1 Introduction

Caseous lymphadenitis (CLA) in small ruminants is caused by Corynebacterium pseudotuberculosis, a Gram-positive facultative intracellular pathogen. It is an easily transmitted infectious disease that commonly infects sheep and goats (Nascimento, 2020). The disease causes inflammation of the lymph node resulting in the formation of caseaous cheesy material in the nodes, abscess formation in superficial and internal lymph nodes as well as in internal organs (Cetinkaya et al., 2002). CLA cases have been reported by many countries such as Malaysia (Komala et al., 2008), Brazil (Guimarães et al., 2009), Egypt (Al-Gaabary et al., 2010), northern Norway (Hektoen, 2012), France and Spain (De La Fuente et al., 2017). The severity of CLA is expected to associate with biofilm formation of the causative organism.

Biofilm is a complex and structured microbial community that tends to attach to inert and living surfaces in the presence of extracellular polymeric substances (EPS) and causes a wide range of diseases. Bacteria within the biofilm become more resistant to antimicrobial treatment compared with planktonic counterparts because the bacterial community in the biofilm differs from its planktonic counterpart in the gene and protein expression patterns that typically results in distinct metabolic and antimicrobial resistance profiles (Giaouris et al., 2013). According to National Institutes of Health (NIH), among all microbial and chronic infections, 65% and 80%, respectively, are associated with the biofilm formation. Over the past few decades, many aspects of biofilms have been understood including basic life cycle, antimicrobial susceptibility pattern (Costerton et al., 1999), essential enzymes for biofilms (Yahya et al., 2014), biofilm heterogeneity (Yahya et al., 2017), chemical composition of EPS matrix (Costerton et al., 1999; Yahya et al., 2018) and whole-cell proteome expression (Giaouris et al., 2013; Zawawi et al., 2020).

Since the last few years, previous works have addressed the important genes for vaccination and the protein interaction networks in C. pseudotuberculosis. Hassan et al. (2014) adopted a novel integrative strategy combining substractive proteomics and modelomics. They successfully identified four essential and non-homologous proteins as potential vaccine targets. On the other hand, Folador et al. (2016) identified 181 essential proteins, among which 41 are non-host homologous from the inferred protein interaction networks. However, until now, there is still a lack of experimental data on C. pseudotuberculosis biofilm. Thus, the present work was performed to determine the biochemical composition of C. pseudotuberculosis biofilm using Raman spectroscopy. Biofilm morphology and biofilm susceptibility towards antimicrobials were investigated using field emission scanning electron microscopy (FESEM) and microplate biofilm assay respectively.

2 Materials and methods

3 Results

3.1

3.1 Differential Raman spectra of C. pseudotuberculosis biofilm across different growth stages

Fig. 1 shows microscopic images and averaged Raman spectra of C. pseudotuberculosis biofilm at 24 h, 48 h and 72 h. Microscopic images were compared with the corresponding averaged Raman spectra in order to determine the correlation between density and biochemical composition of C. pseudotuberculosis biofilm. Almost the entire surface of silicon substrate was covered by C. pseudotuberculosis cells at 48 h and 72 h, indicating the biofilm maturity. Across all stages, C. pseudotuberculosis biofilm showed Raman spectral peaks at 615 cm⁻¹ (CCC symmetric bend phenyl ring), 668 cm⁻¹ (Valine) and 825 cm⁻¹ (Ring breath Tyr). The Raman spectral peaks at 1400 cm⁻¹ (COO— sym.), 1450 cm⁻¹ (COO— sym.), 1581 cm⁻¹ (Ring breath Trp.), 1650 cm⁻¹ (COO— asym.) and 1725 cm⁻¹ (C⚌O str.) were observed to appear in both 48-h-old and 72-h-old biofilms, but not in 24-h-old biofilm. These five Raman spectral bands may specifically associate with the mature stage of C. pseudotuberculosis biofilm.

Close

Fig. 1

Microscopic images at 100x magnification and the corresponding averaged spectra of C. pseudotuberculosis biofilm. a) 24 h-old biofilm; b) 48 h-old biofilm and c) 72-h biofilm. Spectral regions that commonly show organic molecules in biofilms: 600–900 cm⁻¹; 1100–2000 cm⁻¹. Functional groups: 615 cm⁻¹ (CCC symmetric bend phenyl ring), 668 cm⁻¹ (Valine), 825 cm⁻¹ (Ring breath Tyr), 838 cm⁻¹ (phosphodiester symmetric str.), 1400 cm⁻¹ (COO— sym.), 1450 cm⁻¹ (COO— sym.), 1581 cm⁻¹ (Ring breath Trp.), 1650 cm⁻¹ (COO— asym.) and 1725 cm⁻¹ (C⚌O str.).

Export to PPT

3.2

3.2 Heterogeneity of C. pseudotuberculosis biofilm at 48 h and 72 h

Fig. 2 shows the Raman spectra of C. pseudotuberculosis taken at three different locations of the C. pseudotuberculosis biofilm. Raman spectral peaks in the range 1000–9000 cm⁻¹ indicated the silicon substrate. There were no differences in all Raman spectra of C. pseudotuberculosis at 24 h. However, inconsistent Raman spectral bands at 835 cm⁻¹ (phosphodiester symmetric str.) and 1725 cm⁻¹ (C⚌O str.) were observed in 48 h-old biofilm and 72 h-old biofilm, showing the biochemical heterogeneity.

Close

Fig. 2

Diverse Raman spectra within the specific stage of C. pseudotuberculosis biofilm. a) 48 h-old biofilm and b) 72 h-old biofilm. Functional groups: 838 cm⁻¹ (phosphodiester symmetric str.) and 1725 cm⁻¹ (C⚌O str.). Different colours in each panel indicate Raman spectra taken at three different locations within single biofilm samples.

Export to PPT

3.3

3.3 Morphology of C. pseudotuberculosis biofilm

Fig. 3 shows the morphology of C. pseudotuberculosis biofilm at a magnification of 5000x. Rod-shaped cells of C. pseudotuberculosis were clearly observed in the 24-h-old biofilm. There was only one layer of biofilm that was formed on the surface. The biofilm cells were also encapsulated in a fibrous extracellular matrix.

Close

Fig. 3

FESEM image of C. pseudotuberculosis biofilm at 5000x magnification. a) Biofilm cells; b) Extracellular matrix.

Export to PPT

3.4

3.4 Pellicle formation at air–liquid interface

The pellicle formation was compared between C. pseudotuberculosis biofilm and S. typhimurium biofilm. Fig. 4 shows C. pseudotuberculosis pellicle biofilm at air–liquid interface. It was found that C. pseudotuberculosis biofilm formed an intact pellicle similar to that formed by S. typhimurium biofilm.

Close

Fig. 4

Formation of pellicle biofilm at air–liquid interface. a) S. typhimurium; b) C. pseudotuberculosis. Adequate staining with 0.5% crystal violet, 25% methanol was achieved after 5 min.

Export to PPT

3.5

3.5 Inhibitory action of antibiotics against C. pseudotuberculosis biofilm

The biofilm susceptibility towards antibiotics was compared between C. pseudotuberculosis biofilm and S. typhimurium biofilm. Fig. 5 shows the antibiofilm activities of antibiotics. Four test concentrations of streptomycin (3.12 µg/ml, 12.5 µg/ml, 25 µg/ml, 100 µg/ml) significantly (p < 0.05) inhibited C. pseudotuberculosis biofilm. The same effect was also observed in S. typhimurium biofilm. Three test concentrations of tetracyclin (3 µg/ml, 12.5 µg/ml, 50 µg/ml) significantly (p < 0.05) inhibited C. pseudotuberculosis biofilm but not S. typhimurium biofilm. Two test concentrations of nalidixic acid (25 µg/ml, 50 µg/ml) significantly (p < 0.05) inhibited C. pseudotuberculosis and S. typhimurium biofilms while 12.5 µg/ml nalidixic acid significantly (p < 0.05) inhibited C. pseudotuberculosis biofilm but not S. typhimurium biofilm.

Close

Fig. 5

Inhibitory effects of antibiotics on C. pseudotuberculosis biofilm and S. typhimurium biofilm. Positive control: overnight inocula added with IP-protected antibiofilm cocktail; negative control: overnight inocula added with fresh broth. Each bar represents mean ± standard deviation. Significant differences (p < 0.05) when compared with control group are shown by *.

Export to PPT

3.6

3.6 Inhibitory action of common chemicals against C. pseudotuberculosis biofilm

The biofilm susceptibility towards common chemicals was compared between C. pseudotuberculosis biofilm and S. typhimurium biofilm. Fig. 6 shows antibiofilm activities of common chemicals. Two test concentrations of EDTA (250 mM and 500 mM) significantly (p < 0.05) inhibited C. pseudotuberculosis and S. typhimurium biofilms. All test concentrations of DMSO (1.56%, 3.13%, 6.25%, 12.5%, 25%, 50%) significantly (p < 0.05) inhibited C. pseudotuberculosis biofilm but not S. typhimurium biofilm. NaOH and ethanol did not show any significant inhibitory actions against C. pseudotuberculosis biofilm and S. typhimurium biofilm.

Close

Fig. 6

Effect of common chemicals on C. pseudotuberculosis biofilm and S. typhimurium biofilm. Positive control: overnight inocula added with IP-protected antibiofilm cocktail; negative control: overnight inocula added with fresh broth. Each bar represents mean ± standard deviation. Significant differences (p < 0.05) when compared with control group are shown by *.

Export to PPT

4 Discussion

Proteinaceous components are important for all stages of biofilm formation. They play crucial roles in initial attachment to surface, stabilization of the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, and development of three-dimensional biofilm architectures. The present study showed the presence of Raman spectral peaks associated with protein groups (615 cm⁻¹, 668 cm⁻¹, 828 cm⁻¹ and 1581 cm⁻¹) in C. pseudotuberculosis biofilm (Fig. 1). Raman spectral peak at 1581 cm⁻¹ (Trp. proteins) may represent a component of extracellular matrix of C. pseudotuberculosis biofilm (Ivleva et al., 2009).

Multiple lines of evidences have shown that lipids are present on the plasma membrane and extracellular matrix which are important for the architectural stability of the microbial complex. In most cases, planktonic and biofilm cells exhibit different profiles of sterol and sphingolipid. Herein, Raman spectral peaks at 668 cm⁻¹ (CN+(CH3)3 str., lipids) and 1650 cm⁻¹ (C⚌C and C⚌O, lipids) were found to be present in C. pseudotuberculosis biofilm (Fig. 1). The Raman spectral peak at 668 cm⁻¹ has been shown to be present in Pseudomonas aeruginosa biofilm (Jung et al., 2014).

It has been established that the dense extracellular matrix surrounding mature biofilms is composed primarily of glycoproteins and carbohydrates along with lipids and nucleic acids. Carbohydrates function in providing structure to biofilms, allowing stratification of the bacterial community and establishing gradients of nutrients and waste products. Raman spectral peaks associated with carbohydrate group (1400 cm⁻¹, 1450 cm⁻¹, 1650 cm⁻¹ and 1725 cm⁻¹) were identified in the present study (Fig. 1). These Raman spectral peaks may also represent components of extracellular matrix of C. pseudotuberculosis biofilm (Ivleva et al., 2009).

Mature biofilms from both clinical and environmental settings have been extensively investigated due to its harmful impacts and antimicrobial resistance phenotype. In most experiments, the 48-h-old biofilm and older ones are considered as mature biofilms. Herein, the mature C. pseudotuberculosis biofilms were characterized by the Raman spectral peaks associated with proteins, carbohydrates and lipids groups (Fig. 1). This result may indicate the domination of extracellular matrix of the mature C. pseudotuberculosis biofilm by high composition of protein fraction and relatively lower composition of saturated fatty acids and polysaccharides (Ramirez-Mora et al., 2019).

Physiological heterogeneity in the biofilm is due to the multilayer microbial communities and three dimensional structures that are established on the surface. In the present study, the heterogeneity in C. pseudotuberculosis biofilm based on altered Raman spectra was observed at 48 h and 72 h (Fig. 2). Spectra taken from different positions within single biofilm colonies could reveal the formation of more than one layer in the colonies (Kelestemur et al., 2018). Thus, changes in Raman spectral features are useful to explain heterogeneity in different developmental stages of C. pseudotuberculosis biofilm (Sandt et al., 2007).

FESEM analysis is useful for biofilm study because it provides information on the size, shape, production of extracellular matrix and localization within the biofilm of single bacteria. In the present study, FESEM images showed that the extracellular matrix surrounded the C. pseudotuberculosis biofilm cells and attached them to surface (Fig. 3). It is believed that extracellular matrix may play an important role in establishing C. pseudotuberculosis biofilm on surfaces (Combrouse et al., 2013; Ivleva et al., 2009; Jung et al., 2014; Ramirez-Mora et al., 2019).

Biofilm tends to form pellicle at air–liquid interface in aerobic environment. The pellicle gives a more cloudy appearance to the culture than planktonic population. Glucose-rich, cellulose-like polymer is essential for the formation of a pellicle at the air–liquid interface. In the present study, C. pseudotuberculosis biofilm successfully formed the pellicle at air–liquid interface like S. typhimurium biofilm (Fig. 4). According to Jolly (1965), the pellicle formation contributes to the pathogenesis of Corynebacterium ovis. Thus, the pellicle formation observed herein may also play role in the pathogenesis of C. pseudotuberculosis.

Streptomycin controls the bacterial infection by inhibiting the binding of formyl-methionyl-tRNA to the 30S subunit during protein synthesis. In the present study, streptomycin effectively inhibited C. pseudotuberculosis biofilm (Fig. 5). This finding contradicts Olson et al. (2002) that demonstrated the resistance of C. pseudotuberculosis biofilm against streptomycin with the minimum biofilm eradication concentration (MBEC) values greater than 256 µg/ml. The disparity in this case is probably due to different growth medium used in the respective experiment whereby nutrient broth was used in the present study whilst tryptic soy broth supplemented with fetal bovine serum was used in the study performed by Olson et al. (2002). The present study used only nutrient broth because C. pseudotuberculosis biofilm was being compared with a veterinary model pathogen namely S. typhimurium to investigate the pellicle formation and antibiofilm activity.

Inhibition of bacterial growth by DMSO is known to involve membrane perturbation. The present study revealed the inhibitory action of DMSO against C. pseudotuberculosis biofilm (Fig. 6). Inhibition of C. pseudotuberculosis biofilm by four disinfectants namely iodine, chlorhexidine, chlorine and quaternary ammonia has previously been reported (Sá et al., 2013). The present finding provides the first evidence for antibiofilm activity of DMSO against C. pseudotuberculosis and also suggests the potential use of DMSO as a surface disinfectant to control microbial surface attachment (Siddiqui et al., 2016).

Based on the significantly effective test concentrations, discrepancies in antibiofilm susceptibility pattern between C. pseudotuberculosis biofilm and S. typhimurium biofilm were observed. In Fig. 5, C. pseudotuberculosis biofilm was less susceptible to streptomycin and more susceptible to tetracyclin, as compared with S. typhimurium biofilm. On the other hand, C. pseudotuberculosis biofilm was less susceptible to EDTA and more susceptible to DMSO, as compared with S. typhimurium biofilm (Fig. 6). These discrepancies remain not well understood, however, the possible explanations are possibly due to the presence of outer membrane proteins in Gram-negative pathogen (Rollauer et al., 2015) and species-dependent antimicrobial effects (Fernandes et al., 2020).

5 Conclusions

We have demonstrated the biochemical composition and morphology of C. pseudotuberculosis biofilm. The pellicle formation of C. pseudotuberculosis biofilm is similar to that of S. typhimurium biofilm while the antibiofilm susceptibility pattern of C. pseudotuberculosis is slightly different from that of S. typhimurium. To our knowledge, the present study represents the first effort to extensively characterize the biofilm formation by C. pseudotuberculosis that may mediate the persistence of CLA in small ruminants. A detailed investigation on how these antimicrobials could inhibit C. pseudotuberculosis biofilm at gene and protein expression levels need further attention.