Review
Use of MALDI-TOF mass spectrometry for identification of bacteria that are difficult to culture

https://doi.org/10.1016/j.mimet.2012.10.014Get rights and content

Abstract

Rapid and reliable detection and identification of bacterial species are necessary for diagnosis and efficient treatment. Until recently, bacterial identification in clinical laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The identification of anaerobic bacteria, fastidious and slow growing bacteria using conventional methods is time consuming, expensive and complicated. Many anaerobes grow poorly or are nonreactive in most diagnostic systems. Unambiguous diagnosis of active tuberculosis is a time-consuming process, requiring as long as 12 weeks for positive identification of the organism. This long time frame presents challenges for case identification. Early identification of pathogenic bacteria is very important for the disease control. Recently, bacteriologists have focused their attention on the use of mass spectrometry (MS) for bacterial identification, especially Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF). Use of MALDI-TOF-MS is described in this review, with a special emphasis on the successful identification of groups of bacteria, which are difficult to culture. MALDI-TOF-MS is a powerful, rapid, precise, and cost-effective method for identification of intact bacteria, compared to conventional phenotypic techniques or molecular biology. Our review suggests that identification of anaerobes, fastidious bacteria and slow growing bacteria, has been improved by the arrival of MALDI-TOF-MS in clinical laboratories.

Introduction

Accurate bacterial identification is important in case of outbreaks of infectious diseases and play crucial roles in diagnosis and efficient treatment. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a new technology for routine identification of bacteria in clinical microbiology laboratories (Bizzini and Greub, 2010, Croxatto et al., 2012, Emonet et al., 2010, Sauer and Kliem, 2010, Seng et al., 2009, Seng et al., 2010, Sogawa et al., 2011). In 1975 Anhalt and Fenselau used pyrolysis MS for the characterization of microorganisms (Anhalt and Fenselau, 1975). They observed that unique mass spectra were produced from bacterial extracts of different genera and of different species. In the 1980s, the development of soft ionization matrix-assisted laser desorption ionization mass spectrometry allowed the analysis of relatively large biomarkers (Bizzini and Greub, 2010, Heller et al., 1987, Karas et al., 1985). Because MALDI-TOF-MS detects a large spectrum of proteins, the technique is able to discriminate between closely related species and to classify organisms at the species level (Fox, 2006, Murray, 2010). Much of the work using MALDI-TOF-MS for microbial identification has focused on demonstrating that reproducible mass spectra can be obtained using intact cells and developing algorithms for interpretation and comparison of these spectra (Bright et al., 2002, Demirev et al., 1999, Fenselau and Demirov, 2001, Haag et al., 1998, Holland et al., 1996, Jarman et al., 2000, Krishnamurthy and Ross, 1996, Krishnamurthy et al., 2000, Lay, 2001, Pribil and Fenselau, 2005, Saenz et al., 1999, Vargha et al., 2006, Wahl et al., 2002).

MALDI-TOF-MS is a fast and inexpensive technology that has the potential to replace or complement conventional phenotypic identification for most bacterial strains isolated in clinical microbiology laboratories. In a short time the technique has been widely adopted and is integrated into many clinical microbiology laboratories (Carbonnelle et al., 2007, Carbonnelle et al., 2010, Croxatto et al., 2012, Seng et al., 2009, Welham et al., 1998, Wunschel et al., 2005). By testing colonies, it takes only a few minutes to have a correct identification which makes not only possible to identify the microorganisms at the species levels but sometimes at the sub-species and strains levels, allowing the detection of epidemic lineages. In addition, some antibiotic resistance might be detected and also some bacterial toxins (Seng et al., 2010). Recently, Bizzini and Greub (2010) reported that, for routine bacterial isolates, correct identification by MALDI-TOF-MS at the species level was obtained in 84.1–93.6% of instances. In one of these studies, a protein extraction step clearly improved the overall valid identification yield, from 70.3% to 93.2%.

Databases are continuously updated, and MALDI-TOF-MS represents a fast and inexpensive technology for bacterial species identification. This technique works well for many bacterial species but not so well for others such as Streptococci and Staphylococci. Interestingly, in their previous study, Rees and Voorhees (2005) discussed the simultaneous analysis of multiple target microorganisms using MALDI-TOF-MS with bacteriophage amplification. Following infection of target bacteria with specific bacteriophages, proteins contained in the progeny phage are utilized as a secondary biomarker for the target bacterium. Identification of each bacterium was made based on the presence of the secondary bacteriophage biomarkers.

Two MALDI-TOF mass spectrometers, MALDI BioTyper by Bruker Daltonics and Shimadzu have entered to the market as bacterial identification tools. The Bruker instrument provides its own solution, MALDI BioTyper (software, bioinformatic and database), whereas the Shimadzu instrument uses its own software (Launchpad). Peaks that are shared by a minimum number of strains from the same species, in order to build a reference peak signature, called a super-spectrum (Emonet et al., 2010). Recently Cherkaoui et al. (2010) compared two MALDI-TOF-MS methods (Bruker Daltonics and Shimadzu) with conventional phenotypic identification for 720 bacterial isolates under routine clinical laboratory conditions. The Bruker MS system gave high-confidence identifications for 680 of 720 isolates, of which 674 (99.1%) were correct; and the Shimadzu MS system gave high-confidence identifications for 639 of 720 isolates, of which 635 (99.4%) were correct.

Bacterial identification is routinely based on phenotypic tests, including Gram staining, culture and growth characteristics, and biochemical pattern (Carroll and Weinstein, 2007). Complete identification using these tests takes longer time for bacteria, which are difficult to culture. Such time-consuming procedures affect proper treatment of patients with respect to antibiotic and supportive treatments. The potential advantages that MALDI-TOF-MS offers over other techniques for microbial characterization include minimal sample preparation, rapid results, and negligible reagent costs. Rapid identification of the bacteria that are difficult to culture such as anaerobic bacteria, fastidious bacteria, slow growing Mycobacteria, and archaea is of clinical importance, although it is challenging. There are two steps in dealing with poorly culturable organisms—growth and identification. MALDI-TOF-MS speed up step 2 but not step 1; thus identification will still be slow depending on the slow growth. Species identification of anaerobic bacteria from serious infections (e.g., blood cultures) is important because information about virulence, potential resistance to certain antimicrobial agents, and primary site of infection can be obtained. For identification of bacteria using MALDI-TOF-MS, mass signal pattern analysis algorithms and libraries of bacterial reference mass spectra can be applied. For classification, clustering methods are conventionally used, and results are visualized as dendrograms (Sauer and Kliem, 2010). Several experimental procedures have been developed to generate samples from a wide range of different bacterial genera. Usually, a single bacterial colony is enough for MALDI-TOF-MS analysis, but in many cases culturing or enrichment of bacteria is required to obtain sufficient material (Chong et al., 1997, Sauer and Kliem, 2010). Moreover, as several studies have shown, the MALDI method outperforms conventional assays and can also be applied to human pathogens such as Bacteroides fragilis, which is frequently misidentified with phenotypical identification procedures (Nagy et al., 2009, Sauer and Kliem, 2010); the MALDI method can be extended for the analysis of unknown bacteria, as well as bacteria difficult to culture. Firstly, we will discuss about the bacteria which take longer time to grow in the culture media and conventional identification approaches with difficulties and limitations; and then this review will focus on the use of MALDI-TOF-MS for the rapid identification of the group of bacteria which are difficult to culture such as anaerobic bacteria, fastidious bacteria, slow growing Mycobacteria and archaea.

Section snippets

MALDI-TOF-MS technique, speed and cost for identification of bacteria

Each mass spectrometer consists of three functional units: an ion source, to ionize and transfer analyte ions into the gas phase; a mass analyzer, to separate ions by their mass-to-charge ratio (m/z); and a detection device, to monitor ions (Sauer and Kliem, 2010). MALDI-TOF is known since 1996 but this is a new technology for the identification of bacteria to clinical or microbiological laboratories. This technique is a soft ionization method, which allows desorption of peptides and proteins

Anaerobic bacteria

Bacterial culture had been the most important technique for diagnosing bacterial infection. The clinical importance of ‘uncultivable’ anaerobic bacteria is now well recognized. Among the various molecular assays available, 16S rRNA sequencing method is a conventional technique for detecting uncultivable bacteria (Woo et al., 2008). These bacteria are poorly identified using phenotypic methods. Anaerobic bacteria do not grow on solid media in room air (10% carbon dioxide and 18% oxygen);

Identification of anaerobic bacteria using MALDI-TOF-MS

To date, different studies have demonstrated the use of MALDI-TOF-MS for routine identification of anaerobic bacteria. These organisms are poorly identified using phenotypic methods with a lack of specificity and ambiguous or false identification (Croxatto et al., 2012).

Table 1 shows the use of MALDI-TOF-MS for the identification of different anaerobic bacteria with high MALDI-TOF-MS accuracy. B. fragilis and related species are important opportunistic anaerobic pathogens causing severe

Bartonella spp. identification

Bartonella species, which belong to the α-2 subgroup of Proteobacteria, are fastidious Gram-negative bacteria that are highly adapted to their mammalian host reservoirs. Due to their poor chemical reactivity, these fastidious bacteria are poorly characterized using routine phenotypic laboratory tests. Identification is usually achieved using molecular techniques that are time-consuming and expensive. Fournier et al. (2009) have reported identification of Bartonella species using automated

Use of MALDI-TOF-MS for the identification of Mycobacterium species

The slow growth rate of mycobacteria makes it difficult to isolate them from samples rich in other microorganisms. Incubation times of 6 weeks to 5 months are mostly needed for the detection of slow-growing mycobacteria (Levy-Frebault and Portaels, 1992, Rautiala et al., 2004). Classical methods for identification of Mycobacterium species rely on morphology and biochemical profiles. Speciation of a Mycobacterium isolate using these standard methods is a lengthy process. With the rise of

Identification of archaea and extremophilic bacteria using MALDI-TOF-MS

Archaea and a number of groups of environmentally important bacteria, e.g., sulfate-reducing bacteria, anoxygenic phototrophs, and some thermophiles, are difficult to characterize. Previously Krader and Emerson (2004) evaluated MALDI-TOF-MS as a rapid method for identifying different groups of archaea and extremophilic bacteria. The method was rapid and required a minimum of sample processing. They have tested 28 archaea (10 genera, 20 species) and 42 extremophilic bacteria (25 genera, 37

Conclusions

MALDI-TOF-MS is a powerful tool for the classification of a group of bacteria that are difficult to culture. MALDI-TOF-MS-based identification, which will become more effective with future spectra database improvement, will be likely responsible of a burden of emerging anaerobes in clinical microbiology. The performance of the MALDI-TOF-MS systems for species identification is faster and less expensive than those of the commercial phenotypic systems. Although a higher number of species can be

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