D - 4025
Contact information: Dr. Markus Kostrzewa Bruker Daltonik GmbH Permoserstrasse 15 04318 Leipzig, Germany Email:
[email protected]
Rapid Identification of Bacteria Causing Urinary Tract Infections by MALDI-TOF MALDI-TOF MS
48th Annual ICAAC/IDSA 46th Annual Meeting Washington, DC – October25-28, 2008
G. SCHWARZ1, T. MAIER1, M. KOSTRZEWA1, C. BOOGEN2, and U. WELLER2 1Bruker
Daltonik GmbH, Bremen/Leipzig, Germany, 2Lab. Dr. C. Boogen, Cologne, Germany
Abstract
Introduction
Methods
Results
Background: MALDI-TOF MS has become a fast and reliable method for identification of microorganisms after cultivation. However, the method has not been widely applied to specimens taken from patients, directly. In order to develop a more rapid identification method we analyzed bacteria collected from infected urine, directly. As state-of-the-art instruments have a high sensitivity (as low as 102 CFU / target spot) and with urinary tract infections involving bacterial counts of >104 /ml urine our “proof-of-concept” study shows reliable identification results in significantly reduced time. Methods: Clean catch urine samples submitted to a routine laboratory for bacteriologic analysis were screened for leucocytes and nitrite, 37 samples were selected. Urine (1ml) was centrifuged for 30 seconds at 2000g to reduce load of leucocytes. The supernatant was centrifuged at 15500g for 5 minutes. The pellet was dried by a cotton swab and washed once with de-ionized water. Proteins were extracted from pellets by adding formic acid and acetonitrile. 1 µL was applied to a polished steel target and overlaid with 1 µL Matrix solution (HCCA). MALDI-TOF mass spectrometry was performed using a Bruker microflex LT instrument equipped with the BioTyper 2.0 software, to identify all raw data sets. Results: Compared to the culture results, 33 samples where correctly identified by this rapid method. The complete process takes about 1.5h. 4 samples that were not identified by the direct MALDI approach resulted in 2 or more organisms in the culture workup. The time required for identification was shortened at least by 24h compared to traditional procedures. Conclusions: These results show that it is possible to directly and rapidly identify bacteria from patients with urinary tract infections, thus enabling the physician to select an antibiotic more accurately before testing for AST.
The identification of microorganisms grown on culture plates by MALDI-TOF MS fingerprinting is getting a routine method in many laboratories. Direct analysis of clinical samples will further increase its benefits for patient care by shortening time to identification, significantly. Here, we present the MALDI-TOF identification of bacteria causing urinary infections which were isolated directly from urine, without cultivation.
Urine (1ml) was centrifuged for 30 seconds at 2000g to remove leucocytes. Supernatant was centrifuged at 15500g/5 minutes to collect the bacteria. The pellet was dried and washed once with de-ionized water. Cell content was extracted using formic acid/acetonitrile. Extracts (1µl) were applied to a MALDI target and overlaid with 1µL matrix solution (HCCA). Profile spectra were measured in a microflex LT instrument and analyzed with the MALDI Biotyper 2.0 software and the according reference database (3290 entries).
37 urine samples were analyzed. In 33 cases analysis resulted in identifications which were confirmed by classical tests. The four samples that were not identified by the direct MALDI approach were found to contain two microorganisms after culturing. The complete direct identification process took about 1.5h from centrifugation to database matching result. Thereby, the time required for identification was shortened at least by 24h compared to traditional culture-dependent procedures.
Urine (1 ml)
I
II
Centrifuge (30 s, 2000g) Transfer supernatant in new cup
Table 1: Identification results from urine samples Biochemical ID
Identical MALDI ID
Deviating MALDI ID
No MALDI ID
Enterococcus faecalis
4
0
0
Escherichia coli
17
0
0
Klebsiella pneumoniae
6
0
0
Pseudomonas aeruginosa
2
0
0
Streptococcus anginosus
1
0
0
Streptococcus agalactiae
3
0
0
Mix E. coli/E. faecalis
0
0
1
Mix P. vulgaris/E. faecalis
0
0
1
Mix P. mirabilis/E. coli
0
0
1
Mix P. aeruginosa/E. coli
0
0
1
∑ 37
33
0
4
A
Centrifuge (5 min., 15500g)
Conclusions
Dry pellet with cotton swab Wash once with de-ionized water Standard Bruker extraction protocol for bacterial profiling
B
Spot 1 µl onto MALDI target, overlay with HCCA matrix
Measurement Of profile spectra
Data interpretation
C
ID Figure 1: Flow chart of the microorganism identification directly from urine by the MALDI Biotyper system.
1
Instrument status
2
Instrument status
3
Instrument status
• MALDI-TOF MS profiling enables a very quick and reliable identification of bacteria directly from infected urine.
Analyte
Analyte
Organism
Score
Organism
Score
Analyte
Analyte
Organism
Score
Organism
Score
Name
ID
(best match)
Value
(second best match)
Value
Name
ID
(best match)
Value
(second best match)
Value
474 Ko
6aca4b41-0cbd4206-a4bbe21058466e79
474
38cd6db6-11f64cde-a65466a131af0b5b
( +++ )
Escherichia coli DH5alpha BRL
2.484
Escherichia coli ATCC 25922 THL
2.275
( +++ )
Escherichia coli ATCC 25922 THL
2.218
Escherichia coli MB11464-1_CHB
2.024
Figure 2: Identification of a bacterium from infected urine, (I) after cultivation on agar plate, (II) directly from urine; (A) mass spectrum, (B) mirror view of the best match against the Bruker database, (C) ID Score output. Score is slightly higher for the cultivated bacterium but still sufficient for reliable ID after direct enrichment from urine.
• Shortened analysis time enables an early selection of a more appropriate treatment of patients. • Development of algorithms for mixture detection may further enhance the potential of the method.
MALDI Biotyper
