B. fragilis

“No Reservations” 

Acceptance and accuracy of MALDI- 

TOF methods for identification and 

typing of aerobic and anaerobic bacteria 

Elisabeth Nagy 

Institute of Clinical Microbiology, University of Szeged, Hungary, 

ASM 2011, New Orleans, Louisiana 

1

Gram-negative aerobic bacterium isolated from blood culture 

gentamicin 

amikacin 

tobramycin 

netilmicin 

If it is an Escherichia coli all aminoglycosids can be used !!! 

If it is a Salmonella sp., despite of the susceptibility to 

aminoglycosides in vitro , they are ineffective in vivo !!!! 

2

Only 12 of 23 Bacteroides 

species are in the database 

3

Possibilities for species determination in 

routine laboratories 

Classical methods using phenotypic characteristics 

Automation of biochemical tests 

Use of short-chain fatty acid production (anaerobic 

bacteria) 

DNA-based identification 

(DNA-DNA hybridization) 

16S rRNA gene sequencing (whole DNA sequencing ) 

Protein-based identification (based on small ribosomal 

proteins) by mass spectrometry 

4

History 

1975: the first use of MS for bacterial identification 

(Anhalt et al. Anal Chem 1975) 

Limited mass range (

Protein-based, “real-time” species 

identification MALDI-TOF MS 

Microflex LT TM 

Bruker Daltonics 

MALDI BioTyper: 

software, 

bioinformatics, 

database 

Species identification within 10 

minutes if it is included in the 

database 

Launchpad software 

SARAMIS database 

AXIMA 

Shimadzu 

6

m/z 

Technical description of 

MALDI-TOF MS 

Emonet et al. CMI 16: 1604-13, 2010 

7

An anaerobic clincal isolate 

Identification view: sample MSP stick 

spectrum (upside) and reference MSP 

stick spectrum (downside) 

Gel overview: view 

of sample MSP 

MALDI-TOF Biotyper 

Taxonomy tree 

view: Based on 

NCBI taxonomy 

browser 

MSP results and 

log(score) values 

8

Sample preparation 

Direct transfer of a colony of bacteria or yeasts onto the target 

or 

Pretreatment: 

One colony is suspended in 300 μl bidistilled water 

900 μl ethanol is added and mixed thoroughly 

Centrifugation for 2 min – the pellet is dried 

Re-suspend in up to 50 μl 70% aqueous formic acid and 50 μl 

acetonitrile 

Centrifugation for 2 min and 1 μl supernatant is spotted on the target 

plate and dried at room temperature 

(in the case of some pathogens special sample preparation is needed) 

Pretreated and untreated samples are overlaid with 1 μl matrix 

solution 

Measurements are usually made on multiple parallels 

9

Identification of clinically relevant pathogens (1) 

Enterobacteriaceae (Conway, et al. J Mol Microbiol Biotechnol 2001) 

Genetically distinct E. coli strains could be identified and distinguished 

from several other genera/species of Enterobacteriaceae 

Non-fermenters (Mellmann et al. J Clin Microbiol 2008 and 2009) 

- 248 culture collection strains 

belonging in 37 genera were used to 

set up the database 

- MALDI-TOF identified 67 of 78 

(85.9%) clinically relevant 

non-fermenters concordant with the 

sequencing 

- Mean log(score) values for 60 

blind-coded samples identified by 8 

laboratory were evaluated 

97.3% of all log(score) values were >2 

10

Identification of clinically relevant pathogens (2) 

Staphylococci (Carbonnelle et al. J Clin Microbiol 2007; Szabados et 

al. J Med Microbiol 2010) 

Enterococci (Eigner et al. Clin Lab 2009) 

Streptococci (Friedrichs et al. J Clin Microbiol 2007) 

Neisseria spp (Ilina et al. J Mol Diagn 2009) 

Listeria monocytogenes (Barbuddhe et al. Appl Environ Microbiol 

2008) 

Mycobacteria (Pignone et al. J Clin Microbiol 2006) 

Etc. 

11

1. retrospective study 

2. prospective study 

J Clin Microbiol 48: 900-907 (2010) 

12

~5000 clinical isolates; 2009-2010 

Conclusions: > 90% of all clinical isolates, 

94% of Gram-negative bacteria and 

89.8% of Gram-positive bacteria were identified at a species level 

13

Use of MALDI-TOF MS for typing of bacteria 

Appl Enviroment Microbiol 74:5402 (2008) 

Besides the rapid identification of 146 different Listeria 

strains, 16 L. monocytogenes were typed and the PFGE 

profile gave a result identical with that of MALDI-TOF MS 

14 

II. 

I. 

III.

- By analysis of MS of 25 MRSA isolates belonging in 

the 5 major HA-MRSA clonal complexes, 13 m/z 

values were found which discriminated them. 

- 60 further clinical isolates were tested. 15 different 

MALDI types could be differentiated. 

Int J Med Microbiol 301:64-68 (2011) 

15

Use of MALDI-TOF MS for identification of 

anaerobic bacteria 

Difficult to isolate from mixed infections in pure 

culture 

Most of them are slow-growing (small colonies) 

Classical methods are time-consuming 

Automated phenotypic methods need a large 

inoculum (difficult to achieve) 

Many clinically important anaerobes are inactive 

16

Shah et al. CID 35: S58-64 (2002) 

Through the use of intact cell MALDI-TOF MS, Porphyromonas 

strains could be differentiated 

Porphyromonas 

culture collection 

strains 

Prevotella intermedia belonged in 3 different subgroups and P. 

nigrescens was more homogeneous by MS 

17

Intens. [a.u.] 





Identification of Bacteroides strains by MALDI Biotyper 

4 

x10 

3 

2 

1 

0 

4000 

2000 

0 

4000 

3000 

2000 

1000 

0 

x104 

1.00 

0.75 

0.50 

0.25 

0.00 

x104 

1.5 

1.0 

0.5 

0.0 

Bacteroides fragilis ATCC 25285\0_M11\1\1SLin 

Bacteroides massiliensis DSM 17679_DSM\0_A12\1\1SLin Raw 

Bacteroides eggerthii DSM 20697_DSM\0_K11\1\1SLin Raw 

Bacteroides nordii DSM 18764_DSM\0_I3\1\1SLin Raw 

Bacteroides intestinalis DSM 17393_DSM\0_A22\1\1SLin Raw 

3000 4000 5000 6000 7000 8000 

m/z 

18 

Nagy et al: CMI 15: 796-802 (2009)

Species determination of 277 Bacteroides isolates with commercial 

identification kits and by MALDI Biotyper 

Species Number of isolates 

with an identification score with discrepant species selected for 16S rDNA 

>2.0 by MALDI-TOF MS identification by biochemical tests sequencing* 

Bacteroides fragilis 179 5 2 

Bacteroides thetaiotaomicron 43 5 4 

Bacteroides ovatus 15 6 1 

Bacteroides vulgatus 

Bacteroides uniformis 

20 

5 97.5% 

1 

3 8.3% 

1 

2 

Bacteroides eggerthii 1 0 

Bacteroides nordii 4 3 1 

Bacteroides salyersiae 1 0 

Bacteroides massiliensis 2 0 

Inconclusive identification by 

MALDI-TOF MS 7 7 

*Only strains with a log(score) 2.0-2.5 were selected for sequencing 

Nagy et al. CMI 15: 796-802 2009 

19

Comparative species determination of selected Bacteroides strains 

Sample number Species determined by 

_______________________________________________________________________________________________ 

Biochemical tests MALDI-TOF MS 16S rDNA sequencing 

1 B. ovatus B. fragilis B. fragilis 

2 B. uniformis B. fragilis B. fragilis 

3 B. ovatus B. thetaiotaomicron B. thetaiotaomicron 

4 B. uniformis B. thetaiotaomicron B. thetaiotaomicron 

5 B. uniformis B. thetaiotaomicron B. thetaiotaomicron 

6 B. ovatus B. thetaiotaomicron B. thetaiotaomicro 

7 B. uniformis B. ovatus B. ovatus 

8 B. thetaiotaomicron B. vulgatus (log(score) 2.023) Bacteroides sp (new?) 

9 B. ovatus B. uniformis B. uniformis 

10 B. ovatus B. uniformis B. uniformis 

11 B. ovatus B. nordii B. nordii 

_______________________________________________________________________________________________ 

12 B. thetaiotaomicron II*(log(score)>2.5) P. (B.) distasonis 

13 P. (B.) distasonis II P. (B.) distasonis 

14 P. (B.) distasonis II(log(score)>2.5) P. (B.) distasonis 

15 P. (B.) distasonis II(log(score)>2.5) P. (B.) distasonis 

16 B. thetaiotaomicron II B. eggerthii 

17 B. merdae II B. goldsteinii 

18 Bacteroides sp II B. intestinalis 

*II = Inconclusive identification Nagy et al. CMI 15: 796-802 2009 

20

Routine identification of anaerobic bacteria by MALDI-TOF 

Stingu et al: Oral Microbiology Immunology 2008 

84 oral biofilm isolates (including 9 reference strains) were tested 

MALDI-TOF was superior to biochemical identification 

P. intermedia/nigrescens (37 strains) could be distinguished by MALDI-TOF MS 

Veloo et al: CMI 2010 

79 clinical isolates representing 19 genera 

Comparison of Bruker and Simadzu system with 16S rRNA gene sequencing 

Correct identification at a genus level 61% versus 71% and at a species level 51% 

versus 61% 

When species not present in the database were excluded 75% versus 76.7% 

21

Routine identification of anaerobic bacteria by MALDI-TOF 

196 anaerobic bacteria isolated from clinically relevant 

samples (blood cultures, abdominal, pelvic, oral, soft tissue infections) during 

2009 and 2010 were blindly tested 

MALDI-TOF MS identified 166 (84.6%) at a species level with a 

log(score) >2.000 

184 (93.8%) at a genus level 

After 16S rRNA gene sequencing, it turned out that the species was not 

included in the database for 10 isolates 

In cases of discrepant phenotypic identification (ATB, API and 

conventional methods), 16S rRNA gene sequencing supported the 

MALDI-TOF identification in 97% of the cases. 

Soki J, Nagy E, Backer S : Publication in preparation 

22

Dendogram of Bacteroides/Parabacteroides strains according to MALDI-TOF 

1000 

900 

800 

700 

MSP Dendrogram 

600 500 

Distance Level 

400 

300 

200 

100 

x_Bac_HU29105 B. ovatus PNU 99.5% B. thetaiotaomicron 

x_Bac_HU29104_4 B. ovatus 99.7% PNU B. eggerthii 

x_Bac_HU40347_2 B. vulgatus PNU B. vulgatus 

x_Bac_HU33866_2_18 B. thetaiotaomicron PNU B thetaiotaomicron 

x_Bac_HU26686_6 B. thetaiotaomicron PNU B. thetaiotaomicron 

x_Bac_HU36220 P. distasonis PNU P. distasonis 

x_Bac_HU33120_3_13 

B. uniformis 

PNU 

B. unifomis 

x_Bac_HU32405_3 PNU B. fragilis 



x_Bac_HU35225 PNU B. fragilis 

x_Bac_HU27912 PNU B. fragilis 

x_Bac_HU28715 B. fragilis PNU Bacteroides sp. 


x_Bac_HU33866_2_19 PNU 

B. fragilis 

x_Bac_HU34886_3 

99.5% 

PNU 

B. capillosus 

x_Bac_HU26938_2 PNU 

99.5% B. thetaiotaomicron 

x_Bac_HU32759 PNU 

x_Bac_HU33488 PNU 







0 

MALDI-TOF 16S rDNA 

Phenotypic identification 



x_Bac_HU33120_3_12 PNU 




16S rRNA gene sequencing confirmed the identification based on MS 

23

Comparative identification of 17 Gram-positive 

anaerobic clinical isolates 

Biochemical identification Analyte Organism Score 

MALDI Biotyper 

Name (best match) Value 

Finegoldia magna x HU 29644_1 PNU not reliable identification 1.238 

Clostridium perfringens x HU66505 PNU Clostridium perfringens 2.473 





Clostridium difficile x HU59744 PNU Clostridium difficile 2.552 

Clostridium sp x HU56448 PNU Clostridium innocuum 2.3 




Clostridium clostridioforme x HU52042 PNU not reliable identification 1.406 


Clostridium tertium x HU46926 PNU Clostridium tertium 2.351 

Clostridium innocuum x HU46588 PNU Clostridium innocuum 2.481 


Clostridium fallax x HU33416 PNU not reliable identification 1.313 

16S rRNA 

F. magna 

C. innocuum 

C. hathewayi 

B. ureolyticus 

24

Use of MALDI-TOF MS to distinguish cfiApositive 

and cfiA-negative B. fragilis strains 

(typing of B. fragilis) 

Carbapenem resistance in B. fragilis is due in most cases to the 

presence of the cfiA gene and its expression (IS elements are 

needed in the promoter region) 

This metallo-beta-lactamase confers resistance to all betalactam 

antibiotics 

Only B. fragilis strains belonging in Division II harbor the cfiA 

gene 

Different studies indicate an increased resistance of B. fragilis 

to carbapenems and an increased frequency of silent harboring 

of the gene 

25

Selection of well-characterized B. fragilis strains 

Strains PCR detection of 

cfiA 

IS element(s) Imipenem MIC 

(mg/L) 

1 B. fragilis TAL 3636 + IS942 >32 

2 B. fragilis CZE60 + - 16 

3 B. fragilis 1672 + IS1186, (IS4351) >32 

4 B. fragilis BF8 + (IS1186, IS4351) 1 

5 B. fragilis R19811 + IS614B 32 

6 B. fragilis O21 + IS1186, (IS4351) >32 

7 B. fragilis 15470 - nt

Gel/stack view of MSP: zoom 4000 to 6000 Da 

02 - CZE60 

05 - R19811 

01 - TAL3636 

03 – 1672 

06 – O21 

04 – BF8 

07 15470 

08 – 15476 

09 – 15361 

10 – 72232_1 

11 – 638R 

12 9343 

cfiA+ 

cfiA- 

Nagy E, et al.: presented during the Congress of ASA, Philadelphia (2010)

02 - CZE60 

05 - R19811 

01 - TAL3636 

03 – 1672 

06 – O21 

04 – BF8 

______________ 

07 15470 

08 – 15476 

09 – 15361 

10 – 72232_1 

11 – 638R 

12- 9343 

Gel/stack view of MSP (zoom 4600 to 4850 Da) 

cfiA + 

cfiA - 


4 

x10 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

4688 

4711 

4817 

4826 

4650 4700 4750 4800 4850

B. fragilis strains tested blindly for cfiA positivity by MALDI-TOF 

Gel/stack view: zoom 4000–5200 Da 

06 PNU 

11 PNU 

12 PNU 

13 PNU 

14 PNU 

15 PNU 

16 PNU 

18 PNU 

19 PNU 

01 PNU 

02 PNU 

04 PNU 

05 PNU 

07 PNU 

08 PNU 

09 PNU 

10 PNU 

17 PNU 

20 PNU 

21 PNU 

22 PNU 

23 PNU 

24 PNU 

26 PNU 

27 PNU 

28 PNU 

31 PNU 

33 PNU 

34 PNU 

Putative 

cfiA+ 

Putative 

cfiA- 

29

Retrospective analysis of mass spectra of 145 

B. fragilis isolates 

9 of 145 B. fragilis strains had characteristic peaks for Division II (cfiA-positive) 

Strains Characteristic peaks CfiA (PCR) Imipenem MIC (mg/L) 

B. fragilis A20 4826, 9375, 9649 +

Dendogram of B. fragilis isolates according to MALDI- 

TOF MS measurements 

41 cfiA-positive isolates 

Division II 

207 cfiA-negative isolates 

Division I 

Wybo et al. JCM (2011) 

31

Identification and typing of bacteria in 

the future by MALDI-TOF MS? 

The method is easy to use 

Very rapid and the running costs are low 

The equipment is suitable for multiple usage 

However, 

Extensive development of the databases is needed 

Standardization should be carried out to obtain 

comparable results 

32

The studies carried out on the identification and typing of 

anaerobic bacteria in our laboratory were supported by research 

grants from: 

Hungarian National Research Foundation (OTKA-K-69044) 

Hungarian Office of Science and Technology (GVOP-0304) 

ESCMID grant for the Study Group for Anaerobic Infections (ESGAI) 

and were performed in cooperation with Bruker Daltonik 

GmbH, Bremen, Germany 

Szeged, Hungary: Edith Urbán, József Sóki, Noémi Bartha 

Bremen, Germany: Simone Backer, Tomas Maier, Markus Kostrzewa 

33

Sincere apologies to authors whose 

work could not be cited here. 

34

B. fragilis

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