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Frequency, epidemiological, clinical, molecular and laboratorial profile of Staphylococcus aureus isolated from patients with Pneumonia associated with the ventilator in an university hospital in southern of Brazil
Universidade Estadual de Londrina (UEL)
Department of Pathology
Marcia Regina Eches Perugini
Marcia Regina Eches Perugini
Englush, Portuguese, Spanish
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
Bacteria of the genus Staphylococcus aureus are extremely virulent, resistant and are associated with several infections, both community and hospital. Among the strains of S. aureus the MRSA (Methicillin-Resistant Staphylococcus aureus) are often associated with infectious processes. In the hospital environment, it is believed that up to 40% of the cases of pneumonia are related to this pathogen. In this retrospective observational, longitudinal study, 108 samples collected from tracheal secretion from patients diagnosed with ventilator-associated pneumonia (pav) by S. aureus admitted during the years 2015 and 2016 to the university hospital of Londrina will be analyzed.
What is the aim of the project?
Find the genes involved in the resistance process associated with therapeutic failure and compare the laboratory/molecular findings with the clinical evolution of the patient
What techniques and methods are used?
FENOTYPICAL METHODS The samples included in this study were collected by the respiratory physiotherapy team and sent to the microbiology laboratory for infectious diagnosis. Following manipulation and report releasing, the isolates were stored for further studies. Previously stored samples from patients with clinical and laboratorial diagnosis of pneumonia were selected and reactivated in tripticaseine soy broth (TSB) and isolated in salted mannitol agar. Suspensions with a cell density of 0.5 and 2.0 McFarland (approximately 1.5x108 and 6.0x108 colony forming units per mL), measured employing a turbidimeter, were prepared after incubation for 18 – 24 h in an incubator at 37oC. All isolates were identified using automated methods, with Vitek®2 (bioMeriéux-USA), Phoenix® (Becton, Dickinson) and Microscan® (Siemens-California) systems, depending on the analyzed period. The determination of the antimicrobial sensitivity profile was performed for the following drugs: cefoxitin (30μg); eritromicin (15μg); clindamicin (2μg); teicoplanin (30μg); gentamicin (10 μg); amikacin (30 μg); tetracycline (30 μg); linezolid (30 μg); ciprofloxacin (10 μg); rifampicin (5 μg) e sulfamethoxazole + trimetoprim (25 μg). Disc diffusion methodolgy was used and the results were evaluated and interpreted according to what is described on CLSI, 2018. The vancomycin MIC (minimum inhibitory concentration) was determined using e-test® strips (bioMeriéux-USA). The tapes were impregnated with vancomycin in concentrations ranging from 0.16 μg / mL to 256 μg / mL. After incubation for 18-24h at 37oC, the MIC value was evaluated following the manufacturer's recommendations and CLSI, 2018. GENOTYPICAL METHODS The extraction of total DNA (Deoxyribonucleic acid) was performed by enzymatic lysis technique as described by Sambrook, Fritsch and Maniatis (1990). The genotyping of the strains, identification of the CC (cell culture) was executed by RTq-PCR ( Polymerase chain reaction quantitative real time) as described by Lilliebridge et al. (2011). The isolates were submitted to RTq-PCR tests for amplification of six internal fragments, housekeeping (arcC, aroE, gmK, pta294, tpi36, and tpi241). The CC of each sample were determined according to the MLST (Public databases for molecular typing) database (http://saureus.mlst.net) and the methodology proposed by Lilliebridge et al. (2011).
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- If the project is clinical
- the student will take active part in the clinical examination
- If the project is clinical
- the student will be allowed to work with patients
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
It is hoped that the student will develop the test (molecular biology testing), evaluate the medical records and perform a clinical case discussion. The student will see that the data collected will be tabulated and the statistical analysis will be done using the program IBM SPSS 20.0 SOFTWARE (SPSS, CHICAGO, IL, USA) In other words, the student will be involved from the sample to the processing of the results and their respective discussion, in order to guarantee the student a more practical and complete learning.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes. The tutor will indicate books and scientific papers for reading before and during the internship. In addition, the student will also participate in scientific meetings during the time of the internship
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report - The student will prepare an abstract - The student’s name will be mentioned in a future publication - The student will have the opportunity to present the results together with the supervisor at a conference
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Determination and interest in learning. Subjects passed: Microbiology and antimicrobial infection control
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields - Dental medicine students (IADS members)
- CAMPBELL; S. J. et al. Genotypic characteristics of Staphylococcus aureus isolates from a multinational trial of complicated skin and skin structure infections. Journal of Clinical Microbiology; v. 46; n. 2; p. 678–684; 2008.
- JOSE; J. D. ; LUCIO; D. S. ; PERUGINI; M. R. E. ; STIPP-ABE; A. T. ; FONTANA; L. M. S. ; PERUGINI; V. H. ; CAPOBIANGO; J. D. . Prevenção de pneumonia associada à ventilação mecânica em neonatologia. Journal of Infection Control ; v. 4; p. 1-5; 2015.
- LILLIEBRIDGE; R. A. et al. The utility of high-resolution melting analysis of SNP nucleated PCR Amplicons-an MLST based Staphylococcus Aureus typing scheme. PLoS ONE; v. 6; n. 6; 2011.
- MILHEIRIÇO; C.; OLIVEIRA; D.C.; de LENCASTRE; H. Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob Agents Chemother 2007;51:3374-7.
- PERUGINI; V. H. ; JOSE; J. D. ; LUCIO; D. S. ; FONTANA; L.M.S. ; BELEI; R. A. ; CAPOBIANGO; J. D. ; RIBEIRO; M. A. G. ; CARRARA-MARRONI; F. E. ; VESPERO; E. C. ; STIPP-ABE; A. T. ; PERUGINI; M. R. E. . Impacto de um bundle nas taxas de pneumonia associada à ventilação mecânica (PAV) em uma unidade de terapia intensiva pediátrica em Londrina-PR. SEMINA. CIÊNCIAS BIOLÓGICAS E DA SAÚDE (ONLINE) ; v. 36; p. 259-266; 2015.
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