Within recent years there has been a considerable increase in the number of literature data reporting non-diphtherial corynebacteria (also known as diphtheroids) as the causative agents of opportunistic and nosocomial infections in humans (De Miguel-Martinez et al. 1996; Yoon et al. 2011; Bernard 2012b; Nhan et al. 2012; Qin et al. 2017; Santos et al. 2017; Kang et al. 2018). These bacteria are members of the microbiota of the skin and mucous membranes (Qin et al. 2017; Santos et al. 2017; Kang et al. 2018). The potential association between coryneforms and clinical infections can be attributed to immunosuppression, severe underlying medical disorders or invasive procedures (Nhan et al. 2012; Cacopardo et al. 2013; Kimura et al. 2017; Qin et al. 2017). In order to shed more light on the role of diptheroids as medically relevant microorganisms, their inherent low virulence should be confronted to the increasingly reported multidrug resistance (Yoon et al. 2011; Bernard 2012b; Kimura et al. 2017; Qin et al. 2017), and the ability to adhere to biotic and abiotic surfaces and/or to form biofilms (Kwaszewska et al. 2006; Souza et al. 2015a; Souza et al. 2015b; Qin et al. 2017; Kang et al. 2018).
The biofilm-producing bacteria are protected both against antibiotics (Fux et al. 2005; Lebeaux et al. 2014), and the host innate and adaptive immune responses (Souza et al. 2015b). Biofilm infections are associated with simultaneous activation of both arms of the host immune response. Neither of them, however, can eliminate the biofilm pathogen, but instead, in synergy, causes collateral surrounding tissue damage due to the release of phagocytic enzymes, oxidative radicals and/or due to formation of immune complexes (antigen-antibody) (Costerton et al. 1999; Jensen et al. 2010). Moreover, during a biofilm-related infection, planktonic bacteria released from the biofilm can spread into the bloodstream or in the vicinity the source of the infection. These strategies favor microbial persistence and chronicization of the infectious process which significantly complicates the therapy of biofilm-associated infections (Costerton et al. 1999; Lebeaux et al. 2014).
Although studies focused on the investigation and comparison of immune mechanisms stimulated in response to planktonic and biofilm types of bacterial growth have already been undertaken for medically important bacteria such as
Hence, the aim of the study was the investigation of the impact of
After confirmation of the ability to produce biofilm of a given strain, its planktonic and biofilm culture was performed in order to obtain PCMs and BCMs used subsequently to stimulate the Jurkat T cell lines. This was done for each of ten
Planktonic and biofilm cultures of the reference strains:
Planktonic cultures of the strains tested were grown in glass tubes (with the inoculum of McFarland 0.5 turbidity, approximately corresponding to 1.5 × 108 CFU/ml) in RPMI 1640 medium supplemented with 10% FCS, and gently agitated for 48 hours at 37°C. Biofilms were grown from the bacterial inoculum (McFarland 0.5 turbidity) of each strain in RPMI 1640 medium supplemented with 10% FCS in 24-well plates (volume 1000 µl, Nunc, USA) for 48 hours at 37°C. Both types of bacterial cultures resulted in the different number of bacterial cells following the incubation. For
The whole protein content was measured in the supernatants of all bacterial strains investigated before inoculation of the medium for growth of Jurkat T cells. For this purpose, the absorbance was measured in the BioPhotometer (Eppendorf BioPhotometer, Germany) at a wavelength of 280 nm. The absorbance values ranged from 0.018 to 0.720 (planktonic cultures) and from 0.022 to 0.776 (biofilm cultures). The protein content expressed by the optical density (OD) was higher in the biofilm culture of each investigated isolate when compared to its planktonic culture. These supernatants were subsequently used to stimulate cytokine production in Jurkat T cells.
Differences were considered statistically significant with
All experiments were performed in triplicate in three independent experiments.
The range of OD values for
Stimulation of the Jurkat T cells line with ten clinical strains of
The level of two cytokines, namely IL-1β and IL-12p70, produced by Jurkat T cells was significantly (
Concentrations of cytokines produced by Jurkat T cells in response to the to the PCM and BCM of planktonic and biofilm cultures of clinical
We observed a statistically significant (
A similar tendency was observed when the planktonic and biofilm types of growth of
Levels of cytokines (pg/ml) produced by Jurkat T cells in response to the PCM and BCM derived from mono- and mixed cultures of the reference
Stimulation of Jurkat T cells by the PCM and BCM from the mono- and mixed cultures of
Many potentially pathogenic bacteria grow in a planktonic state that can be attributed to acute infections and in biofilms that are associated with chronic infections. Bacteria growing within the biofilm communities secrete an extracellular matrix, form complex structures, demonstrate diverse metabolic activity and are phenotypically distinct from their planktonic counterparts. Therefore, it can be expected that the host immune response may contribute to different outcomes associated with these two disease types (Secor et al. 2011; Brady et al. 2018). To the best of our knowledge, the immune response manifested by the cytokine release after stimulation of immune cells with the planktonic and biofilm diphtheroids has not been studied yet. In our study, the planktonic and biofilm cultures of
The assessment of Jurkat T cell inflammatory responses in our study revealed a statistically significant (
These results are in line with the previously published results regarding other potentially pathogenic bacteria. Secor et al. (2011) revealed that
An unexpected result obtained in the present study was the level of anti-inflammatory IL-10, which was significantly (
On the other hand, according to what has been published by Saraiva and O’Garra (2010), induction of IL-10 often occurs together with pro-inflammatory cytokines, although pathways that induce IL-10 may negatively regulate these pro-inflammatory cytokines. Gutierrez-Murgas et al. (2016) reported a murine model of a catheter-associated
Another part of this study was to investigate the immunomodulatory effect of
Stimulation of the Jurkat T cell line by the PCM and BCM derived from the mono- and mixed cultures of the reference
It should also be noted that the interpretation of the results obtained is burdened with some limitations. The conditions used in
Hence, the unravelling of the interplay between the immune system and the coryneform bacteria as well as other bacteria that occurs within the biofilm communities requires further studies, which would shed more light on the increasing role of these medically important microorganisms. The results presented here can be considered as a starting point in this investigation.