Mannitol-Salt Agar and Slide Coagulase Test: Are they ... · Mannitol-Salt Agar and Slide Coagulase...
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Mannitol-Salt Agar and Slide Coagulase Test: Are they enough to identify Staphylococcus aureus? Matilde Teles 1 , Marina Majar 1 , Fernando Branca 1 , Carmén Iglesias 1 , Alexandra Estrada 1 1 Clinical Pathology – Hospital de Braga - Portugal Antibiotics have been associated with an improvement in the treatment of infectious diseases but its misuse has led to the development of resistant and multi-resistante strains. So it has become challenging to treat infectious diseases. (1) It is important to promptly identify the pathogen and to test its susceptibility to antibiotics in order to correctly treat the disease. Staphylococcus spp. have the ability to grow on 10% NaCl. They can also be coagulase-positive and usually pathogenic (for example, Staphylococcus aureus), or coagulase-negative which are mostly part of human microbiome (for example, Staphylococcus haemolyticus). (2) The growth and production of yellow colonies with a yellow halo on Mannitol-Salt Agar is described as a presumptive way of identify S. aureus. It is also described as a differentiator between Coagulase-Positive and Coagulase-Negative strains. (3) Slide Coagulase Test is a fast test that is usually used to identify S. aureus, instead of Tube Coagulase Test because it is known for being accurate and allows early release of results.(4) (1) PAIVA, José Artur et al., .Programa de Prevenção e Controlo de Infecções e Resistência aos Antimicrobianos – 2014,. Direcção Geral de Saúde, Lisboa: Novembro de 2014. (2) SCHLEIFER, Karl-Heinz, BELL, Julia A.(2009). Family VIII. Staphylococcaceae. In BRENNER, Don, KRIEG, Noel, STALEY, James. Bergey’s Manual of Systematic Bacteriology, Volume III. Springer, pp. 392-421. (3) Shittu, A et al. (2006). Identification and molecular characterization of mannitol salt positive, coagulase-negative staphylococci from nasal samples of medical personnel and students. Journal of Medical Microbiology. 55, 317-324. (4) Griethuysen, A. et al. (2001). International Multicenter Evaluation of Latex Agglutination Tests for Identification of Staphylococcus aureus. Journal of Clinical Microbiology . 39, 86-89. (5) Kateete, D. et al. (2010). Identification of Staphylococcus aureus: Dnase and Mannitol Salt agar improve the efficiency of the tube coagulase test. Annals of Clinical Microbiology and Antimicrobials. 9, 23-29. Introduction Materials and Methods Results Discussion Bibliography Hand Exsudate Sample Seeding • Blood Agar • Mannitol-Salt Agar Cultural Exam • S. aureus? • Pastorex® Staph Plus (BioRad) Vitek2® • Species identification • Antimicrobial Susceptibility MicroScan WalkAway® 96 Plus • Confirmation Tube Coagulase Test • Discriminative • Coagulase positive VS Coagulase negative PCR (Roche®) • Definitive result Colonies Growth on Solid Culture Media Pastorex® Staph Plus Vitek2® AMY - PIPLC - dXYL - ADH1 + BGAL - AGLU - APPA - CDEX - AspA - BGAR - AMAN - PHOS - LeuA - ProA - BGURr - AGAL - PyrA + BGUR - AlaA - TyrA - dSOR - URE - POLYB - dGAL + dRIB+ lLATk + LAC + NAG + dMAL + BACI + NOVO - NC6.5 + dMAN + dMNE - MBdG - PUL - dRAF - O129R + SAL - SAC + dTRE + ADH2s - OPTO + MicroScan WalkAway® 96 Plus CV - NOV - VP - BE - PGT + LAC + ARA - INU - RAF - MS + PGR - OPT + PYR + URE - TRE + BAC + NACL + RBS - NIT + IDX - PHO - ARG + MAN + MNS - HEM PRV - SOR - Left plate: Growth of big greyish, beta-hemolytic colonies on Blood Agar Right plate: Growth of Yellow colonies surrounded by an yellow halo on Mannitol-Salt Agar. Left: Negative control s h o w i n g n o agglutination. Right: Sample result, showing agglutination which is interpreted as a Coagulase-Positive sample. Tube Coagulase Test PCR Species Identification: Staphylococcus haemolyticus (99% probability) Species Identification: Staphylococcus haemolyticus (99,99% probability) Lower tube: Control with Methicillin Susceptible S. aureus , showing Coagulase Positive activity Middle tube: Control with Methicillin Resistant S. aureus, showing Coagulase Positive activity Upper tube: Sample, showing Coagulase Negative activity The nucleotide sequence obtained presented 100% homology with the sequence of S, haemolyticus JCM 2416 (seq. NR_118998.1). Although MannitolSalt Agar is used to presump6vely S. aureus (3), some Staphylococcus CoagulaseNega6ve have the ability to ferment mannitol and, in this way, they can lead to false posi6ve results with this method. The Pastorex® Staph Plus Test is easy to execute allowing rapid results on S. aureus but one study showed that S. haemoly1cus can lead to falseposi6ve results by this method, probably due to the produc6on of Capsular Polysaccharide 8. (4) As previously wriIen in the literature, no phenotypic test alone can iden6fy S. aureus. Automa6c methods and PCR may be needed to confirm the results, once the difference has important clinical implica6ons. (4)(5)
Transcript of Mannitol-Salt Agar and Slide Coagulase Test: Are they ... · Mannitol-Salt Agar and Slide Coagulase...
Mannitol-Salt Agar and Slide Coagulase Test: Are they enough to identify Staphylococcus aureus?
Matilde Teles1, Marina Majar1, Fernando Branca1, Carmén Iglesias1, Alexandra Estrada1
1 Clinical Pathology – Hospital de Braga - Portugal
Antibiotics have been associated with an improvement in the treatment of infectious diseases but its misuse has led to the development of resistant and
multi-resistante strains. So it has become challenging to treat infectious diseases. (1) It is important to promptly identify the pathogen and to test its
susceptibility to antibiotics in order to correctly treat the disease.
Staphylococcus spp. have the ability to grow on 10% NaCl. They can also be coagulase-positive and usually pathogenic (for example, Staphylococcus aureus),
or coagulase-negative which are mostly part of human microbiome (for example, Staphylococcus haemolyticus). (2) The growth and production of yellow
colonies with a yellow halo on Mannitol-Salt Agar is described as a presumptive way of identify S. aureus. It is also described as a differentiator between
Coagulase-Positive and Coagulase-Negative strains. (3) Slide Coagulase Test is a fast test that is usually used to identify S. aureus, instead of Tube Coagulase
Test because it is known for being accurate and allows early release of results.(4)
(1) PAIVA, José Artur et al., .Programa de Prevenção e Controlo de Infecções e Resistência aos Antimicrobianos – 2014,. Direcção Geral de Saúde, Lisboa: Novembro de 2014. (2) SCHLEIFER, Karl-Heinz, BELL, Julia A.(2009). Family VIII. Staphylococcaceae. In BRENNER, Don, KRIEG, Noel, STALEY, James. Bergey’s Manual of Systematic Bacteriology, Volume III. Springer, pp. 392-421. (3) Shittu, A et al. (2006). Identification and molecular characterization of mannitol salt positive, coagulase-negative staphylococci from nasal samples of medical personnel and students. Journal of Medical Microbiology. 55, 317-324. (4) Griethuysen, A. et al. (2001). International Multicenter Evaluation of Latex Agglutination Tests for Identification of Staphylococcus aureus. Journal of Clinical Microbiology. 39, 86-89. (5) Kateete, D. et al. (2010). Identification of Staphylococcus aureus: Dnase and Mannitol Salt agar improve the efficiency of the tube coagulase test. Annals of Clinical Microbiology and Antimicrobials. 9, 23-29.
Introduction
Materials and Methods
Results
Discussion
Bibliography
Hand Exsudate Sample
Seeding
• Blood Agar • Mannitol-Salt
Agar
Cultural Exam
• S. aureus? • Pastorex®
Staph Plus (BioRad)
Vitek2®
• Species identification
• Antimicrobial Susceptibility
MicroScan WalkAway®
96 Plus
• Confirmation
Tube Coagulase
Test
• Discriminative • Coagulase
positive VS Coagulase negative
PCR (Roche®)
• Definitive result
Colonies Growth on Solid Culture Media
Pastorex® Staph Plus
Vitek2®
AMY - PIPLC - dXYL - ADH1 + BGAL - AGLU - APPA - CDEX - AspA - BGAR - AMAN - PHOS - LeuA - ProA - BGURr - AGAL - PyrA + BGUR - AlaA - TyrA - dSOR - URE - POLYB - dGAL + dRIB+ lLATk + LAC + NAG + dMAL + BACI + NOVO - NC6.5 + dMAN + dMNE - MBdG - PUL - dRAF - O129R + SAL - SAC + dTRE + ADH2s - OPTO +
MicroScan WalkAway® 96 Plus
CV - NOV - VP - BE - PGT + LAC +
ARA - INU - RAF - MS + PGR - OPT +
PYR + URE - TRE + BAC + NACL + RBS -
NIT + IDX - PHO - ARG + MAN + MNS -
HEM PRV - SOR -
Left plate: Growth of big greyish, beta-hemolytic colonies on Blood Agar
Right plate: Growth of Y e l l o w c o l o n i e s
surrounded by an yellow halo on Mannitol-Salt
Agar.
Left: Negative control s h o w i n g n o agglutination.
Right: Sample result, showing agglutination
which is interpreted as a C o a g u l a s e - P o s i t i v e
sample.
Tube Coagulase Test PCR
Species Identification: Staphylococcus haemolyticus (99% probability) Species Identification: Staphylococcus haemolyticus (99,99% probability)
Lower tube: Control with Methic i l l in Susceptible S. aureus, showing Coagulase Positive activity
Middle tube: Control with Methicillin Resistant S. aureus, showing Coagulase Positive activity
Upper tube: Sample, showing Coagulase Negative activity
The nucleotide sequence obtained presented 100% homology with the sequence of S, haemolyticus JCM 2416 (seq. NR_118998.1).
Although Mannitol-‐Salt Agar is used to presump6vely S. aureus (3), some Staphylococcus Coagulase-‐Nega6ve have the ability to ferment mannitol and, in this way, they can lead to false posi6ve results with this method. The Pastorex® Staph Plus Test is easy to execute allowing rapid results on S. aureus but one study showed that S. haemoly1cus can lead to false-‐posi6ve results by this method, probably due to the produc6on of Capsular Polysaccharide 8. (4)
As previously wriIen in the literature, no phenotypic test alone can iden6fy S. aureus. Automa6c methods and PCR may be needed to confirm the results, once the difference has important clinical implica6ons. (4)(5)
