Identification of Staphylococcus in samples from patients with periodontitis

by NGS.

Luis Enrique Romero Cruz, Ernesto Arturo Rojas Jiménez, Héctor Martínez
Gregorio, Mario Enrique Díaz Pineda, Alejandro Rodríguez Aguilar, Luis Alberto
Brito Elías, Octavio Hernández Rosas, Miriam Rodríguez Sosa, Nelly Jazmín
Pacheco Cruz, Erick Alan Romero Macías, Sareni Ruiz Montoya, David Sánchez
Marín, María Guadalupe Figueroa Méndez, Alina Uribe García, Claudia Fabiola
Méndez Catalá, Erick Monroy Pérez, Gloria Luz Paniagua Contreras, Felipe Vaca
Paniagua, Clara Estela Díaz Velásquez.
Universidad Nacional Autónoma de México. FES-Iztacala. Avenida de los barrios 1,
los reyes Iztacala C.P. 54090 Tlalnepantla, México.
(55) 56231333 ext. 39860. cdiaz@comunidad.unam.mx
INTRODUCTION
Bacteria are a group of microorganisms that have a high clinical impact due to the
degree of virulence, resistance capacity and the wide range of diseases that cause
them.
Otherwise, periodontitis is an infectious disease that causes the destruction of the
supporting tissues of the tooth. For Mexico it is reported up to a prevalence of 70%
according to the American Academy of Periodontics (AAP, DATE). In periodontal
lesions, several organisms have been identified which are associated with this
disease, we can mention Staphylococcus aureus, Parvimonas micra, Prevotella
intermedia; as well as bacteria of the called red complex, such as Porphyromonas
gingivalis, Tannerella forsythia and Treponema denticola (Fritschi, et al, 2008).
Staphylococcus is a genera of anaerobic facultative, gram positive bacteria that is
characterized by having a microscopic growth that resemble grape clusters, first
described by Rosenbach in 1884 (Todar, 2015). Taxonomically this genus is
grouped in the family Staphilococcaceae and although this family includes three
recognized genera, the genus Staphylococcus is the most clinically important (Holt,
et al, 1989).
In this genera, Staphylococcus aureus is the cause of different serious diseases
such as osteomyelitis, endocarditis and septicemia. However, there are other
species of the same genera of less importance as S. epidermidis, S. Warneris,
which is part of the microbiota present in the skin and are rarely pathogenic.
Currently the most common method for the determination of and diagnosis of
infections by bacteria is performed by different biochemical tests based on the
metabolism of these organisms, however, these tests may not be reliable because
of the plasticity of the metabolism of bacteria. Another aspect that has become
important in recent years is the resistance that some strains have developed
against antibiotics, making it more difficult to treat the diseases they cause (Sherry,
2010).
The use of next-generation sequencing technologies has allowed to study in more
detail the genome of bacteria, in order to understand the molecular mechanisms
that give them resistance to drugs, virulence factors, as well as the interaction and
flow of genetic information through plasmids that allow recombination between
bacteria, creating recombinant and heterogeneous strains. This phenomenon has
been observed even in bacteria of different genera within the same
microenvironment (Chang & Cohen 1974).
GENERAL OBJECTIVE
Determine the species, genes of virulence and resistance, of the genus
Staphylococcus present in periodontal lesions of Mexican patients by massive
sequencing in parallel.
PARTICULAR OBJECTIVES
1. Isolate Staphylococcus strains from periodontal lesions using selective cut
media
2. Isolate gDNA from these cultures and prepare libraries for sequencing
3. Bioinformatic analysis of the sequences
METHODS
Twelve samples were obtained from pharyngeal exudates of patients with
periodontitis, the samples were then seeded by cross-striae method in solid culture
media of blood agar, Sabouraud and S-110 and incubated at 37 ° C for 24 hours.
After confirming the fermentation of mannitol in S-110 medium, the coagulase test
and Gram staining were performed to confirm the presence of Staphylococcus.
The extraction of gDNA was done by affinity column and at the end the
performance, purity and integrity of the DNA was evaluated, using Fluorometry,
nanophotometer and agarose gel electrophoresis. Once the quality was evaluated,
the making of libraries for sequencing was preformed, doing the gDNA
tagmention and adding universal adapters and molecular barcodes. Then, the
quality of the libraries was evaluated using a Bioanalyzer profile
The sequencing was performed using the Illumina myseq platform and the data
generated by the sequencer were evaluated using the FASTQC software. Finally
the data was sent and processed by the Center for Genomics Epidemiology.
REFERENCE
1. Todar, K. (2015). Staphylococcus.
2. Holt, J. G., Williams, S. T., & Holt. (1989). Bergey's manual of systematic
bacteriology, Vol. 4. Lippincott Williams & Wilkins.
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species in vitro: replication and expression of Staphylococcus plasmid genes
in Escherichia coli. Proceedings of the National Academy of Sciences, 71(4),
1030-1034.
4. Sherris, J. (2010). Microbiología médica. K. J. Ryan, & C. G. Ray (Eds.).
McGraw-Hill.
5. Fritschi, B. Z., Albert-Kiszely, A., & Persson, G. R. (2008). Staphylococcus
aureus and other bacteria in untreated periodontitis. Journal of dental
research, 87(6), 589-593.