Rapid identification and antimicrobial susceptibility testing directly from blood cultures Mauro M.V., Dodaro S., Perugini D., Greco F., Tenuta R., Varisano L., Giraldi C. Microbiology and Virology Department, «SS Annunziata Hospital», Cosenza - Italy

INTRODUCTION AND PURPOSE The bacterial suspensions were dissolved in saline E-test: direct method versus standard procedure Sepsis is a health emergency and a collaboration solution at a concentration of 0.5 Mc Farland. This For the antimicrobial susceptibility by direct method in between the microbiologist and clinicians is necessary inoculum was used on Vitek 2 for microorganism Gram-negative strains (reading at 6 hours), in respect to for a successful outcomes of patients suffering from identification and susceptibility and were tested against the antimicrobial susceptibility evaluated with disk bacteraemia. Rapid diagnosis of the etiology of different antibiotics with disk diffusion methodology on diffusion by subculture we have obtained a correlation infection is imperative, with mortality increasing nearly Muller Hinton agar (E-test) using a shorter incubation equal to 90.5%. We detected 3 mE which concern the 8% for every hour of inappropriate antimicrobial time (6 h for Gram negative isolates and 8 h from MIC of Piperacillin/tazobactam for n.1 E.coli and n.2 therapy administrated to patients with sepsis. Gram positive isolates). The drugs tested in the study K.pneumoniae which was higher than a dilution by rapid figure 3a figure 3b Therefore it is evident that results must be produced were: Piperacillin/tazobactam, Cefotaxime, Gentamicin, method in respect to subcultures, the interpretation of rapidly. The Turn Around Time (TAT) of blood cultures Amikacin, Ertapenem, Imipenem, Meropenem and sensitivity was however confirmed (fig 3a, 3b). For the (BC) is influenced by: 1) the time that a positive signal Colistin for Gram negative bacteria, Cefoxitin, antimicrobial susceptibility by direct method in 20 is emitted by the automatic instrument; 2) from the Vancomycin, Daptomicyn and Linezolid for S.aureus and 17 Enterococcus spp (reading at 8 hours), time necessary for bacterial identification (ID) and Staphylococcus aureus, Ampicillin, Vancomycin and in respect to the antimicrobial susceptibility evaluated antimicrobial susceptibility test (AST). To reduce TAT, Teicoplanin for Enterococcus spp. These results were with disk diffusion by subculture, a concordance equal to in our laboratory, early organism identification and compared to the standard procedure: VITEK 2 and 100% was found (fig. 4a, 4b). detection of antimicrobial resistance directly from disk diffusion by subcultures. The following error blood cultures have been introduced (fig. 1). Our goal classes have been established: Very Major Error figure 4a figure 4b is to give information to the clinicians as soon as (VME) variation of interpretation for S to R, Major Error possible so that an appropriate antimicrobial therapy (ME) variation of interpretation for R to S, mE (minor CONCLUSIONS can be given to the patient which leads to a positive Error) not included in the first two categories. The long TAT which characterizes traditional blood impact upon patient care because the mortality rate is culture methods encourages the microbiologist to decreased, the period of hospitalization is reduced, RESULTS introduce rapid diagnostic techniques which will bring and overall hospital-associated costs are also reduced. We analyzed 220 blood cultures from which we isolated to a correct therapeutic intervention. the following microorganisms: n. 33 E.coli, n. 15 Our study shows that performing identification and MATERIALS AND METHODS K.pneumoniae, n. 3 P.mirabilis, n. 3 S.maltophilia, n. 2 antibiotic susceptibility directly from blood cultures with A total of 220 monomicrobial blood cultures, including E.cloacae, n. 2 E.aerogenes, n. 2 A.baumannii, n. 2 VITEK2 provided excellent results for Gram-negative 71 Gram negative and 149 Gram positive isolates, R.picketii, n. 1 A.hydrophila, n. 1 P.aeruginosa, n. 1 strains but it is not reliable for Gram-positive strains. were detected by direct methods and standard C.braakii, n. 1 Salmonella group B, n. 1 C.jejuni, n. 20 The antibiotic susceptibility testing evaluated with disk procedure. Briefly, the direct method consists in S.aureus, n. 112 CoNS, n. 17 Enterococcus spp., n. 3 diffusion directly from blood cultures provided excellent drawing 8 ml of BC broth which were centrifugated in a anaerobic microorganims, n.1 S.pneumoniae (table 1). results in Gram-negative and Gram-positive strains. tube with a separator gel at 3000 rpm for 15 minutes. table 1 Currently, considering the results obtained, our VITEK 2: direct method versus standard procedure workflow is based upon reporting quickly to the clinical Gram-negative: 68 of 71 (95.7%) Gram-negative rods . the results of the microscopic examination which were correctly identified at the species level and provides guidance on the drugs to be tested by E-test. antimicrobial susceptibility by VITEK2 performed This allows, within 12-24 hours, to define the directly from positive blood culture. The 3 strains not microorganism identification (Gram positive or Gram identified were: n.1 E.cloacae, n. 1 E.coli and n. 1 negative) and Antimicrobial Susceptibility Test R.pickettii, therefore it was not possible to evaluate correlated, guiding the clinician towards a timely and AST. targeted therapy. Our systematic approach of rapid Gram-positive: none of Gram-positive bacteria have diagnostic methods reduces the TAT that, in serious been identified by VITEK2 performed directly from pathologies like sepsis, is very important to ensure the positive blood culture therefore it was not possible positive outcome to the patient. figure 1 evaluate AST.(fig. 2). figure 2 For further information send mail to c.giraldi54@gmail