. 61 of Federal Statistical Survey “Data on Cohorts of HIV-Infected Patients”.

About the authors

Yuliyа V. Zakharova

Kemerovo State Medical University

Author for correspondence.
Email: yvz@bk.ru
ORCID iD: 0000-0002-3475-9125

D. Sci. (Med.), Associate Professor, Professor, Department of microbiology and virology

Russian Federation, Kemerovo

Larisa Yu. Otdushkina

Kemerovo State Medical University

Email: yvz@bk.ru
ORCID iD: 0000-0003-4126-4312

Assistant Professor, Department of microbiology and virology

Russian Federation, Kemerovo

Alina A. Markovskaya

Kemerovo State Medical University

Email: yvz@bk.ru
ORCID iD: 0000-0002-5001-7068

Assistant Professor, Department of epidemiology, infectious diseases and dermatovenerology

Russian Federation, Kemerovo

Yuri V. Nesvizhsky

I.M. Sechenov First Moscow Medical University (Sechenov University); G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology

Email: yvz@bk.ru
ORCID iD: 0000-0003-0386-3883

D. Sci. (Med.), Professor, Department of microbiology, virology and immunology; main researcher

Russian Federation, Moscow; Moscow

Stanislav S. Afanasiev

G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology

Email: yvz@bk.ru
ORCID iD: 0000-0001-6497-1795

D. Sci. (Med.), Professor, main researcher

Russian Federation, Moscow

Lyudmila A. Levanova

Kemerovo State Medical University

Email: yvz@bk.ru
ORCID iD: 0000-0002-5977-9149

D. Sci. (Med.), Associate Professor, Head, Department of microbiology and virology

Russian Federation, Kemerovo

References

Supplementary files