How MALDI-TOF mass spectrometry can aid diagnosis of hard-to-identify pathogenic bacteria

It is less than a decade ago that MALDI-TOF (matrix-assisted laser-desorption/ionization time-of flight) mass spectrometry arrived in the clinical microbiology laboratories and has been announced as a revolution [1]. Today it already is established as the standard identification technology for cultivated microorganisms in most clinical laboratories in Europe and on a victory march in the Americas and Asia. The fundamental advantages of MALDI-TOF MS over traditional identification methods, that is, dramatically shortened time to result, reduced consumable costs, and improved quality of results, are generally acknowledged and have already changed workflow in many laboratories. In contrast to molecular methods, which are considered as gold standard for microbial identification but generally are expensive, need considerable manual interaction and very often are time consuming, MALDI-TOF MS is very fast with a simple sample preparation technique, but offers a similar quality of information.

One important microorganism group which benefits from the new technical approach is the anaerobic bacteria. Previously, identification of these bacteria in the routine clinical microbiological laboratories was mainly performed by biochemical tests with limited accuracy and taxonomic resolution, in particular for the less frequently occurring ones. Recently, several studies have demonstrated the superior quality of MALDI-TOF MS for identification of clinically relevant, however often biochemically inactive anaerobes [2–4]. As anaerobic bacteria usually are involved in mixed infections, they generally need sub-culturing to achieve pure culture. Most of them should be incubated longer on agar plates because of their slow growth. With the possibility of direct identification of anaerobes from the primary plates due to the minimal biomass needed for the MS-based identification, the TTR benefit here is particular impressive. Different studies are showing that MALDI-TOF MS could introduce identification accuracy similar to 16S rDNA sequencing, but at clearly lower costs. The cost aspect of mass spectrometry depends on the number of samples which are analyzed per year, but several studies have proven that the technique is very cost-effective for the average clinical microbiology laboratories as well [5].

In the everyday practice MALDI-TOF MS can offer more than just species identification and thereby it may have additional benefits, in particular for the analysis of fastidious microorganisms. Even the identification spectra acquired during the routine measurement can offer information which is not available from other technical approaches. This is highlighted by the finding that two divisions of Bacteroides fragilis, an important anaerobic pathogen, can be clearly differentiated by their mass spectra [6,7]. This will, in the future, enable the seamless secure identification of those isolates which harbor the cfiA gene, responsible for the carbapenem resistance of this pathogen. Similarly, a significant improvement of diagnostic has been reported for Haemophilus influenzae, a fastidious bacterium causing severe localized respiratory tract infection such as otitis media, sinusitis, bronchitis, pneumonia and often is the causative agent of acute exacerbation of chronic obstructive pulmonary disease. Using biochemical identification systems, this highly pathogenic microorganism can hardly be differentiated from the phylogenetically closely related Haemophilus haemolyticus, an organism with much lower pathogenicity. A thorough optimization of the reference database enabled the secure identification and differentiation of both species by mass spectrometry [8]. Further, it has been demonstrated that differentiation of the particular virulent Haemophilus influenzae type b which is associated with severe invasive disease such as meningitis and epiglottitis is also feasible using MALDI-TOF MS profiles [9]. The time consuming MLST-based typing of skin flora member Propionibacterium acnes showed that some phylotypes of this anaerobic bacterium may be associated with different infections such as acne, prosthetic joint infection, post-neurosurgical infection, endocarditis or osteomyelitis. In a recent study the differentiation of the main phylotypes of Propionibacterium acnes directly from MALDI-TOF mass spectra has been demonstrated [10]. The clinical significance of this information, besides epidemiology purposes, is not yet evident, however this might change with more knowledge gained in the future.

Several bacteria have been reported during the last years for the first time to be found in patients as potentially important pathogens, in particular if biochemical tests do not cover them, but MALDI-TOF MS and a broad reference database enables their identification. Recent examples are Lactococcus garviae, a fish pathogen which was found as causative agent of infective endocarditis [11], or Wohlfahrtiimonas chitiniclastica, a microorganism associated to insects causing infections in humans [12]. Rare blood culture isolates, which could be identified earlier only with the aid of sequencing such as Cupriavidus pauculus, Delftia tsuruhatensis, or Ochrobactrum anthropii are identified by MALDI-TOF MS [13]. These findings are challenging the classification of microorganisms as ‘clinically not relevant’. The utilization of MALDI-TOF MS in diagnostic laboratories might teach us that the relevance of many bacteria for human health is underestimated and sometimes is more depending on the status of the ‘host’ (patient) than on the bacterium itself.

Resistance detection is a further area where currently MALDI-TOF MS is thought to improve diagnostics in future. Again, shortened time to result is one of the greatest expectations. Bacteroides fragilis strains which have been indicated to carry the cfiA gene have been confirmed to exhibit carbapenemase activity by detection of carbapenem hydrolysis products in an in vitro assay [14]. Here, MALDI-TOF MS offers the chance of genotypic detection and phenotypic confirmation of resistance in a few hours. Additional fast MALDI-TOF assays for resistance detection in development might further extend the potential of rapid combined MALDI-TOF MS based workflows [15,16] Such assays observe the progress of protein synthesis by incorporation of stable isotopes under influence of an antibiotic or, more classical, their increase of biomass during growth using MALDI-TOF MS as an early and specific readout of resistance or even antibiotic susceptibility. Hope exists that such assays will lead to more resistance testing also for rare and slow growing bacteria which nowadays frequently are not tested at all.

A further application of MALDI-TOF MS can be the direct identification of bacteria from positive blood cultures of monobacterial septic patients, which enables the laboratory to provide early preliminary results to the clinicians and help selection of antibiotic usage according to the known inherited resistance of some pathogens [17]. The broad coverage of bacterial species enables such early identification not only for the frequently occurring but also rare pathogens. To combine growth in selective enrichment broth and MALDI-TOF MS identification may help rapid identification of epidemiologically important carriage of Salmonella [18].

Timely species level identification of the slow growing Mycobacterium tuberculosis complex as well as non-tuberculosis mycobacteria has significant clinical relevance. Using an optimized protein extraction method and a specially developed robust library for the identification by mass spectrometry of Mycobacterium spp, grown in liquid culture or on the surface of traditional solid medium can help in the reliable identification of this difficult to identify group of bacteria [19]. Further a more recent study has proven that MALDI-TOF MS could discriminate Mycobacterium abscessus subspecies bollettii, abscessus and massiliense deferring in antibiotic resistance and clinical relevance [20].

With the improvement of databases for MALDI-TOF MS identification the term ‘hard-to-identify bacteria’ might even disappear or may at least keep true only for few species in the clinical routine. Here, MALDI-TOF mass spectrometry based identification will supported by whole genome sequencing and benefit from knowledge generated by this upcoming technology. Bacteria previously considered as ‘difficult’ will get ‘normal’, up to now hardly known species will be observed in routine clinical microbiological laboratories more frequently. This will enable researchers to learn about them, for example, their pathogenicity, resistance patterns, treatment options and epidemiology. Thereby, the knowledge about many pathogens will increase and enable better and sooner therapy for affected patients.

Financial and competing interests disclosure

M Kostrzewa is an employee of Bruker Daltonik GmbH, a manufacturer of MALDI-TOF mass spectrometry systems.

E Nagy has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Notes on contributors

Markus Kostrzewa

Elisabeth Nagy