Effects of Solid-Medium Type on Routine Identification of Bacterial Isolates by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium. P rompt and accurate identification of bacterial isolates is an objective of the clinical microbiology laboratory and is paramount for patient care. Conventionally this has been achieved using macro-and microscopic observation of morphology and biochemical analysis. These methods often require isolation of individual colonies from polymicrobial cultures and subculture prior to isolate identification. These steps can add significant time to identification of isolates. Molecular methods provide reliable results, though many are expensive to perform, time-consuming, and technically demanding (6). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a rapid, accurate, and cost-effective method of identification, applicable to a wide range of bacterial isolates (9,12,13,16,17,19).A major benefit of MALDI-TOF technology is the ability to obtain identifications using a single colony, eliminating the need for subculture and further incubation. To identify most isolates, a single colony can be picked from solid culture media using a swab or toothpick and smeared directly onto a polished steel target plate for identification (direct method) (1, 9, 14, 17, 18). Alternatively, isolates with formidable cell walls or those that produce excess exopolysaccharide matrix (i.e., mucoid pseudomonads) may require processing through a short (approximately 5-min) form...

Optimizing Identification of Clinically Relevant Gram-Positive Organisms by Use of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System

“…In a previous study, Anderson et al (32) found that MALDI-TOF MS identification was slightly inferior if organisms were grown on CNA agar compared to blood agar. That study tested a collection of 20 staphylococci, while our study assayed a more diverse group of 28 Grampositive organisms, which might account for the differences observed.…”

Optimization of Routine Identification of Clinically Relevant Gram-Negative Bacteria by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and the Bruker Biotyper

“…Using our optimized procedure, the Bruker MALDI Biotyper system made only two erroneous identifications across all studies, (7,26,27). Many clinical specimen types, such as stool and respiratory specimens, are plated to a variety of selective and differential media to minimize the overgrowth of normal flora and facilitate the recovery of pathogens.…”

“…The ability to analyze isolates directly on these medium types, rather than subculturing to enriched medium first, can improve the time it takes to identify these isolates. It is intuitively understood that incubation on different media may alter the MALDI-TOF spectral profile for a given isolate (26,28,29), and others have noted that Hektoen enteric medium results in poor performance or requires a full extraction (13,30). However, we found that this medium type, as well as other selective media, such as MacConkey and OFPBL, reduced the rate of identification but induced no misidentifications, verifying that MALDI-TOF analysis can be performed from primary subcultures on selective agar.…”

Mass spectrometry: a revolution in clinical microbiology?

“…Other problems could also be noted: 1) the use of MS is challenged by the high costs of the instruments and by the long period of maintenance (up to 2 days) requiring another solution during this period; 2) this technology must be completely adapted to the new constraints of diagnostic laboratories, in particular, the traceability of all the tests; 3) Anderson et al has demonstrated the effects of some selective solid-medium type on the rate of identification of bacterial isolates by MS [87] . For example, Staphylococcus spp.…”