Study of the ultrastructure of Enterococcus faecalis and Streptococcus mutans incubated with salivary antimicrobial peptides

Abstract Objectives Enterococcus faecalis has been associated with root canal infections, while Streptococcus mutans has a central role in the etiology of dental caries. One of the main reasons of endodontic failure has been associated to the presence of E. faecalis and the formation of biofilms. S. mutans inhabits the oral cavity, specifically the dental plaque, which is a multispecies biofilm formed on the hard surfaces of the tooth. The biofilm formation is the main factor determining the pathogenicity of numerous bacteria. Natural antimicrobial peptides in the saliva protect against pathogenic bacteria and biofilms. The aim of this study was to assess the ultrastructural damage induced by salivary peptides in bacteria involved in biofilms has not been previously studied. Material and methods Enterococcus faecalis and S. mutans incubated with cystatin C, chromogranin A, or histatin 5 were morphologically analyzed and counted. The ultrastructural damage was evaluated by transmission electron microscopy (TEM). Results A decrease in bacterial numbers was observed after incubation with cystatin C, chromogranin A, or histatin 5, compared to the control group (P <  0.001). Ultrastructural damage in E. faecalis and S. mutans incubated with salivary peptides was found in the cell wall, plasma membrane with a decreased distance between the bilayers, a granular pattern in the cytoplasm, and pyknotic nucleoids. Conclusions This study demonstrated that salivary peptides exert antibacterial activity and induce morphological damage on E. faecalis and S. mutans. Knowledge on the ultrastructural damage inflicted by salivary antimicrobial peptides on two important bacteria causing dental caries and root canal infections could aid the design of new therapeutic approaches to facilitate the elimination of these bacteria.

Enterococci can resist various intracanal treatment procedures, (Hancock et al., 2001;Sundqvist, Figdor, Persson, & Sjögren, 1998), this is attributed to the expression of different virulence factors, (Sedgley, Lennan, & Appelbe, 2005) enterococci invade dentinal tubules, (Portenier, Waltimo, & Haapasalo, 2003), to resist alkaline pH (Sedgley, Molander, & Flannagan, 2005), and possess the ability to form biofilms (Svensater & Bergenholtz, 2004). In fact, biofilms are the major cause for primary and secondary root canal infections and therefore, the success of endodontic treatment relies on their effective eradication (Nair, 2006). The National Institute of Health estimates that 60% of all human bacterial infections are due biofilms (Lewis, 2001). Biofilms are bacterial communities associated to surfaces and surrounded by an extracellular matrix (Donlan, 2001), that protects cells from antibiotics and immune cell attack Stewart (2002Stewart ( , 2015. Bacteria readily develop resistance to antimicrobials by several mechanisms, including mutations, activation of efflux pumps, and through the enzymatic inactivation of the antimicrobials (Munita & Arias, 2016). Bacteria that are not resistant to antibiotics can be tolerant by forming persistent biofilms, leading to chronic infections (Lebeaux, Ghigo, & Beloin, 2014). The World Health Organization (WHO) has considered that antibiotic resistance has become a critical public health concern that without coordinated proactive actions among all countries, by 2050, will cause more deaths than cancer (WHO, 2000). Antimicrobial peptides (AMPs) are promising candidates for the treatment of oral infections caused by biofilms, such as caries, periodontitis, implant-associated infections and root canal infections (Lijun et al., 2011;Liu, Xu, Huo, Wei, & Ling, 2014;Mohamed, 2013;Hua et al., 2010).
AMPs effectively kill bacteria, by multifactorial mechanisms, targeting different subpopulations in the biofilms. Moreover, they also can interrupt several stages of biofilm formation, including cellsubstrate adhesion and biofilm maturation, and can also kill bacteria in mature multi-species biofilms promoting their detachment (Pletzer, Coleman, & Hancock, 2016). Furthermore, AMPs, have attracted much attention for their potential to be applied as antimicrobial agent, due their potent antimicrobial activities against a broad spectrum of microorganisms and also due their low bacterial resistance (Aoki, Kuroda, & Ueda, 2012;Guaní-Guerra, Mendoza, Lugo-Reyes, Santos-SO, & Ter an, 2010;Jenssen, Hamill, & Hancock, 2006;Parachin, Mulder, Viana, Dias, & Franco, 2012). Natural AMPs molecules are found in the oral cavity and exert antimicrobial activities against oral pathogenic bacteria and biofilms (da Silva et al., 2012). Despite the high microbial load in the oral cavity, abrasions, cuts, and minor surgical procedures rarely lead to infections, indicating that the host's defense mechanisms, including AMPs, are highly effective (Zasloff, 2002).
In the current study, we analyzed the ultrastructural damage in E. faecalis and S. mutans induced by salivary peptides on bacteria involved in the production of biofilms. We hypothesized that cystatin C, chromogranin A, or histatin 5 will effectively kill bacteria, allowing them to target different subpopulations of biofilms.

| Bacteria incubation with cystatin C, histatin C, or chromogranin A
For experimental and control group assays, 50 μl of E. faecalis and S. mutans (2 × 10 5 CFU/per well) in PBS 7.2 was inoculated into 60 wells of microtiter plates containing the following: Enterococcus faecalis: Growth inhibitory effects of the peptides in E. faecalis and S. mutans was determined by recording the colony forming units (CFU). Microtiter plates were incubated at 37 C, with shaking for 1 hr (Mackay, Denepitiya, Iacono, Krost, & Pollok, 1984). In order to evaluate the bacterial viability, 10 μl of the samples were diluted 1:1000, S. mutans and E. faecalis were cultured on Mueller Hinton Agar (BD Bioxon Milan, Italy) under microaerophilic, conditions and facultative anaerobiosis respectively, and incubated at 37 C during 24 hr.
After that CFU/ml was determined.

| Transmission electron microscopy
In order to determine changes in the ultrastructure of bacteria induced by the peptides, a suspension of 3 × 10 6 CFU was incubated

| Specimens analysis by electron microscopy
Changes in cell walls and plasma membranes of E. faecalis of S. mutans incubated with cystatin C, histatin 5, or chromogranin C and with cystatin C and chromogranin C, respectively, were evaluated using approximately 100 bacteria per condition at a magnification of 7500×. Data were obtained using the image processing package Fiji/ ImageJ. The width of the bacterial cell wall and the distance between bilayers in the plasma membrane were determined.

| Statistical analysis
Statistical analysis was performed using the Instat-statistical software  The study of the ultrastructural damage in bacteria involved in the formation of biofilms, induced after their incubation with salivary peptides, showed extensive damage in different bacterial structures. This knowledge would allow for the design of compounds addressed at specific targets; for instance, we reported in this study that cystatin A, chromogranin A, and histatin 5 induce pyknotic nucleoids, a mechanism resembling an apoptosis-like death in E. feacalis and in S. mutans.
We observed breaks in the cell wall and in the plasma membrane of S. mutans and E. faecalis, these breaks could allow the peptide to enter the bacterial cytoplasm causing extrusion of the cytoplasmic content, which could partially explain the death of the bacteria. In E. faecalis and S. mutans, as well as in other Gram-positive bacteria, the primary interrelation of the peptide would occur between anionic peptides and teichoic acid. After this primary interaction, the peptides bind to the cell surface allowing their subsequent binding, which enables them to pass through the external membranes (teichoic and lipoteichoic acids) and interact with the bacterial plasma membrane (Brogden, 2005). Different mechanisms have been proposed, such as the perpendicular orientation of the peptide followed by penetration into the cell membrane, which leads to the formation of transmembrane pores and cell death due to fluid loss and disruption of the cell membrane (Lee, Chen, & Huang, 2004).However, the mechanisms by which salivary peptides execute their antibacterial activity is not well understood.
Our results showed granular patterns in the cytoplasm, pyknotic nucleoids, total lysis of bacteria, loss of membrane integrity, detachment of peptidoglycan and swollen cytoplasm with a decrease in electro-density in S. mutans and E. faecalis incubated with the

| CONCLUSIONS
The present study showed that salivary peptides induce extensive damage in E. faecalis and S.s mutans. Our findings support the hypothesis that the use of antimicrobial peptides as therapeutic alternatives are valuable tools for the control of these bacteria.

ACKNOWLEDGMENTS
This research was funded by from PAPITT, DGAPA, UNAM, Mexico City, grant #IN218419 and partially by Universidad Anahuac México Campus Norte. Ethical Approval was given by the Ethics committee of the Medicine Faculty UNAM with the reference number C54-11.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.