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 Introduction

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)

Materials and Methods

Hand MicroScan Tube Cultural PCR Exsudate Seeding Vitek2® WalkAway® Coagulase Exam (Roche®) Sample 96 Plus Test • Blood Agar • S. aureus? • Species • Confrmation • Discriminative • Defnitive result • Mannitol-Salt • Pastorex® identifcation • Coagulase Agar Staph Plus • Antimicrobial positive VS (BioRad) Susceptibility Coagulase negative

Results Colonies Growth on Solid Culture Media Pastorex® Staph Plus

Left plate: Growth of big Left: Negative control greyish, beta-hemolytic s h o w i n g n o colonies on Blood Agar agglutination. Right plate: Growth of Right: Sample result, Y e l l o w c o l o n i e s showing agglutination surrounded by an yellow which is interpreted as a halo on Mannitol-Salt C o a g u l a s e - P o s i t i v e Agar. sample.

Vitek2® MicroScan WalkAway® 96 Plus

AMY - PIPLC - dXYL - ADH1 + BGAL - AGLU - APPA - CDEX - AspA - BGAR - AMAN - PHOS - CV - NOV - VP - BE - PGT + LAC + LeuA - ProA - BGURr - AGAL - PyrA + BGUR - ARA - INU - RAF - MS + PGR - OPT + AlaA - TyrA - dSOR - URE - POLYB - dGAL + PYR + URE - TRE + BAC + NACL + RBS - dRIB+ lLATk + LAC + NAG + dMAL + BACI + NOVO - NC6.5 + dMAN + dMNE - MBdG - PUL - NIT + IDX - PHO - ARG + MAN + MNS - dRAF - O129R + SAL - SAC + dTRE + ADH2s - HEM PRV - SOR - OPTO +

Species Identifcation: Staphylococcus haemolyticus (99% probability) Species Identifcation: Staphylococcus haemolyticus (99,99% probability)

Tube Coagulase Test PCR

Lower tube: Control with Methicillin Susceptible S. aureus, showing Coagulase Positive activity The nucleotide sequence obtained presented 100% homology with the sequence of Middle tube: Control with Methicillin Resistant S, haemolyticus JCM 2416 (seq. NR_118998.1). S. aureus, showing Coagulase Positive activity Upper tube: Sample, showing Coagulase Negative activity

Discussion

Although Mannitol-Salt Agar is used to presumpvely S. aureus (3), some Staphylococcus Coagulase-Negave have the ability to ferment mannitol and, in this way, they can lead to false posive 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. haemolycus can lead to false-posive results by this method, probably due to the producon of Capsular Polysaccharide 8. (4) As previously wrien in the literature, no phenotypic test alone can idenfy S. aureus. Automac methods and PCR may be needed to confirm the results, once the difference has important clinical implicaons. (4)(5) Bibliography

(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.