Role of prophages in Staphylococcus aureus virulence and pathogenicity

Prophage, a temperate phage embedded in a bacterium's genome, affect bacterial fitness in multiple ways, including infectivity, toxin secretion, virulence regulation, surface modification, immune evasion, and microbiome competition. Studies have revealed that prophages significantly impact the distribution and spread of virulence and antimicrobial resistance. Lysogenic conversion by prophages introduces novel accessory functions to bacteria, enhancing bacterial fitness, host adaptation and persistence in different econiches. Prophages can also be triggered by stress conditions, such as exposure to ultraviolet (UV) radiation, antibiotics, or other chemical agents. Antibiotic-mediated prophage induction is known to cause a high frequency of prophage mobilization, implying that certain clinical strains carrying virulent prophages may have unintended consequences from antibiotics. For example, the hlb-converting prophage (also known as ϕSa3int) of Staphylococcus aureus encodes exotoxins and immune modulatory molecules that can inhibit human innate immunity, increasing the bacterium's pathogenicity. This property contributes to chronic infections and inflammation, such as chronic rhinosinusitis (CRS). Moreover, sub-lethal concentrations of fluoroquinolones, trimethoprim, and β-lactams are known to trigger prophage induction in S. aureus, accelerating the dissemination of prophage-encoded virulence factors to avirulent strains. In this study, our first goal was to identify and describe prophages found in S. aureus isolates recovered from CRS patients and to examine their relationship to CRS disease phenotype and severity. We also aimed to determine whether these prophages could produce active reinfecting phage particles under sub-lethal concentrations of commonly used antibiotics and steroids. Furthermore, we explored links between inducible and non-inducible prophage in S. aureus and factors such as biofilm formation, metabolic activity, and CRS disease severity. Finally, we investigated the genomic and phenotypic plasticity of S. aureus and changes in its extracellular proteome following the acquisition of the ϕSa3int prophage, which was one of the most frequently found prophages in CRS with nasal polyp (CRSwNP) patients. To achieve our goals, we first used various computational tools to identify prophage regions in S. aureus genomes (N = 66) primarily isolated from CRS patients' sinonasal cavities. We then detected virulence and antibiotic resistance genes within the prophage regions of these bacteria. To measure the disease severity of CRS patients, we used computed tomography Lund Mackay scores. We determined antibiotic resistance patterns using the broth microdilution method and identified the minimum inhibitory concentration (MIC). Using sub- MIC concentrations of antibiotics and steroids, we induced the prophages and assessed their infectivity, biofilm biomass and metabolic activity in relation to prophage inducibility. Furthermore, we observed the beta-hemolysis activity of the isolates on sheep blood agar to understand its prevalence in human-adapted S. aureus. Moving forward, we then, induced a ϕSa3int prophage from one of the high biofilm-forming isolates (SA333) and transduced it into another Sa3int-prophage-free S. aureus (SA222, relatively low biofilm forming) isolate to obtain a laboratory-generated 'lysogen'. We confirmed the successful integration of ϕSa3int prophage into hlb-gene and stable lysogenic conversion by short- and long-read sequencing. We then compared the growth kinetics, biofilm biomass, and metabolic activity between the parent and the laboratory-generated lysogen by establishing growth curves, crystal violet and resazurin assays. Finally, we identified and quantified exoproteins secreted by parent strains and lysogens using mass spectrophotometry to understand the virulence factors encoded and secreted by ϕSa3int prophage carrying S. aureus. All S. aureus clinical isolates obtained from CRS patients (N = 57) and control patients (N = 9) carried at least one prophage (average = 3.6 prophages/isolate), with prophages contributing up to 7.7% of the bacterial genome. Based on the completeness scores, we found that nearly 85% (56/66) of S. aureus clinical isolates had at least one intact prophage that were likely inducible. Prophages belonging to type 3 integrase (ϕSa3int-type) were the most prevalent (40%), followed by ϕSa2int (14%). The prophages harbored a distinct set of virulence genes: ϕSa3int-group often encoded human immune evasion cluster genes like sak, scn, chp, and sea, while ϕSa2int-group often harbored leukocidins like lukE/D. Intact prophages were more frequently found in S. aureus isolated from CRS with nasal polyp (CRSwNP) patients than in CRS without nasal polyp (CRSsNP) patients (p = 0.0021). Similarly, intact prophages belonging to the ϕSa3int-group were more frequent in CRSwNP than in CRSsNP (p = 0.0008). Spontaneous prophage induction (SPI) was observed in around 26% (17/66) of the S. aureus clinical isolates, while mitomycin C dependent induction was observed in almost 52% (34/66) of the clinical isolates. Most of the S. aureus clinical isolates showing prophage induction harbored at least one intact prophage(s). Exposure of exponentially growing bacteria to sub-inhibitory concentrations of antibiotics enhanced the prophage induction compared to SPI in almost 50% of active lysogens. Among antibiotics tested, ciprofloxacin was the most potent prophage inducer inducing prophages from 51% of the isolates, followed by amoxicillin, doxycycline, mupirocin, clindamycin, and azithromycin, all of which enhanced the release of prophage in > 40% of the isolates. There was no correlation between S. aureus harboring active prophages and inactive prophages with their biofilm biomass and metabolic activity. However, the disease severity score of patients harboring inducible prophage within S. aureus was significantly lower, implying the role of active lysogeny in CRS disease. In addition, beta-hemolysin activity was absent in almost 92% of S. aureus isolated from the sinonasal cavities of chronic rhinosinusitis patients. Integration of a ~43.8 kb ϕSa3int prophage in one of the hemolysin-producing clinical isolates (SA222) down-regulated the beta-hemolysin expression, implying the role of Sa3int-type prophages in the disruption of beta-hemolysin production. There was no change in bacterial growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells, and the metabolic activity in a biofilm after ϕSa3int prophage integration. However, the acquisition of ϕSa3int prophage significantly altered the expression of various secreted proteins, both bacterial and prophage encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory-generated lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3%), including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB), and downregulation of 17 exoproteins (44.7%), including beta-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins are known for human immunomodulation which helps S. aureus escape human innate immunity and cause chronic infection. In summary, our research has expanded the understanding of prophage distribution in S. aureus among patients with chronic rhinosinusitis (CRS) and their potential impact on the development of the disease. We identified diverse types of prophages in S. aureus within a limited geographic area and among a specific population suffering from CRS. This suggests the presence of a range of prophages that contribute to the adaptability and virulence of the bacteria. Our findings also shed light on the prevalence of active lysogeny in clinical S. aureus isolates and the impact of commonly used antibiotics on prophage mobilization. This can affect both virulence and the spread of antimicrobial resistance. Therefore, our research underscores the importance of minimizing the unnecessary use of antibiotics and the potential hazards associated with exposure of bacteria to sub-lethal antibiotics. Such exposure can promote not only antimicrobial resistance but also accelerates the development of virulent strains. Finally, we also caution against poly-lysogeny, which can worsen the pathogenicity of an isolate through an accumulation of auxiliary phage-encoded traits.