Deisy Tatiana Sánchez ZapataManuela Rincón MuñozFacultad de Medicina

2013

Micrografía electrónica de barrido de Staphylococcus aureus resistente a meticilina (MRSA, marrón), rodeada de restos celulares.

Gram-positive bacteria.

Round (cocci)- grape like clusters.

40 different species• 9 2 subspecies• 1 3 subspecies

Staphylococci grows on almost every bacteriological Medium.

Facultative anaerobic cocci.

Growth rate 5 - 25 °C.

Catalase.2 H2O2  2 H2O + O2

Presence of B lactamase.

Facultative anaerobic Gram-positive coccal bacterium.

S. aureus reproduces asexually by binary fission.

Catalase-positive differentiate

Responsible for many infections, it can also be a commensal. nosocomial.

It can survive from hours to months dry environmental surfaces.

Infection hyaluronidase destroys tissues.

Skin and mucous membrane infections benign

Folliculitis, furunculosis, conjunctivitis, cellulitis, deep abscesses, osteomyelitis,

meningitis, sepsis, endocarditis, pneumonia

Narrow-spectrum beta-lactam antibiotic of the penicillin class.

Previously used to treat infections gram-positive bacteria.

Replaced by Oxacillin, flucloxacillin, dicloxacillin.

Inhibition synthesis of bacterial cell walls.

Inhibits cross-linkage between the linear peptidoglycanpolymer chains that make up a major component of the cell wall of Gram-positive bacteria.

Binds to transpeptidase used to cross-link in peptidoglycan synthesis.

Resistance difficult treatment dangerous.

Characteristics

• Performance exponential• Duration• Specificity high• Detection capability det. Single DNA molec.• Fidelity clone w/out mutations

Molecular diagnosis for MRSA nasal

Rapid detection and screening of MRSA

Colonization

No. cepas de referencia 16S rRNA un mec A b luk S fem A Control interno

Staphylococcus spp. cepas ( n  = 7)1. S. aureus (ATCC 33591) + + - + +2. S. aureus (ATCC 43300) + + - + +3. S. aureus (ATCC 25923) d + - + + +4. S. aureus (ATCC 49775) + - + + +5. S. aureus (ATCC 51153) e + - - + +6. S. epidermidis (ATCC 14990) + - - - +7. ECN resistentes a la meticilina e + + - - +

Las bacterias Gram-positivos ( n  = 8)1. Streptococcus spp. Grupo A (ATCC 19615) e - - - - +

2. Streptococcus spp. Grupo B (ATCC 12401) e - - - - +

3. Streptococcus spp. Grupo G e - - - - +4. Bacillus subtilis (ATCC 6633) e - - - - +5. Listeria monocytogenes (ATCC 7644) e - - - - +6. Enterococcus faecium LMG 16192 c - - - - +7. Enterococcus faecalis (ATCC 29212) e - - - - +8. Corynebacterium spp. correo - - - - +

Patógenos entéricos gram-negativos ( n  = 15)1. Escherichia coli (EHEC) e - - - - +2. E. coli (EPEC) e - - - - +3. E. coli (ETEC) e - - - - +4. Klebsiella pneumoniae (ATCC 10031) e - - - - +5. Shigella sonnei (ATCC 25931) e - - - - +6. Shigella flexneri (ATCC 12022) e - - - - +7. Shigella boydii (ATCC 9207) e - - - - +8. Proteus mirabilis (ATCC 29245) e - - - - +9. Salmonella typhi correo - - - - +10. Pseudomonas aeruginosa (ATCC 27853) e - - - - +

11. Yersinia enterocolitica (ATCC 23715) e - - - - +12. Vibrio cholerae (O1 clásica) e - - - - +13. Citrobacter freundii (ATCC 8090) e - - - - +14. Gardnerella spp. correo - - - - +15. Candida albicans (ATCC 10231) e - - - - +

Primers Secuencia del cebador (5'-3 ')

Objetivo Gene Gen Banco número de

acceso

Tamaño del producto

16S ARNr-F GCA AGC ATC GTT CGG AAT T

16S rRNA D83356 597 pb

16S ARNr-R CTT AAT GAT GGC AAC TAA GC

mec A-F ACG TCA AGT AGA TGC ATA TAA

mec A NC_003923M 293 pb

mec A-R CTT TTA AGT TCT GCG ATT GC

Luks -F CAG GAG GTA ATG TTG CAT TT

luk S AB186917 151 pb

Luks -R ATG TCC AGA CAT TAA TTT ACC

fem A-F CGA TCC ATA TTT ACC ATA TCA

fem A CP000255 450 pb

fem A-R ACG ATC CTC TTC GTT TAG TT

IC-F AGC GTC TGT CAT GAG A

hem M AF227752 759 pb

IC-R ATT CTC AGA TAT GTG TGG

Molde de ADN, que van desde 100 ng a 1 pg.

REFERENCE

WHAT THEY SAID? AGREE DESAGREE

 Rossney , Herra

and et al.

“The Thermostabilized PCR simpler and easier to use than real-time PCR”

Klatser , Kuijper and et al.

“The thermostabilized PCR has been used similarly for mycobacteria and Salmonella typhimurium”

Gillet , Issartel and et al.

“The Luks gene partly encodes PVL toxin, which is the main virulencefactor responsible for nosocomial “

Zhang K, McClure and et al.

“Detection of PVL toxin has been described and has proved useful for identifying strains of MRSA often associated with epidemics”

• The molecular nasal diagnosis to identify MRSA , is important for the epidemiological level especially in nosocomial diseases.

• The thermostabilized PCR assay has many advantages: does not require cold chain, the less margin for error, makes a high-performance rapid analysis. The thermostabilized PCR assay is a very useful technique to identify MRSA one important pathogen in nosocomial infections

• Molecular methods like PCR facilitate early detection of MRSA patients and helps in infection control and prevention.

• Comparing other methods like chromogenic media with molecular methods explain why molecular methods take de lead in the implementation of antibiotics and reduced hospital bed stays.

Gracias….