Rapid Identification of Bacterial Species Directly from Enrichment Broth By MALDI-TOF Mass Spectrometry

Rapid identification of bacterial species causing an infection has shown to have positive impact on the clinical outcome. MALDI-TOF MS is a viable and rapid method for identification of bacterial species not only on agar plates but also directly from positive blood culture bottles and can thus shorten the turnaround time significantly. The aim of this study was to evaluate MALDI-TOF MS on liquid enrichment medium as a possible method for rapid identification of bacterial species by comparing it to routine culturing procedures. The broth was harvested when growth was suspected and analyzed using MALDI-TOF MS. An overall sensitivity of 70.4% was achieved when comparing MALDI-TOF MS and routine procedures. In conclusion, the direct identification from the broth showed a substantial degree of correlation to the routine procedures, where identification was performed from the colonies growing on the agar plates. Biomedical Journal of Scientific & Technical Research Cite this article: Malin J, Johanna Haiko, Baharak S. Rapid Identification of Bacterial Species Directly from Enrichment Broth By MALDI-TOF Mass Spectrometry. Biomed J Sci&Tech Res 10(1)-2018. BJSTR. MS.ID. 001897. DOI: 10.26717/ BJSTR.2018. 10.001897. 2/4 Volume 10Issue 1: 2018 at 35°C and thereafter treated similarly to clinical samples. Sample preparation. According to routine procedures, the bacterial colonies growing on the agar plates were subjected to MALDI-TOF MS analysis, and this result was compared with the direct MALDI-TOF MS result. For direct identification by MALDI-TOF MS, 1ml of FAB was harvested and spun down at 21380g for 1min. Pellet was resuspended and washed in 1ml 0.01M PBS-buffer at PH 7.4 (0.80% NaCl, 0.14% Na2HPO4xH2O, 0.02% KCl, 0.02% KH2PO4) and spun down as above. Supernatant was discarded and 2μl of the pellet was transferred to the stainless-steel target (Bruker Daltonics GmbH) and smeared to achieve even distribution. Sample (either pellet for direct detection or colony for routine detection) was allowed to dry before application of 1μl of 70% formic acid (Sigma Aldrich). Each sample was overlaid with 1μl of HCCA matrix solution (2.5mg α-cyano-4-hydroxycinnamic acid [Bruker Daltonics] suspended in 47.5% LC-MS grade H2O [CHROMASOLVTM], 50% LC-MS grade pure acetonitrile [CHROMASOLVTM], 2.5% pure trifluoracetic acid [Sigma Aldrich]). Each sample was analyzed in duplicate. Interpretation of analysis. The spectra achieved were automatically analyzed against the Bruker database using the software Bio typer 3.0 (Bruker Daltonics GmbH). Scores were interpreted with lower cut-off rates than manufacturer’s recommendations (1.7-3.0) with reference to earlier studies and evaluations [12-15]. Scores ≥1.7 were interpreted as species-level identification and 1.5-1.69 for genus-level identification. Scores <1.5 were interpreted as unreliable identification. Samples resulting in No Peaks Found (NPF) or scores <1.5 were reanalyzed once and the results with higher scores were used. Statistical analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated using the GraphPad Quick Calcs Web site: https:// www.graphpad. com/quickcalcs/clinTest1/. Results A total of 218 samples, both clinical samples (n=178) and clinical isolates (n=40), were analyzed during this study. Within these, a total of 228 bacterial and Candida species were identified during routine analysis, i.e., identification of colonies by MALDI-TOF MS. Of these microorganisms, 77 were identified as gram-negative rods, 33 as gram-positive rods, 99 as gram-positive cocci and 19 as Candida species. All these samples were also analyzed by direct MALDI-TOF MS from FAB, and the results showed 53.3% agreement of gram-negative rods and 59.3% agreement of gram-positive cocci (Table 1). Among gram-positive rods and Candida species, only 27.3% and 10.5% agreement were achieved, respectively. Polymicrobial samples rarely yielded hits for every microorganism present. In just 3/49 cases two unique bacteria were found with hits >1.5, all three with a different amount (2-5) of microbial species present. For polymicrobial samples the probability of detection was 21% less than that of the monomicrobial samples and isolates (Table 2). In no case did a true negative sample yield a positive result but a mismatch was calculated as a type of false positive. Polymicrobial samples yielded 2 false positives, regarded as mismatches, where Staphylococcus sciuri (score 1.60) was identified by direct MALDI-TOF MS instead of Enterococcus Faecium and Candida Albicans which grew on routine culture. Additionally, Enterococcus faecium (score 1.57) was identified by direct MALDI-TOF MS instead of Pseudomonas Aeruginosa and Candida Albicans on routine culture. These false positive results led to marginally lower specificity and PPV (98.4% and 98.2%). Monomicrobial samples and isolates yielded marginally higher rates of sensitivity (70.9%) when including slow growing microorganisms such as Candida Albicans and Propionibacterium Acnes. Sensitivity was markedly increased when excluding these from the calculations (Table 3). Table 1: Microorganisms present in all samples with respective MALDI-TOF MS score. Positive findings Findings scoring ≥2.0-1.5 (%) Findings scoring <1.5 and NPF (%) Mismatch (%) Gram negative rods (n=77) 41 (53.3) 36 (46.8) 1 (1.3) Enterobacteriaceae (n=52) 34 (65.4) 18 (34.6) Other gram negative rods(n=25) 7 (28.0) 18 (72.0) Gram positive rods (n=33) 9 (27.3) 24 (72.7) Gram positive cocci (n=99) 58 (58) 41 (41) 1 (1.0) Enterococcaceae (n=36) 20 (55.6) 16 (44.4) Staphylococcaceae (n=41) 27 (65.9) 14 (35.1) Streptococcaceae (incl. Gemella) (n=22) 11 (50.0) 11 (13.7) Candida species (n=19) 2 (10.5) 17 (89.5) Total (n=228) 101 (44.3) 118 (51.7) 2 (0.9) Table 2: Agreement between routine procedure and direct MALDI-TOF MS on FAB. Monomicrobial Samples, Isolates and Polymicrobial Samples Positive Routine Negative Routine Mono. Incl. P. acnes and Candida albicans Poly Mono+Poly. Incl. P. acnes and Candida albicans All Pos.MALDI on FAB 78 34 112 2 NPF/<cutoff 32 15 47 108 Biomedical Journal of Scientific & Technical Research Volume 10Issue 1: 2018 Cite this article: Malin J, Johanna Haiko, Baharak S. Rapid Identification of Bacterial Species Directly from Enrichment Broth By MALDI-TOF Mass Spectrometry. Biomed J Sci&Tech Res 10(1)-2018. BJSTR. MS.ID. 001897. DOI: 10.26717/ BJSTR.2018. 10.001897. 3/4 Sensitivity 70.9% 69.4% 70.4% Specificity 100.0% 98.2% 98.2% PPV 100.0% 94.4% 98.2% NPV 77.1% 87.8% 69.7% Table 3: Agreement between routine procedure and direct MALDI-TOF MS on FAB, monomicrobial samples and isolates. Monomicrobial Samples and Isolates Positive Routine Negative routine Incl. P. acnes and Candida albicans Excl. P. acnes and Candida albicans All Pos. MALDI on FAB 78 68 0 NPF /<cutoff 32 13 108 Sensitivity 70.9% 84.0% Specificity 100.0% 100.0%