Introduction
Osteomyelitis is a progressive infection of the bone, resulting in destruction of bone tissue and bone necrosis, eventually developing into a chronic or persistent condition [
1]. Chronic osteomyelitis is the prominent type of osteomyelitis with high recurrent rates, which may result from tenacious biofilms formed by
Staphylococcus aureus (
S. aureus),
Streptococcus pyogenes,
Streptococcus pneumonia, mycobacteria, and even fungi [
2]. Particularly,
S. aureus is the most commonly isolated pathogen in any type of osteomyelitis [
3]. The low metabolic rates, adaptive stress responses, and decelerated rates of cell division of the deeply embedded bacteria, factors which contribute to resistance against antimicrobial agents, can be attributed to biofilms that act as diffusion barriers against those agents [
4].
S. aureus, one of the most ubiquitous microorganisms in nature, accounts for more than 50% of the osteomyelitis cases [
5]. The annual incidence of invasive bone and joint methicillin-resistant
Staphylococcus aureus (MRSA) infections accounts for 2.8 to 43% of all invasive MRSA infections which make up 1.6 to 29.7 cases per 100,000 osteomyelitis cases [
6]. In addition to the
mecA gene which induces resistance to almost all β-lactam antibiotics, it is the capacity of
S. aureus to form multilayered biofilms that enables it to thrive in the host, often leading to chronic conditions [
7].
The YycFG two-component regulatory system (TCS), specific to low G+C Gram-positive bacteria such as
Bacillus subtilis,
S. aureus,
Enterococcus faecalis, and
Streptococcus mutans, mediates the synthesis of biofilms that allows bacteria to rapidly adapt to physical, chemical, and biological stresses. YycG is a sensor protein with histidine kinase activity, and YycF is a cognate response regulator that cooperates with the former. Mutations in the YycFG TCS have been associated with resistant and persistent infections [
2,
8]. In
B. subtilis, the genes
yycFG originate from a part of large operon that comprises
yycFGHIJ [
9]. Besides, the
yycH and
yycI genes have been revealed to interact with
yycG expression [
10]. Biofilm organization is associated with the genes of
ica locus including the
icaABCD genes which encode the vital protein polysaccharide intercellular adhesion (PIA) [
11]. In particular,
icaA as an operon for enzyme in PIA synthesis encoding
N-acetylglucosaminyltransferase and
icaD play an essential role for biofilm synthesis [
12,
13]. In this study, we tracked the MRSA from chronic osteomyelitis cases and methicillin-sensitive
Staphylococcus aureus (MSSA) strains to investigate the potential roles of YycFG TCS components.
Discussion
Sequencing of 16S rRNA amplicons indicated that the infection in chronic osteomyelitis specimens was polymicrobial in nature. The bacterial species detected included five phyla (
Actinobacteria,
Bacteroidetes,
Firmicutes,
Fusobacteria,
Proteobacteria). Polymicrobial infections have been observed in previous studies of chronic osteomyelitis of the jaw [
18,
28]. In the present study, the phylum
Bacteroidia made up the major part (82.69%) of the diseased tissues’ microbiota. In terms of microbial diversity, the genus
Porphyromonas was the most abundant (Fig.
1a). The second most abundant phylum in the chronic osteomyelitis specimens was
Firmicutes which made up 13% of the microbiota. It has been speculated that the deeper osteomyelitis tissues adapt to the anaerobic environment. This and the polymicrobial nature of the infection make it difficult to identify most of the causative microbes with routine culture-dependent methods [
29], underscoring the role of genetic sequencing. Moreover, further analysis of the phylogenetic distribution of bacteria in MSSA osteomyelitis specimens would shed light on the interaction between MRSA and the microbiota in chronic osteomyelitis.
The methicillin-resistant
Staphylococcus aureus (MRSA) strains on the other hand have been shown to be crucial in such recalcitrant and persistent infections [
2]. Therefore, the potential mechanism involved in the pathogenicity of MRSA deserves further investigation. In recent years, an increasing number of studies have investigated the genetic pathways involved in the drug resistance of MRSA strains [
30,
31]. However, there is no evidence for any direct interaction between these pathways and the biofilm formation seen in a methicillin-resistant clinical isolate. Among drug-resistant regulatory networks, the two-component signal transduction systems (TCS) are essential for bacterial adaptation, survival, and virulence which contribute to antibiotic resistance [
32,
33]. Typically, TCS signal transduction comprises of a membrane-associated histidine kinase and a cytoplasmic response regulator. The histidine kinase recognizes an environmental change, e.g., in pH, osmolarity, or oxidation reduction, and auto-phosphorylates at a conserved histidine residue. Following the transfer of the phosphorylated moiety to the response regulator, the latter can control transcription of target genes. The metabolic processes controlled by TCS include quorum sensing, sporulation, and bacteriocin production in a wide variety of bacteria [
34,
35].
In the present study, MRSA isolates from chronic osteomyelitis specimens revealed upregulated levels of
yycF,
yycG, and
yycH genes when compared to the MSSA ATCC29213 strain (Fig.
3a), underscoring their role in MRSA pathogenicity and antibiotic resistance. It was revealed that mutants of YycHI are selected for leading to reduced WalRK activation which was associated with activation and impaired cell wall turnover [
30]. In
S. aureus, WalR is a critical transcriptional regulator which has been shown to influence the expression of genes associated with amino acid biosynthesis, central metabolism, and virulence [
36,
37]. In the present study, the MSSA strain showed altered growth pattern with a significantly delayed (4 h) entry into the log phase compared to the MRSA strains (Fig.
2b), suggesting that YycFG affects growth rates.
Polysaccharide intercellular adhesion (PIA), a β-1, 6-linked
N-acetyl-glucosamine homopolymer that triggers the accumulation of bacterial biofilm, is crucial to the cellular adhesion and pathogenesis of
S. aureus [
23]. PIA is synthesized by enzymes encoded by the
ica locus [
38], and the MRSA strains in this study show an upregulation of
icaA transcripts (Fig.
3a), indicating an important role of intercellular polysaccharide in
S. aureus pathogenesis [
39]. We found that the biofilm formation decreased by 65% in MSSA compared with MRSA (Fig.
2c, d). This phenotype fits with the easily disrupted biofilm and decreased intercellular polysaccharide matrix accumulation seen in MSSA.
Regarding the association between intercellular polysaccharides layer and biofilm aggregation of
S. aureus isolates, the AFM is a useful tool to observe bacterial biofilm formation [
40]. With accumulated intercellular polysaccharides matrix and enhanced biofilm biomass (Fig.
4a, b), the bacterial adhesion force in MRSA was significantly higher (9.2 ± 0.11 nN) compared to that of MSSA (1.76 ± 0.28 nN, Fig.
4c). Apart from cell adhesion, PIAs are also essential for shaping
S. aureus biofilm architecture [
23]. After the introduction of 0.5% glucose in the culture media, the MRSA cells were covered with reticular intercellular polysaccharides matrix while MSSA biofilms were devoid of these intercellular polysaccharides and had “blank” areas (Fig.
3b). Further studies are needed to validate our findings and elucidate the role of the YycH pathway in MRSA virulence. Within the limitations of this study, the potential mechanisms of the interactions between biofilm organization and expression of YycFG two-component systems should be considered in further investigations. On the other hand, the current study includes the use of single MSSA strain and lacking use of deletion mutants to study the roles of YycFG two-component pathway involved in biofilm formation and bacterial virulence. Therefore, additional information about the in vitro and in vivo studies needs to be explored in the future.