# Patent

## Title
Means for identifying a strain isolated from a clinical sample at the species and/or subspecies level

## Abstract

The invention relates to a method for identifying a strain isolated from a clinical sample, at the species and/or subspecies level, using MALDI-TOF-MS analysis comprising a step of classifying the germ in a group before performing the MALDI-TOF-MS analysis.

## Background
This application is a Continuation-In-Part of International Application No. PCT/IB2008/001058, filed 8 Jan. 2008, which designated the U.S. and claims priority of EP Application No. 07290019.4, filed 8 Jan. 2007, the entire contents of each of which are all hereby incorporated herein by reference.


The invention relates to means for identifying a strain isolated from a clinical sample. It more particularly relates to a method for a precise and rapid identification at the species level and/or at the sub-species level of a clinical isolate by using Matrix assisted Laser Desorption Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS). It also relates to the databases used for this identification.
MALDI-TOF-MS is a technique used to examine the profile of proteins detected directly from intact bacterial cell surface.
This is a soft ionization method based on relative molecular masses allowing desorption of peptides and proteins from whole different cultured microorganisms. Ions are separated and detected according to their molecular mass and charge. The species are identified by their mass:charge ratio (m/z). This approach yields reproducible spectrum within minutes, consisting of series of peaks from 500 to 20000 m/z. Each peak corresponds to a molecular fragment released from the cell surface during laser desorption.
MALDI-TOF-MS has already been used for characterization of bacteria. However, among the various components identified in a spectra, only a few are present in a large population of germs of a given genospecies, the other are either isolate specific or vary upon growth conditions (media, temperature of incubation, . . . ) and cannot be used to identify the species or sub-species of a bacteria, and more generally of a germ.
The lack of a global strategy to identify within the spectra those peaks that can discriminate between various genospecies has hampered the use of MALDI-TOF-MS in routine clinical microbiology laboratories for the diagnostic of genospecies. Indeed these peaks have to correspond to components that are present in the majority of the isolates of a given genospecies and that are present in large quantity in the cell wall. Considering the possible variability inside a genospecies, it is likely that the characteristic of a genospecies does not rely on the presence of a single peak but rather on the presence of a set of peaks that are more or less conserved among the genospecies.
The inventors have found that such problems could be overcome (i) by performing a phenotypic analysis of the germ to be identified prior to the MALDI-TOF-MS analysis, and (ii) by carrying out the MALDI-TOF-MS analysis under specific conditions particularly concerning the choice of the matrix solution and reference data bases.
It is then an object of the invention to provide a new method based on MALDI-TOF-MS analysis for identifying a strain isolated from a clinical sample at the species and/or sub-species level.
It is another object of the invention to provide databases established from a set of strains with respect to a given germ.
The invention thus relates to a method for identifying at a species and/or sub-species level a strain isolated from a clinical sample using MALDI-TOF-MS analysis comprising eventually a step of classifying the strain in a group before performing the MALDI-TOF-MS analysis, wherein said analysis comprises the step of retaining in the spectrum of the tested strain the peaks having, compared to the peak with the highest intensity arbitrary set up to 1, a relative intensity which may vary depending on the germ, said relative intensity being then higher than 0.01 for germs such as fungi, and for most of the other germs higher than 0.02, particularly higher than 0.05, more particularly higher than 0.1.
The invention particularly relates to a method wherein said MALDI-TOF-MS analysis comprises the step of comparing the spectrum profile of the strain to be identified with a so-called reference database containing the characteristic MALDI-TOF-MS spectra of strains representative of the genospecies of the group of germs to which belongs said strain to be identified, said database containing for each representative strain, peaks having, compared to the peak with the highest intensity arbitrary set up to 1, a relative intensity which may vary depending on the germ, said relative intensity being then higher than 0.01 for germs such as fungi, and for most of the other germs higher than 0.02, particularly higher than 0.05, more particularly higher than 0.1, as far as they are present in at least 2 sub-cultures, preferably 5 or even 10 or more.
The selection of peaks as carried out according to the invention enables an accurate identification of the germ present in a clinical sample.
The classification step is based on the growth conditions and/or the characteristic of colonies and/or morphology of the bacteria upon microscopic examination and/or the gram stain and/or simple phenotypic tests.
The MALDI-TOF-MS technique is carried out on whole intact cells or on protein extracts obtained after lysis or corresponding to a fraction of the cellular components (cell wall, cytoplasm . . . ). This technique is ideally performed on an isolated colony of the strain to be identified, obtained from a clinical sample cultured on an appropriate growth medium and incubated at an appropriate temperature.
A matrix medium is added to the isolated colonies before performing the MALDI-TOF-MS technique.
For example, suitable matrix media comprise benzoic acid derivatives, such as hydroxyl benzoic acid, particularly 2,5-dihydroxybenzoic acid (DHB).
Other components may be present. For example, the matrix media may further comprise sinnapic acid. Such media are particularly appropriate for the identification of Mycobacterium. 
Each spectrum is the sum of at least 300 laser shots, preferably more than 1 000, and even more than 2 000, from different regions of the well containing the strain to be analyzed, said spectrum being analyzed in a range of m/z from 500 to 50 000, and more preferably from 500 to 20 000, and more preferably from 2000 to 20000.
The profiles are then compared using a software which chooses the best match between the tested strain and the selected peaks of said reference database obtained by analyzing one or several reference strains specific of a given genospecies or genosubspecies. Each strain being a representative of one genospecies or genosubspecies of the group under study, so that the set of strains corresponds to the relevant genospecies or genosubspecies of one group of germs. The spectrum of each reference strain is recorded using the MALDI-TOF-MS technique. As above mentioned, the peak with the highest intensity is arbitrarily set up to 1, and all the other peaks have a value corresponding to the relative intensity of this highest peak. The peaks with a relative intensity being higher than 0.01 for germs such as fungi, and for most of the other germs higher than 0.02, or more particularly above 0.05 or more particularly above 0.1 present are retained, as far as they are present in at least 2 sub-cultures, preferably 5 or even 10 or more. These peaks are the selected peaks.
Considering some possible variation, the software chooses the best match between the tested strain and the database, taking into account a possible error of the m/z value. The range of the error is arbitrary set up to +/−20 or more preferably to +/−10.
The presence and absence of peaks are considered as fingerprints for a particular isolate.
Said method is a powerful tool for the fast identification of a germ, allowing to use it in the routine diagnosis of the laboratory. The requirement of expensive equipment is compensated by the very simple protocol, which requires minimum hands-on time. It is particularly appropriate for reference laboratories or hospitals receiving a high volume of samples.
The high resolution of the MALDI-TOF-MS method as performed according to the invention makes it possible for example to obtain characteristic spectral fingerprints for each species and even sub-species of a given germ.
Advantageously, said method is used for identifying germs selected in the group comprising bacteria, yeasts, fungi, molds such as filamentous fungi, particularly Aspergilli at the species and/or sub-species level.
According to a specific embodiment, the invention relates to the identification of species and/or sub-species of coagulase-negative Staphylococci (CNS in short).
The identification of CNS at the species level is not routinely performed as they are frequently considered as sample contaminations. In addition, the results of phenotypical methods performed on clinically significant strains are disappointing. Commercial identification kits are of limited value in identifying CNS isolates.
SodA sequence determination, despite to be the more accurate method for correct species identification of CNS, is time consuming, expensive, and technically demanding. In addition, although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence based genotypic methods may be a lack of quality of deposited sequences in data banks.
Thanks to the invention, the differentiation at the subspecies level is more accurate, for example for the subspecies of Staphylococci, with MALDI-TOF-MS than with SodA method, as it was shown that SodA gene does not well differentiate between the subspecies S. capitis, S. hominis, S. saprophyticus and S. schleiferi. 
According to another embodiment, the invention relates to the identification of species or subspecies of non fermenting Gram negative bacilli, and other bacterial species of clinical relevance: Streptococci, gram positive bacilli (such as Nocardia, corynebacteria . . . ), enterobactericeae (such as Escherichia coli, Klebsiella, . . . ), mycobacteriaceae . . . . 
In still another embodiment, the invention relates to the identification of yeast species particularly Candida or Saccharomyces species or subspecies such as illustrated by the Examples.
In a further embodiment, the invention provides means to identify at the species or subspecies level molds such as filamentous fungi of the Aspergillus species.
The databases used in the above defined identification method are also part of the invention.
They consist of a set of selected peaks obtained by MALDI-TOF-MS analysis of strains at least partially conserved among strains belonging to the same genospecies.
Preferably, said reference peaks correspond to major, conserved peaks with m/z above 0.01 for fungi, and above 0.02, more particularly above 0.05, more particularly above 0.1 with other germs.
Such databases are advantageously obtained for each group of germs by using a set of strains, each strain being the representative of one genospecies of the group under study, so that the set of strains corresponds to the relevant genospecies of one group of germs. The spectrum of each strain of such group is recorded using the MALDI-TOF-MS technique. Each strain is the representative of a genospecies of this group of germs, and is further designated as reference strain. As above mentioned, the peak with the highest intensity is arbitrarily set up to 1, all the other peaks have a value corresponding to the relative intensity of this highest peak. The major peaks are retained, such peaks preferably having a relative intensity of at least 0.01 with fungi and for most of the other germs at least 0.02, more particularly above 0.05, more particularly above 0.1. The peaks with a relative intensity above either 0.01 or 0.02, more particularly above 0.05 or more particularly above 0.1, depending of the germs present in all sets of data obtained for a given reference strain are then retained, as far as they are present in at least 2 sub-cultures, preferably 5 or even 10 or more.
The set of peaks thus obtained is specific of a genospecies for a given growth condition (medium and duration), and with respect to the characteristics of a given spectrometer.
Other characteristics and advantages of the invention are given hereinafter with respect to the identification of 1) CNS at a species level, 2) non fermenting Gram negative bacilli isolated in cystic fibrosis, 3) yeasts and 4) molds. They are given to illustrate the invention without limiting its scope. It will be therein referred to FIGS. 1 to 7, which represent, respectively:



FIG. 1: MALDI-TOF-MS profiles of 10 isolates of the same strain of S. aureus CIP 7625;
FIG. 2: MALDI-TOF-MS dendrograms of Micrococcacae species, according to databases 1 (Muëller Hinton 24H) and 2 (Muëller Hinton 48H), thus designating the genospecies to be identified in this group of germs;
FIG. 3: m/z value of the peaks with a relative intensity above 0.1, concerning Staphylococci strains;
FIGS. 4 and 5: conservation of the set of peaks of selected strains among strains belonging to the same genospecies;
FIG. 6: aerobic and facultative anaerobic bacteria classification;
FIG. 7: MALDI-TOF-MS profiles of 6 Gram negative bacilli. 



1. Identification of CNS at a Species Level
Material and Methods
Bacterial Strains
The results given hereinafter were obtained by analysing fifty one strains of bacteria belonging to the Micrococcaceae obtained from the collection of Institut Pasteur (CIP) (Paris, France).
The selected strains used to establish the MALDI-TOF-MS databases are given in Table 1










TABLE 1







species
Strain









Staphylococcus aureus
CIP 7625



Micrococcus luteus
CIP 103664



M. lentus
CIP 103430



S. epidermidis
CIP 103563



S. warneri
CIP 103960



S. xylosus
CIP 8166



S. intermedius
CIP 8177



S. haemolyticus
CIP 81.56



S. saprophyticus subsp saprophyticus
CIP 104064



S. saprophyticus subsp bovis
CIP 105260T



S. lugdunensis
CIP 103642



S. hominis subsp hominis
CIP 102642



S. hominis subsp novobiosepticus
CIP 105721



S. capitis subsp capitis
CIP 8153T



S. capitis subsp ureolyticus
CIP 104191



S. caprae
CIP 104519



S. pasteuri
CIP 103831



S. cohni subsp cohni
CIP 8154T



S. cohni subsp urealyticum
CIP 104023



S. scheiferi subsp scheiferi
CIP 103643T



S. scheiferi subsp coagulans
CIP 104370



S. sciuri subsp sciuri
CIP 103824



S. simulans
CIP 8164 T










The strains belonging to the Micrococcacae family are given in Table 2.










TABLE 2







Species
Strain









S. aureus
68 clinical isolates



M. luteus
CIP A270



S. epidermidis
81 clinical isolates



S. warneri
CIP 106511




CIP 8165




6 clinical isolates



S. xylosus
CIP 103720




CIP 104065



S. intermedius
CIP 81.60



S. haemolyticus
CIP 104114




13 clinical isolates



S. saprophyticus subsp saprophyticus
CIP 76.125T




CIP 103545



S. saprophyticus subsp bovis
CIP 105262




CIP 105261



S. saprophyticus spp*
6 clinical isolates



S. lugdunensis
CIP 103584



S. hominis subsp hominis
CIP 81.57




CIP 104689



S. hominis subsp novobiosepticus
CIP 105719T



S. hominis spp*
10 clinical isolates



S. capitis subsp capitis
CIP 103688



S. capitis subsp ureolyticus
CIP 104192T



S. caprae
CIP 104000T




CIP 104520



S. pasteuri
CIP 105540T




CIP 103830




CIP 103832



S. cohni subsp urealyticum
CIP 104024T




CIP 104025



S. scheiferi subsp coagulans
CIP 104366



S. sciuri subsp sciuri
CIP 8162T




CIP 103583




CIP 103825







*the genospecies of these clinical strains were identified using the sequence of the sodA gene which does not discriminate between the 2 subspecies






One hundred and sixteen clinical coagulase negative Staphylococci (CNS) isolates were also studied: 74 strains isolated from blood cultures, 25 strains isolated from children mediastinitis (skin, mediastinal liquid, electrodes), 12 strains isolated from bone infections (Hôpital Necker-Enfants malades, Paris; Hôpital Raymond Poincaré, Garches, France), and 5 strains isolated from urinary tract infections. In addition, 68 clinical strains of Staphylococcus aureus isolated from miscellaneous infections were analyzed.
Phenotypic and Genotypic Identification
The clinical strains of CNS were differentiated from S. aureus strains by conventional phenotypic tests including Slidex latex agglutination test (BioMérieux, Marcy l'Etoile, France) and DNAse test. Tube coagulase tests were performed in case of discordance between the two latest techniques. The precise identification of the CNS was obtained by sequencing an internal fragment of the SodA gene as previously described. Briefly, extraction of genomic DNA from pure cultures of CNS was performed with Quiagene kit (Courtaboeuf, France). Partial SodA gene was amplified and the amplicons obtained were sequenced using ABI Big Dye Terminator v1.1 cycle sequencing ready reaction kit (Applied Biosystems, Foster City, Calif., USA) as previously described by Poyart et al. 2001, J. Clin. Microbiol. 39:4296-4301. The nucleotidic sequences were sent to the GenBank database for species assignment.
Using this strategy, the 116 clinical CNS isolates given in table 2 were identified.
MALDI-TOF-MS Technique
The strains were grown on Muëller Hinton agar or Columbia agar +5% horse blood (BioMérieux), incubated 24 or 48H at 37° C. For each identification, a isolated colony was harvested in 100 μl of sterile water; 1 μl of this mixture was deposited on a target plate (Bruker Daltonics, Bremen, Germany) in 3 replicates and allowed to dry at room temperature. One μl of absolute ethanol was then added in each well, and 1 μl of the matrix solution DHB (2.5 dihydroxybenzoic acid 50 mg/ml, acetonitrile 30%, trifluoroacetic acid 0.1%). After drying, 1 μl of the matrix solution DHB was added. Samples were then processed in the spectrometer MALDI-TOF-MS autoflex (Bruker Daltonics) with the flex control software (Bruker Daltonics). Positive ions were extracted with an accelerating voltage of 20 Hz in linear mode. Each spectrum was the sum of the ions from 5×200 laser shots coming from different regions of the same well, and was analyzed in a range from m/z 1000 to 23000. The analysis was performed with the flex analysis software and calibrated with the protein calibration standard T (Protein I, Bruker Daltonics). The data obtained with the 3 replicates were added to minimise random effect. The presence and absence of peaks were considered as fingerprints for a particular isolate. The profiles were analysed and compared using the software BGP-database available on the website http://sourceforge.net/projects/bgp.
Results
Strains listed in table 1 are those arbitrary selected as being representative of the genospecies.
Ten isolates of each of these selected strains were analyzed by MALDI-TOF-MS as described in the materials and methods section.
For each spectrum, a value corresponding to the intensity was given to each peak.
The peak with the highest intensity was arbitrarily set up to 1, all the other peaks had a value corresponding to the relative intensity of this highest peak (FIG. 1).
Minor peaks (relative intensity below 0.1) were inconstantly present. The analysis was then subsequently concentrated on peaks with a relative intensity above 0.1 that were present in all 10 sets of data obtained for a given strain.
In order to determine the impact of growth conditions on bacterial identification, the above described procedure was performed with all strains listed table 1 and grown on Muëller Hinton or Columbia agar supplemented with 5% horse blood for 24 or 48 hours.
The results are given in Table 3.
The databases were designated 1 through 4, respectively: they were obtained using bacterial cultures on Muëller Hinton 24H (database 1), Muëller Hinton 48H (database 2), columbia horse blood medium 24H (database 3), columbia horse blood medium 24H (database 4). For a given strain, the standard deviation of the m/z value for each of the conserved peak was never above 7. The m/z value of the peaks with a relative intensity above 0.1 and present in all 10 sets of data for each growth condition are reported on FIG. 3.







TABLE 3







Number of peaks for each database*

















database 1
database 2
database 3
database 4

database 1
database 2
database 3
database 4



















S. aureus
11
11 (8)
7 (2)
8 (4)
S. intermedius
10
12 (9)
7 (6)
6 (5)


S. capitis
8
14 (7)
4 (4)
6 (4)
S. lugdunensis
9
12 (5)
5 (3)
9 (4)


capitis











S. capitis
5
 9 (4)
6 (3)
4 (4)
S. pasteuri
11
 12 (11)
10 (10) 
12 (9) 


ureolyticus











S. caprae
5
12 (5)
6 (2)
6 (2)
S. saprophyticus
9
12 (8)
7 (2)
5 (3)







bovis






S. cohni cohni
6
 9 (4)
10 (2) 
11 (5) 
S. saprophyticus
8
10 (6)
7 (4)
11 (4) 







saprophyticus






S. cohni
6
12 (6)
7 (4)
8 (5)
S. schleiferi
7
 8 (6)
4 (4)
10 (5) 


urealyticum




coagulans






S. epidermidis
4
11 (4)
4 (2)
6 (2)
S. schleiferi
9
10 (9)
5 (5)
9 (5)







schleiferi






S. haemolyticus
5
10 (5)
4 (4)
8 (4)
S. sciuri sciuri
8
11 (8)
3 (1)
4 (3)


S. hominis
8
 9 (4)
4 (4)
5 (3)
S. simulans
8
 8 (7)
4 (5)
110 (7) 


hominis











S. hominis
6
12 (6)
8 (2)
8 (3)
S. warneri
9
13 (6)
10 (7) 
14 (6) 


novobiosepticus











S. intermedius
10
12 (9)
7 (6)
6 (5)
S. xylosus
5
 9 (4)
8 (4)
9 (4)


M. lentus**
3
 7 (3)


M. luteus
6
13 (4)
10 (4) 
13 (6) 





*number in brackets is the number of common peaks with those of database 1


**M. lentus did not grow on horse blood medium






Table 3 shows for each selected strain and each database the number of peaks that have been retained. Whereas for a strain, some peaks were conserved regardless of the medium or the time of culture, other peaks either disappeared or appeared upon changing culture conditions. However, for a given medium and time of culture, the set of peaks was strain specific as showed on dendrograms (FIG. 2).
Conservation of the Set of Peaks of Each Selected Strains Among Strains Belonging to the Same Genospecies
To address this point, MALDI-TOF MS was performed using bacteria grown on Muëller Hinton for 24 hours at 37° C. The strains used are the isolates listed table 2.
For each strain, the sole peaks with a value above 0.1 were retained. The profile obtained was then compared for each of these isolates with that of those of database 1.
To perform this task, a software (BGP-database available on http://sourceforge.net/projects/bgp) was developed allowing the rapid identification of the closest set of values in the database with that of the tested strain. This software chooses the best match between the tested strain and the reference strains of the database taking into account a possible error of the m/z value that has been measured. This value was arbitrarily set up to 7.
The tested strain had always the best match with the strain belonging to the same genospecies of the database. All data are reported on FIG. 4 and FIG. 5.
Average Number of Conserved Peaks of the Tested Strains Cultivated According on Muëller Hinton for 24H Compared to the Number of Peaks of Each Database
The same set of data obtained by growing the tested bacteria on Muëller Hinton during 24 hours was tested against databases 2, 3 and 4.
The results are given in table 4.












TABLE 4






Number of






Species
strains
database 1
database 2
database 3
database 4




















S. aureus
68
9.6/11 
8.7/11 
4.5/7  
4.7/8


S. capitis capitis
1
8/8
14/14
4/4
  6/6


S. capitis
1
7/7
7/9
6/6
  4/4


urealyticus







S. caprae
2
5/5
 8/12
6/6
  5/6


S. cohni urealyticus
2
6/6
 9/12
7/7
6.5/8


S. epidermidis
81
3.2/4  
5.1/11 
2.2/4  
3.4/6


S. haemolyticus
14
3.4/5  
5.4/10 
3.3/4  
4.8/8


S. hominis hominis
6
5.6/8  
*5.7/9  
3.5/4  
3.6/5


S. hominis novobio
7
4/6
*7.7/12  
***2.7/7   
*****3.1/8   


S. intermedius
1
10/10
11/12
7/7
  6/6


S. lugdunensis
1
8/8
11/12
5/5
  9/9


S. saprophyticus
3
7.6/9  
9.6/12 
****4/7  
  4/5


bovis







S. saprophyticus
7
6/8
**5.8/10  
5.1/7  
  8/11


saprophyticus







S. schleiferi
1
7/7
6/8
4/4
  10/10


coagulans







S. pasteuri
3
11/11
11/12
9.5/10 
11.6/12


S. sciuri sciuri
3
7/7
8.3/11 
2.3/3  
3.6/4


S. xylosus
2
5/5
9/9
7/8
  6/9


S. warneri
8
6.7/9  
8.8/10 
 8/10
 6.8/14


M. luteus
1
7/7
11/12
 8/10
  11/11





*One strain could not be differentiated be identified at the subspecies level


**3 S. saprophyticus bovis identifications


***6 S. hominis hominis identifications


****1 S. saprophyticus saprophyticus identifications


*****3 strains not differentiated between S. hominis hominis and S. hominis novobiosepticus






Table 4 shows for each genospecies the average number of peaks that were conserved between the tested strains and each of the four databases.
Even though the number of peaks that were conserved when the databases used for the genospecies identification were established using different growth conditions from those used for the tested strains, in all cases the identification at the species level was possible.
The only difference observed by using databases 2, 3 and 4 with those obtained with database 1, is that the identification at the subspecies level was impossible for S. hominis and S. saprophyticus unlike results obtained with database 1.
Altogether, said data demonstrate that by selecting an appropriate set of strains and retaining only the conserved peaks with a m/z above 0.1 a database that can be used for genospecies identification can be engineered. Furthermore the specificity of these peaks is such that genospecies identification remained possible even if the strains to be identified have been grown using different culture conditions than those used to engineer the database.
2. Identification of Non Fermenting Gram Negative Bacilli 
The first step was to build a complete database for all species belonging to the group of non fermenting Gram negative bacilli recovered in human. This database was then validated to identify using MALDI-TOF MS all the clinical non fermenting Gram negative bacilli that have been recovered from Cystic fibrosis (CF) patients in a one year period.
Material and Methods
Bacterial Strains
The reference strains used to engineer the MALDI-TOF MS database belong to 52 species of non fermenting Gram negative bacilli that can possibly be recovered from patients. These strains are listed Table 4.










TABLE 4





Species
Reference strain
Species
Reference strain







Pseudomonas aeruginosa
CIP 76.110
Inquilinus limosus
CIP 108342 T


Pseudomonas fluorescens
CIP 69.13 T
Pandoraea apista
CIP 106627 T


Pseudomonas mosselii
CIP 104061
Bordetella avium
CIP 103348 T


Pseudomonas putida
CIP 52.191 T
Bordetella bronchiseptica
CIP 55.110 T


Pseudomonas stutzeri
CIP 103022 T
Bordetella hinzii
CIP 104527 T


Pseudomonas mendocina
CIP 75.21 T
Alcaligenes faecalis subsp. faecalis
CIP 60.80 T


Pseudomonas alcaligenes
CIP 101034 T
Aeromonas sobria
CIP 74.33 T


Pseudomonas pseudoalcaligenes
CIP 66.14 T
Aeromonas hydrophila subsp. hydrophila
CIP 76.14 T


Pseudomonas oryzihabitans
CIP 102996 T
Aeromonas veronii
CIP 103438 T


Pseudomonas luteola
CIP 102995 T
Aeromonas caviae
CIP 76.16 T


Stenotrophomonas maltophilia
CIP 60.77 T
Delftia acidovorans
CIP 103021 T


Achromobacter xylosoxydans subsp.
CIP 71.32 T
Shewanella putrefaciens
CIP 80.40 T


xylosoxydans

Plesiomonas shigelloides
CIP 63.5 T


Achromobacter xylosoxydans subsp.
CIP 77.15 T
Chryseobacterium indologenes
CIP 101026 T


denitrificans

Elizabethkingia meningoseptica
CIP 60.57 T


Achromobacter piechaudii
CIP 60.75 T
Sphingobacterium multivorum
CIP 100541 T


Burkholderia cepacia
CIP 80.24 T
Sphingobacterium spiritivorum
CIP 100542 T


Burkholderia multivorans
CIP 105495 T
Brevundimonas diminuta
CIP 63.27 T


Burkholderia cenocepacia
CIP 108255 T
Brevundimonas vesicularis
CIP 101035 T


Burkholderia stabilis
CIP 106845 T
Sphingomonas paucimobilis
CIP 100752 T


Burkholderia vietnamlensis
CIP 105875 T
Cupriavidus pauculus
CIP 105943 T


Burkholderia dolosa
CIP 108406
Inquilinus limosus
CIP 108342 T


Burkholderia ambifaria
CIP 107266 T
Pandoraea apista
CIP 106627 T


Burkholderia anthina
CIP 108228 T
Bordetella avium
CIP 103348 T


Burkholderia pyrrocinia
CIP 105874 T
Bordetella bronchiseptica
CIP 55.110 T


Burkholderia gladioli
CIP 105410 T
Bordetella hinzii
CIP 104527 T


Burkholderia gladioli pathovar
ATCC 33664
Alcaligenes faecalis subsp. faecalis
CIP 60.80 T


cocovenenans

Aeromonas sobria
CIP 74.33 T


Burkholderia glumae
NCPPB 2391
Aeromonas hydrophila subsp. hydrophila
CIP 76.14 T


Burkholderia plantarii
ATCC 43733
Aeromonas veronii
CIP 103438 T


Burkholderia glathei
CIP 105421 T
Aeromonas caviae
CIP 76.16 T


Burkholderia andropogonis
CIP 105771 T
Delftia acidovorans
CIP 103021 T


Ralstonia mannitolilytica
CIP 107281 T


Ralstonia pickettii
CIP 73.23 T


Cupriavidus gilardii
CIP 105966 T


Cupriavidus pauculus
CIP 105943 T









The clinical isolates of non fermenting Gram negative bacilli were recovered from the sputum of CF children attending the pediatric department of the Necker-Enfants Malades hospital (Paris, France) between Jan. 1, 2006 and 31/12/06. Briefly, isolates displaying Green or Yellow-Green pigmentation, positive oxidase test, growth at 42° C., growth on cetrimide agar and susceptibility to colimycin were identified as Pseudomonas aeruginosa. Isolates which didn't express those criteria were identified by API 20NE system. The results of the API 20NE tests were interpreted using the APILAB PLUS software package. When the results obtained using this software were not considered as corresponding to a good identification for P. aeruginosa, A. xylosoxydans subsp. xylosoxydans and S. maltophilia, the bacteria were identified by sequencing an internal fragment of the 16S rDNA gene.
All bacterial strains used in the study were stored at −80° C. in trypticase soy broth supplemented with 15% glycerol.
MALDI-TOF-MS. The strains were grown on Mueller-Hinton agar and incubated for 24 h at 37° C. Most of the isolates grew after 24 h but some strains that didn't grow after 24 h were further incubated for 48 h or 72 h. An isolated colony was harvested in 100 μl of sterile water; 1 μl of this mixture was deposited on a target plate (Bruker Daltonics, Bremen, Germany) in three replicates and allowed to dry at room temperature. One microliter of absolute ethanol was then added in each well, and the mixture allowed to dry. One μl of matrix solution DHB (2,5-dihydroxybenzoic acid, 50 mg/ml, 30% acetonitrile, 0.1% trifluoroacetic acid) was then added. Samples were processed in the MALDI-TOF-MS spectrometer (Autoflex; Bruker Daltonics) with flex control software (Bruker Daltonics). Positive ions were extracted with an accelerating voltage of 20 kV in linear mode. Each spectrum was the sum of the ions obtained from 200 laser shots performed in five different regions of the same well. The spectra have been analyzed in an m/z range of 2,000 to 20,000. The analysis was performed with the flex analysis software and calibrated with protein calibration standard T (Protein I; Bruker Daltonics). The data obtained with the three replicates were added to minimize random effect. The presence and absence of peaks were considered as fingerprints for a particular isolate. The profiles were analyzed and compared using the newly developed software BGP database available on the website http://sourceforge.net/projects/bgp.
Results
Engineering of the Non Fermenting Gram Negative Bacilli Database
Reference strains listed Table 4 were used to determine the m/z values obtained by MALDI-TOF-MS that constitute a characteristic spectrum of each bacterial species and could then be used for bacterial identification. FIG. 7 shows the spectrum obtained with 6 species of non fermenting Gram negative bacilli. Ten isolates of each of these selected strains grown on Mueller Hinton 24H were analyzed by MALDI-TOF-MS as described in the material and methods section. For each spectrum, the same strategy as that used for Micrococcacae was adopted. According to this strategy, only those peaks with an intensity above 0.1 that were constantly present in all 10 sets of data obtained for a given strain were retained. For each strain, the m/z value of standard deviation (normalized data) for each conserved peak never exceeded 8.
Table 5 shows for 8 species the values of peaks that have been retained for the database.














TABLE 5





Achromobacter









xyloxosidans
Pseudomonas
Pseudomonas
Pseudomonas
Burkholderia
Burkholderia
Stenotrophomonas
Ralstonia


xyloxosidans
aeruginosa
fluorescens
putida
cenocepacia
gladioli
maltophilia
mannitolylitica


CIP 71.32T
CIP 76.110
CIP 69.13T
CIP 52.191
CIP 108255 T
CIP 105410T
CIP 60.77T
CIP 107281T







1208 +/− 1
4436 +/− 2
4339 +/− 1
3810 +/− 1
3600 +/− 2
4416 +/− 1
4541 +/− 1
3818 +/− 3


1419 +/− 1
4544 +/− 3
6089 +/− 1
4439 +/− 1
4414 +/− 2
4808 +/− 1
4862 +/− 1
4396 +/− 1


2190 +/− 1
5213 +/− 3
6275 +/− 2
6089 +/− 1
4806 +/− 2
5204 +/− 1
5276 +/− 1
4813 +/− 4


2319 +/− 1
5742 +/− 3
6403 +/− 1
6275 +/− 1
5202 +/− 2
5225 +/− 3
5898 +/− 3
5398 +/− 4


2447 +/− 1
6051 +/− 3
6634 +/− 2
6403 +/− 2
5345 +/− 2
6305 +/− 1
6108 +/− 1
6156 +/− 4


2576 +/− 1
6353 +/− 3
7182 +/− 1
6634 +/− 2
5916 +/− 3
6489 +/− 2
7161 +/− 2
7060 +/− 5


2705 +/− 1
6682 +/− 4
7604 +/− 2
7182 +/− 2
6486 +/− 3
6597 +/− 1
7578 +/− 2
7078 +/− 5


2833 +/− 1
6918 +/− 4
7647 +/− 1
7604 +/− 2
6503 +/− 4
6859 +/− 2
9349 +/− 2
7943 +/− 4


2865 +/− 1
7211 +/− 4
9568 +/− 1
7647 +/− 2
7089 +/− 3
6959 +/− 2

9632 +/− 4


2962 +/− 1
7586 +/− 4

9568 +/− 2
7216 +/− 3
7060 +/− 2




4334 +/− 1
7621 +/− 4


7318 +/− 3
7105 +/− 2




4978 +/− 1
8575 +/− 5


7397 +/− 3
7185 +/− 2




5018 +/− 1
9100 +/− 5


7967 +/− 3
7217 +/− 3




5242 +/− 1



9624 +/− 4
7325 +/− 2




6328 +/− 2




7673 +/− 1




6701 +/− 2




7794 +/− 2




7108 +/− 1




8737 +/− 2




7302 +/− 1




9627 +/− 2




9417 +/− 2




10458 +/− 4 




9445 +/− 2









10045 +/− 2 









10101 +/− 2 









The set of peaks retained for each reference strain gives a characteristic spectrum.
Identification of the Clinical Strains
Experiments were carried out to determine whether the above databases could be used for the identification of clinical non fermenting Gram negative bacilli, thus demonstrating that the set of peaks of each selected strain is, at least partially, conserved among isolates of the same species. From January to December 2006, 811 strains of non fermenting Gram negative bacilli were recovered and identified by phenotypical tests or molecular method: 699 P. aeruginosa strains (120 patients) 54 A. xylosoxydans (12 patients), 32 S. maltophilia (12 patients), 9 R. mannitolilytica (1 patient), 14 Bcc (2 patients), 1 B. gladioli, 1 B. hinzii and 1 I. limosus. Of these, MALDI-TOF-MS analysis was performed on a panel of 400 P. aeruginosa strains and on all remaining non P. aeruginosa strains. For each isolate, all m/z values of the spectrum were considered. For each strain, all peaks were retained regardless of their intensity and were then compared with that of the database using the BGP-database software. This software chooses the best match between the tested strain and the database, taking into account a possible error of the m/z value. This value was set up to 10. The strain was identified as belonging to the genospecies of the strain of the database giving the best match. The results are given in Table 6 below which shows the matches obtained for a P. aeruginosa strain provided by the BGP software.







TABLE 6







Profile Name: P. aeruginosa


Match: 13/13










06178603.0, txt




values
P. aeruginosa values







4441
4436



4548
4544



5216
5213



5743
5742



6053
6051



6354
6353



6682
6682



6917
6918



7209
7211



7584
7586



7620
7621



8571
8575



9096
9100











Profile Name: F. orzyhabitans


Match: 7/12










06178603.0, txt




values
F. orzyhabitans values







4441
4435



6053
6053



6505
6503



6682
6680



7497
7498



7584
7581



9123
9126










Table 7 shows the identification obtained for all clinical strains, compared to the second choice of species identification.










TABLE 7








Species





identified by
Comparison with the reference strain
Difference of % of common peaks with respective












Strains
Patients
conventional

Mean % of common
reference strains between the first and the second


(number)
(number)
technics
Reference strain
peaks
choice of species















400
100
P. aeruginosa
P. aeruginosa
86 (ext: 23-100)
35 (ext: 2-67)


54
11
A. xylosoxydans
A. xylosoxydans
91 (ext: 73-100)
75 (ext: 1-5)




subsp.
subsp.

(2nd choice: mostly A. piechaudii or A. xylosoxydans




xylosoxydans
xylosoxydans

subsp. denitrificans)


32
12
S. maltophilia
S. maltophilia
82 (ext: 63-100)
36 (ext: 4-59)


14
2
B. cepacia*
B. cenocepacia
89 (ext: 71-100)
23 (ext: 2-31)




complex





9
1
Ralstonia spp**
R. mannitolilytica
76 (ext: 60-80)
 8 (ext: 2-18)


1
1
B. gladioli
B. gladioli
78***
14***


1
1
B. hinzii
B. hinzii
90
34


1
1
I. limosus
I. limosus
60
35





*All strains except 3(identified as B. cepacia sensu stricto) were correctly identified as B. cenocepacia.


**All strains but one (identified as R. picketti) were correctly identified as R. mannitolilytica.


***this percentage is the same as that obtained with B. gladioli pathovar. cocovenenans






MALDI-TOF-MS correctly identified 100% of P. aeruginosa, 100% of A. xylosoxydans and 100% of S. maltophilia strains. MS identified A. xylosoxydans clinical strains at the subspecies level. MS analysis correctly identified 11 BCC strains as B. cenospacia. For the remaining 3 BCC strains, there was no differentiation between B. cenocepacia and B. cepacia sensu stricto. It should pointed out that the 9 R. mannitolilytica strains except one (identified by MS as R. picketti) were correctly identified by MS.
Conclusion
These results demonstrate that the database engineered according to the invention is also suitable for accurate species identification of non fermenting Gram negative bacilli. With MALDI-TOF-MS, it is thus possible, from one colony of non fermenting Gram negative bacilli (culture conditions, characteristics of the colony), to obtain within minutes a precise identification without additional test. The tested CF clinical strains were accurately identified at the species level except for three B. cenocepacia strains and one R. mannitolylitica. It is interesting to note that the strain identified as B. gladioli matched also with the reference strain of B. cocovenenans, a bacterial species that has been shown to be a junior synonym of B. gladioli. 
MALDI-TOF-MS enables very rapid bacterial identification while conventional biochemical identification by API 20NE often requires more than 24 h-48 h with frequent misidentifications. MALDI-TOF-MS strains identification is therefore a very interesting method for characterization of Gram negative bacilli species and will help understanding their clinical relevance and distribution.
3. Identification of Candida and Saccharomyces at the Species Level
Material and Methods
Yeast Strains.
The reference strains used to engineer the MALDI-TOF MS database belonged to 19 clinically relevant species of Candida, which have been involved in human pathology. The tested strains used to validate the databases were 148 clinical isolates of Candida belonging to 19 species: 25 C. albicans, 3 C. dubliniensis, 25 C. glabrata, 3 C. nivariensis, 20 C. tropicalis, 25 A. parapsilosis, 1 C. orthopsilosis, 6 C. krusei, 3 C. guillermondii, 1 C. haemulonii, 7 C. kefyr, 3 C. pelliculosa, 3 C. lipolytica, 2 C. norvegensis, 4 C. inconspicua, 1 C. lamblica, 7 C. lusitaniae, 1 C. sphaerica, 2 C. rugosa, 4 S. cerevisiae. All strains were identified by phenotypical methodologies (Auxacolor, ID32C BioMerieux) or by Internal Transcribed spacer (ITS) sequencing. All Candida strains used in the study were stored at −80° C. in trypticase soy broth supplemented with 15% glycerol.
C. albicans isolates belonging to the 17 ever well-described clades were chosen to identify different patterns of spectra within the C. albicans species, in order to be able to discriminate them each other and with other species. Indeed, C. albicans population is complex and can be divided in 17 genetical sub-populations using MLST method. Each clade has homogeneous genetical characteristics.
Some species included in the databases cannot be identified with current phenotypical methodology, but only with molecular identification based on ITS or D1/D2 sequencing.
Hence routine classical identification C. nivariensis and C. bracanensis cannot discriminate from C. glabrata, C. famata and C. fermantati from C. guillermondii, C. orthopsilosis and C. metapsilosis from C. parapsilosis. 
Engineering of the Database.
Clinical identification of Candida species in clinical samples is performed from different media, in particular Sabouraud dextrose agar and different chromogenic media able to discriminate several species among the color of the colony. 3 databases were constructed for each reference strain, one based on Sabouraud dextrose agar media (Biorad), one on CANDI2 (BioMerieux) and the third on CHROMAGAR. Discriminating peaks selected and included in the databases were obtained from 10 independent passages of each strain.
MALDI-TOF-MS.
The strains were grown on CANDI2 medium and incubated at 37° C. for 24 hours. One colony was collected with a 1 microliter hoese and mixed in 1 μl of 70% formic acid previously deposited on a target plate in triplicate (Bruker Daltonics, Bremen, Germany). This mixture allowed to dry at room temperature. One microliter of matrix solution SA (sinapinic acid, 10 mg/l, 30% acetonitrile, 0.1% trifluoroacetic acid) was then added and allowed to co-cristallize with the sample. Samples were processed in triplicates in the MALDI-TOF-MS spectrometer (Microflex, Bruker Daltonics) with the flex control software (Bruker Daltonics). Positive ions were extracted with an accelerating voltage of 20 kV in linear mode. The analysis was performed with the flex analysis software and calibrated with protein calibration standard I (Bruker Daltonics). The presence and absence of peaks were considered as fingerprints for a particular isolate. The profiles were analyzed and compared using the newly developed software BGP. Numerical data obtained from the spectrometer acquisition software (peak value and relative intensity for each peak) are sent to the BGP software. This software identifies the number of common peaks between the spectra of the tested strain and each set of peaks specific of a reference strain contained in the database (i.e Candida database). The software determines a percentage for each reference strain (100× number of common peaks between the tested strain and the peaks specific of one reference strain/total number of peaks specific of one reference strain). The identification of the tested strain corresponds to the species of the reference strain having the greater number of peaks after adding each peak of each specie of the first, second and third matches. The greater the difference between the first and second match, the better is the discrimination between species. A difference of at least 10% is required to obtain a good identification.
Results
The set of reference strains has been selected as belonging to clinically relevant Candida species and is given in Table 8 below:










TABLE 8







Species (n = 21)
Reference strains









C. albicans
ATCC 900028, SC5314



C. glabrata
ATCC 2001



C. parapsilosis
ATCC 22019



C. tropicalis
ATCC 750



C. krusei
ATCC 6259



C. guillermondii
ATCC 387



C. nivariensis
NIV1*



C. orthopsilosis
ORT1*



C. haemulonii
HAE1*



C. kefyr
KEF2*



C. pelliculosa
PEL1*



C. lipolytica
LIP1*



C. norvegensis
NOR1*



C. inconspicua
INC2, NOR2*



C. lamblica
LAM1*



C. lusitaniae
LUS 5*



C. sphaerica
SPH1*



C. rugosa
RUG2*



S. cerevisiae
CER1*



C. neoformans var. grubii
H99










Ten passages of each of these selected strains grown on Sabouraud agar with chloramphenicol were analyzed by MALDI-TOF-MS as described in the material and methods section. For each strain, only those peaks with a relative intensity above 0.07 that were constantly present in all 10 sets of data obtained for a given strain were retained. The standard deviation for each conserved peak did not exceed 10 m/z value. The set of peaks was specific of each selected strain.
The results are given in Table 9 hereinafter











TABLE 9








Number of
Correct identification



Species
strains
(%)




















C. albicans
25
25/25 (100%) 



C. glabrata
25
25/25 (100%) 



C. krusei
6
5/6 (83%) 



C. guillermondii
3
3/3 (100%)



C. nivariensis
3
3/3 (100%)



C. orthopsilosis
1
1/1 (100%)



C. haemulonii
1
1/1 (100%)



C. kefyr
7
7/7 (100%)



C. pelliculosa
3
3/3 (100%)



C. lipolytica
3
3/3 (100%)



C. norvegensis
2
2/2 (100%)



C. inconspicua
4
4/4 (100%)



C. lamblica
1
1/1 (100%)



C. lusitaniae
7
6/7 (85%) 



C. sphaerica
1
1/1 (100%)



S. cerevisiae
4
4/4 (100%)



19
144
128/144 (88%)   










Experiments were then carried out to demonstrate that the set of peaks of each selected strain is, at least partially, conserved among isolates of the same species. The database was tested using the set of strains described in the material and methods section. For each isolate, all peaks with intensity >0.01 were retained and were compared with that of the specific peaks of each reference strain included in the database using the BGP-database software, taking into account a possible error of +/−10 m/z value. The percentage of common peaks obtained with each of the reference strains was next analyzed for all 144 tested strains identified with phenotypical identification. Only the first and second best matches were retained. Acceptable identification of a tested strain corresponds to the species having ≧66 of common peaks with the reference strains in the database. A difference of at least 10% between the first and the second match is required.
Using the database of the invention, identification was correct in over 98% of the cases.
4. Identification of Aspergilli at the Species Level
Materials and Methods
Aspergillus Strains
The reference strains used to engineer the MALDI-TOF MS database belonged to 28 clinically relevant species of Aspergillus, which have been involved in human pathology (Table 10 hereinbelow). The tested strains used to validate the databases were 120 clinical and 16 environmental isolates of Aspergillus belonging to 7 sections: 50 A. fumigatus, 7 A. lentulus, 5 Neosartorya pseudofischeri, 2 A. viridinutans, A. fumigatiaffinis and A. hiratsukae, 1 A. fumisynnematus, N. fischeri and N. udagawae (section Fumigati); 13 A. flavus, 2 A. tamarii, 1 A. parvisclerotigenus (section Flaw); 8 A. terreus (section Terre), 9 A. niger, 2 A. foetidus, 1 A. tubengensis (section Nigri); 4 Emericella nidulans, 1 E. quadrilineata, 7 A. sydowii, 3 A. versicolor (section Nidulantes); 11 A. calidoustus, 1 A. pseudodeflectus, 1 A. insuetus (section Usti); 1 A. ochraceus (section Ochracei). All strains were cultured on Sabouraud agar with chloramphenicol and gentamicin (BioRad, Marnes-la-coquette, France). Clinical and environmental isolates were identified by molecular method based on multilocus sequencing using betatubulin and/or calmodulin genes. All the strains used in the study were stored at −80° C. in trypticase soy broth supplemented with 15% glycerol.
Construction of the Database.
A maturation of spores occurred during Aspergillus growth. For all references strains, 2 databases were constructed: one from early spores (less than 2 days of sporulation) and the other one from mature spores (more than 4 days of sporulation). 2 slow-sporulating reference strains (A. fumisynnematus and A. ochraceus) were included in only one reference database. Discriminating peaks selected and included in the databases were obtained from 10 independent passages of each strain.
MALDI-TOF-MS.
The strains were grown at 30° C. on Sabouraud dextrose Agar with chloramphenicol and gentamicin (Biorad, Marnes la coquette) and checked daily for consequent sporulation. Conidiophores and/or spores were collected gently at the surface of the colony and mixed in 1 μl of sterile water previously deposited on a target plate (Bruker Daltonics, Bremen, Germany). This mixture was allowed to dry at room temperature. One μl of absolute ethanol was then added in each well, and the mixture allowed to dry. One μl of matrix solution DHB (2,5-dihydroxybenzoic acid, 50 mg/ml, 30% acetonitrile, 0.1% trifluoroacetic acid) was then added and allowed to co-cristallize with the sample. Samples were processed in duplicates in the MALDI-TOF-MS spectrometer (Microflex, Bruker Daltonics) with the flex control software (Bruker Daltonics). Positive ions were extracted with an accelerating voltage of 20 kV in linear mode. The analysis was performed with the flex analysis software and calibrated with protein calibration standard I (Bruker Daltonics). The presence and absence of peaks were considered as fingerprints for a particular isolate. The profiles were analyzed and compared using the newly developed software BGP. Numerical data obtained from the spectrometer acquisition software (peak value and relative intensity for each peak) are sent to the BGP software. This software identifies the number of common peaks between the spectra of the tested strain and each set of peaks specific of a reference strain contained in the database (i.e Aspergilli database). The software determines a percentage for each reference strain (100× number of common peaks between the tested strain and the peaks specific of one reference strain/total number of peaks specific of one reference strain). The identification of the tested strain corresponds to the species of the reference strain having at least 66% of common peaks with the tested strain in the database. A difference of at least 10% between the first and the second match is required to obtain a correct identification. If the identification is not acceptable after one passage (ie <66% of common peaks with reference strain or <10% of difference between first and second matches), a second run was performed.
Results
A set of 28 reference strains (CBS or IHEM) have been selected as belonging to clinically relevant Aspergillus species They are given in Table 10 below which gives the reference strains used to establish the Aspergilli database.












Section
Species
Reference strains







Fumigati
A. fumigatus
IHEM 1246


(n = 11)
A. lentulus
CBS 116879



Neosartorya pseudofischeri
CBS 208.92



N. fischeri
IHEM 660



A. fumigatiaffinis
CBS 117194



A. fumisynnematus
CNM-CM-4063*



A. viridinutans
CBS 127.56



N. udagawae
CBS 114217



N. hiratsukae
CBS 109356



N. spinosa
CBS 483.65



N. fennelliae
CBS 598.74


Flavi
A. flavus
IHEM 306, IHEM 351


(n = 5)
A. oryzae
CBS 115.33



A. tamarii
FLA 17*



P. alliaceus
CBS 536.65



A. parvisclerotigenus
ASPSP SL 03*


Terrei
A. terreus
IHEM 5857


(n = 1)


Nigri
A. niger
IHEM 2864


(n = 3)
A. tubengensis
NIG 11*



A. foetidus
NIG 15*


Nidulantes
Emericella nidulans
IHEM 3665


(n = 4)
E. quadrilineata
CBS 426.77



A. sydowii
IHEM 566



A. versicolor
IHEM 2983


Usti
A. calidoustus
CBS 121601, IHEM 659


(n = 3)
A. pseudodeflectus
ASPSP 26*



A. insuetus
CNRMA F1-79*


Circumdati
A. ochraceus
OCH 1*


(n = 1)





IHEM: Institut scientifique de santé publique, BCCM-IHEM collection, Brussels, Belgium


CBS: Centraalbureau voor Schimmelcultures, Utrecht, the Nederlands


CNM-CM: Centro Nacional de Microbiologia-Servicio de Micologia, Madrid, Spain


*Reference spectra established with clinical isolates






The set of peaks was specific of each selected reference strain. For each strain, only those peaks with a relative intensity above 0.01 that were constantly present in all 10 sets of data obtained for a given strain we retained the standard deviation for each conserved peak did not exceed 10 m/z value.
It was then determined whether the above database could be used for the identification of Aspergillus species, thus demonstrating that the set of peaks of each selected strain is, at least partially, conserved among isolates of the same species.
The database was tested using the set of 120 clinical and 16 environmental Aspergillus isolates. The characteristics of the 136 isolates of Aspergillus used in this study are given in Table 11. Species identification of each strain was obtained by sequencing betatubulin and/or calmodulin gene as shown in said Table.













TABLE 11










GenBank
GenBank







Identification
Identification







number
number


Isolates number
Specie
Section
Source
Geographical origin
(betatubulin)
(calmodulin)






















OCH
01
A. ochraceus
Circumdati
Sputum
NEM, Paris, France
EF661322



FLA
05
A. flavus

Sputum
NEM, Paris, France
EF661492



FLA
07
A. flavus

Sputum
NEM, Paris, France
EF661492



FLA
08
A. flavus

Sputum
NEM, Paris, France
AY017536



FLA
10
A. flavus

Sputum
NEM, Paris, France
AY017536



FLA
14
A. flavus

Air hospital
NEM, Paris, France
AY017536



FLA
15
A. flavus

Sputum
NEM, Paris, France
AY017536



FLA
16
A. flavus

Sputum
NEM, Paris, France
AY017536



FLA
18
A. flavus
Flavi
Air hospital
NEM, Paris, France
EF661492



FLA
19
A. flavus

Nasopharyngeal lavage
NEM, Paris, France
EF661492



FLA
25
A. flavus

Bronchoalveolar lavage
NEM, Paris, France
EF661492



FLA
22
A. flavus

Bronchoalveolar lavage
NEM, Paris, France
EF661492



FLA
30
A. flavus

Nasopharyngeal lavage
NEM, Paris, France
EF661492



FLA
31
A. flavus

Sputum
NEM, Paris, France
EF661492



ASPSP SL
03
A. parvislerotigenus

Bronchotracheal aspiration
SL, Paris, France
EF203130
EF202077


FLA
17
A. tamarii

Bronchoalveolar lavage
NEM, Paris, France
AY0175410
EU021686


FLA
29
A. tamarii

Bronchoalveolar lavage
NEM, Paris, France
AY0175410
EU021686


CNM
CM3227
A. fumiaffinitis
Fumigati
Bronchoalveolar lavage
CNM, Madrid, Spain
*
*


CNM
CM2280
A. fumiaffinitis

Sputum
CNM, Madrid, Spain
*
*


ASPSP CO
01
A. fumigatus

Bronchoalveolar lavage
CO, Paris, France
AY048754



ASPSP
06
A. fumigatus

Air hospital
NEM, Paris, France
AY048754



FUM
01
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
02
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
03
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
05
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
06
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
07
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
09
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
11
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
12
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
14
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
15
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
16
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
18
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
19
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
23
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
24
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
28
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
29
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
30
A. fumigatus

Skin
NEM, Paris, France
AY048754



FUM
31
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
32
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
33
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
34
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
35
A. fumigatus

Bronchoalveolar lavage
NEM, Paris, France
AY048754



FUM
36
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
37
A. fumigatus

Bronchoalveolar lavage
NEM, Paris, France
AY048754



FUM
38
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
40
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
41
A. fumigatus

Bronchoalveolar lavage
NEM, Paris, France
AY048754



FUM
43
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
44
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
45
A. fumigatus

Bronchoalveolar lavage
NEM, Paris, France
AY048754



FUM
46
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
47
A. fumigatus

Air hospital
NEM, Paris, France
AY048754



FUM
48
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
49
A. fumigatus

Bronchoalveolar lavage
NEM, Paris, France
AY048754



FUM
50
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
51
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
52
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
53
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
54
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
55
A. fumigatus

Bronchoalveolar lavage
NEM, Paris, France
AY048754



FUM
56
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
57
A. fumigatus

Skin
NEM, Paris, France
AY048754



FUM
58
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
59
A. fumigatus

Sputum
NEM, Paris, France
AY048754



FUM
60
A. fumigatus

Bronchoalveolar lavage
NEM, Paris, France
AY048754



ASPSP SL
05
A. fumigatus

Bronchotracheal aspiration
SL, Paris, France
AY048754



CNM
CM4063
A. fumisynematus

Bronchoalveolar aspirate
CNM, Madrid, Spain
*
*


CNM
CM3303
A. hiratsukae

Skin
CNM, Madrid, Spain
*
*


CNM
CM3305
A. hiratsukae

Skin
CNM, Madrid, Spain
*
*


CNM
CM1290
A. lentulus

Sputum
CNM, Madrid, Spain
*
*


CNM
CM3364
A. lentulus

Bronchoalveolar lavage
CNM, Madrid, Spain
*
*


CNM
CM3537
A. lentulus

Sputum
CNM, Madrid, Spain
*
*


CNM
CM3583
A. lentulus

Sputum
CNM, Madrid, Spain
*
*


CNM
CM3599
A. lentulus

Sputum
CNM, Madrid, Spain
*
*


CNM
CM3134
A. lentulus

Sputum
CNM, Madrid, Spain
*
*


CNM
CM3538
A. lentulus

Sputum
CNM, Madrid, Spain
*
*


CNM
CM4518
A. viridinutans

Nail
CNM, Madrid, Spain
*
*


CNM
CM3147
A. viridinutans

Oropharyngeal exsudate
CNM, Madrid, Spain
*
*


FIS
01
N. fischeri

Bronchoalveolar lavage
NEM, Paris, France
AY870729



CNM
CM2270
N. pseudofischeri

Sputum
CNM, Madrid, Spain
*
*


CNM
CM4060
N. pseudofischeri

Sputum
CNM, Madrid, Spain
*
*


CNM
CM3914
N. pseudofischeri

Sputum
CNM, Madrid, Spain
*
*


ASPSP
25
N. pseudofischeri

Sputum
NEM, Paris, France
EF669823



PSE
01
N. pseudofischeri

Bronchoalveolar lavage
NEM, Paris, France
EF669823



CNM
CM4635
N. udagawae

Sputum
CNM, Madrid, Spain
*
*


NID
06
A. nidulans

Bronchoalveolar lavage
NEM, Paris, France
AY573547
EF652400


NID
10
A. nidulans

Bronchoalveolar lavage
NEM, Paris, France
AY573547
EF652400


NID
13
A. nidulans

Sputum
NEM, Paris, France
AY573547
EF652400


ASPSP SL
06
A. nidulans

Sputum
SL, Paris, France
AY573547
EF652400


ASPSP
04
A. sydowii

Air hospital
NEM, Paris, France
EU907905



ASPSP
27
A. sydowii

Air hospital
NEM, Paris, France
EU907905



SYD
04
A. sydowii

Sputum
NEM, Paris, France
EU907905



SYD
01
A. sydowii
Nidulantes
Sputum
NEM, Paris, France
EU907905



SYD
02
A. sydowii

Sputum
NEM, Paris, France
EU907905



SYD
03
A. sydowii

Sputum
NEM, Paris, France
EU907905



ASPSP SL
01
A. sydowii

Bronchotracheal aspiration
SL, Paris, France
EU907905



ASPSP
02
A. versicolor

Air hospital
NEM, Paris, France
EF652291
EF652379


VER
01
A. versicolor

Sputum
NEM, Paris, France
EF652291
EF652379


VER
03
A. versicolor

Sputum
NEM, Paris, France
EF652291
EF652379


NID
08
E. quadrilineata

Bronchoalveolar lavage
NEM, Paris, France
EF652288
EF652376


ASPSP
03
A. foetidus

Air hospital
NEM, Paris, France
AY585533
AM419749


NIG
15
A. foetidus

Skin
NEM, Paris, France
AY585533
AM419749


NIG
01
A. niger

Nasopharyngeal lavage
NEM, Paris, France
AY820002



NIG
03
A. niger

Air hospital
NEM, Paris, France
AY820002



NIG
04
A. niger

Sputum
NEM, Paris, France
AY820002



NIG
05
A. niger
Nigri
Air hospital
NEM, Paris, France
AY820002



NIG
06
A. niger

Sputum
NEM, Paris, France
AY820002



NIG
07
A. niger

Sputum
NEM, Paris, France
AY820002



NIG
16
A. niger

Sputum
NEM, Paris, France
AY820002



NIG
17
A. niger

Sputum
NEM, Paris, France
AY820002



NIG
25
A. niger

Air hospital
NEM, Paris, France
AY820002



NIG
11
A. tubengensis

Bronchoalveolar lavage
NEM, Paris, France
AY820009
EF661150


TER
01
A. terreus

Skin
NEM, Paris, France
EF669520



TER
02
A. terreus

Sputum
NEM, Paris, France
EF669520



TER
05
A. terreus

Sputum
NEM, Paris, France
EF669520



TER
06
A. terreus
Terrei
Sputum
NEM, Paris, France
EF669520



TER
07
A. terreus

Sputum
NEM, Paris, France
EF669520



TER
08
A. terreus

Sputum
NEM, Paris, France
EF669520



TER
10
A. terreus

Sputum
NEM, Paris, France
EF669520



TER
11
A. terreus

Sputum
NEM, Paris, France
EF669520



ASPSP
01
A. calidoustus

Air hospital
NEM, Paris, France
EF591730
EF591716


UST
11
A. calidoustus

Sputum
NEM, Paris, France
EF591730
EF591716


CNRMA
F2-25
A. calidoustus

Bronchoalveolar lavage
SL, Paris, France
EF591730
EF591716


CNRMA
F5-22
A. calidoustus

Sputum
SL, Paris, France
EF591730
EF591716


CNRMA
F6-60
A. calidoustus

Bronchoalveolar lavage
SL, Paris, France
EF591730
EF591716


CNRMA
F6-64
A. calidoustus

Sputum
SL, Paris, France
EF591730
EF591716


CNRMA
F6-66
A. calidoustus
Usti
Sputum
SL, Paris, France
EF591730
EF591716


CNRMA
F7-8
A. calidoustus

Sputum
SL, Paris, France
EF591730
EF591716


CNRMA
F7-13
A. calidoustus

Air hospital
SL, Paris, France
EF591730
EF591716


CNRMA
F7-15
A. calidoustus

Air hospital
SL, Paris, France
EF591730
EF591716


CNRMA
F7-17
A. calidoustus

Air hospital
SL, Paris, France
EF591730
EF591716


CNRMA
F1-79
A. insuetus

Bronchoalveolar lavage
SL, Paris, France
EU076372



ASPSP
26
A. pseudodeflectus

Air hospital
NEM, Paris, France
EF591732





*refer to Alcazar-Fuoli et al., AAC 2008






Tableau 12 gives results concerning the identification of Aspergilli by MALDI-TOF-MS based on the presence≧66% of the peak of the first match of the database specie and>10% difference between the first and the second match, after the first and the second passage

















Concordance between
Concordance between


Section


MALDI-TOF-MS and MLS
MALDI-TOF-MS and MLS


(number of

Number
with a single passage
with two passages


strains)
Species
of strains
(%)
(%)





















Fumigati
A. fumigatus
50
Early spores
Late spores
Early spores
Late spores


(n = 71)


44/50 (88%)
44/50 (88%)
47/50 (94%)
48/50 (96%)*













A. lentulus
7
21/21
(100%)
/



Neosartorya pseudofischeri
5






N. fischeri
1






A. fumigatiaffinis
2






A. fumisynnematus
1






A. viridinutans
2






N. udagawae
1






N. hiratsukae
2
















Flavi
A. flavus
13
12/16
(75%)
16/16
(100%)


(n = 16)
A. tamari
2







A. parvisclerotigenus
1
















Terrei
A. terreus
8
8/8
(100%)
/


(n = 8)


















Nigri
A. niger
9
10/12
(83.3%)
12/12
(100%)


(n = 12)
A. tubengensis
1







A. foetidus
2






Nidulantes
Emericella nidulans
4
13/15
(86.6%)
15/15
(100%)


(n = 15)
E. quadrilineata
1







A. sydowii
7







A. versicolor
3






Usti
A. calidoustus
11
12/13
(92.3%)
13/13
(100%)


(n = 13)
A. pseudodeflectus
1







A. insuetus
1
















Circumdati
A. ochraceus
1
1/1
(100%)
/


(n = 1)


















Total
24
136
121/136
(89%)
134/136
(98.4%)





*1 non-sporulating A. fumigatus strain non identified and 1 poorly-sporulating A. fumigatus strain identified as A. fumigatus (50% of matching peaks) with 10% difference with the second match after 1 month of culture






For each isolate, all peaks with intensity >0.01 were retained and were compared with that of the specific peaks of each reference strain included in the database using the BGP-database software, taking into account a possible error of +/−10 m/z value. Then the percentage of common peaks were obtained. Only the first and second best matches were retained. Acceptable identification of a tested strain corresponds to the species having ≧66% of common peaks with the reference strains in the database. A difference of at least 10% between the first and the second match is required.
Using the database of the invention, identification was correct in 121 of 136 isolates (89%) after the first passage and in 134/136 (98.4%) after the second passage. In table, results of MALDI-TOF-MS identification are presented for each section. No strains was misidentified, leading a specificity of 100%.
Conclusion
These results demonstrate that the MALDI-TOF database developed and validated according to the invention allows precise identification of an extensive number of Aspergillus species currently isolated in clinical settings, including the recently described species, such as A. lentulus, N. pseudofischeri, A. calidoustus, E. quadrilineata (sensitivity: 98.4%, specificity: 100%).
The database includes the reference spectra from early-spores and late-spores for each species but 2. This originality allows identification of Aspergillus isolates whatever the maturity of the tested isolate.
The panel of reference strains which were included in the study, was concordant with the distribution of Aspergillus species isolated in patients. In addition, the recently-described species which cannot be identified using traditional phenotypic approaches (non-A. fumigatus species of the section Fumigati, E. quadrilineata in the Section Nidulantes, and A. insuetus or A. pseudodeflectus in the section Usti), have been studied and included.

## Claims

1. A method for identifying a germ strain isolated from a clinical sample, at least one of the species and subspecies level, using MALDI-TOF-MS analysis comprising a step of classifying the germ in a group before performing the MALDI-TOF-MS analysis, wherein said analysis comprises retaining in the spectrum of a tested strain, peaks having, compared to a peak with the highest intensity arbitrary set up to 1, a relative intensity higher than 0.05.

2. The method of claim 1, wherein the classification step is based on at least one of growth conditions, a characteristic of colonies, morphology of bacteria upon microscopic examination, gram stain and phenotypic tests.

3. The method of claim 1, comprising performing the MALDI-TOF-MS technique on whole intact cells or on protein extracts obtained after lysis or corresponding to a fraction of cellular components.

4. The method of claim 1, comprising adding a benzoic acid derivative as matrix medium to isolated colonies before performing the MALDI-TOF-MS analysis, said benzoic acid derivative being advantageously selected in the group comprising DHB, and optionally further contains sinnapic acid fior the identification of Mycobacterium.

5. The method of claim 1, wherein each spectrum is the sum of at least 300 laser shots, coming from different regions containing the strain to be analyzed, said spectrum being analyzed, in a range of m/z from 500 to 50 000.

6. The method of claim 5, comprising using at least 1000 laser shots.

7. The method of claim 6, comprising using at least 2000 laser shots.

8. The method of claim 5, wherein said spectrum is analyzed in a range of m/z from 500 to 20000.

9. The method of claim 8, wherein said spectrum is analyzed in a range of m/z from 2000 to 20000.

10. The method of claim 1, wherein the strain to be identified is selected in the group comprising bacteria.

11. The method of claim 10, wherein the bacteria is a coagulase-negative Staphylococcus.

12. The method of claim 10, wherein the bacteria is a non fermenting Gram negative bacilli.

13. The method of claim 1, wherein the strain to be identified is selected in the group comprising yeasts.

14. The method of claim 1, wherein the strain to be identified is selected in the group comprising molds, such as filamentous fungi.

15. A method for identifying a germ strain isolated from a clinical sample, at least one of the species and subspecies level, using MALDI-TOF-MS analysis comprising a step of classifying the germ in a group before performing the MALDI-TOF-MS analysis, wherein said analysis comprises the step of comparing the spectrum profile of strains to be identified obtained by performing said MALDI-TOF-MS analysis with a database containing the characteristic MALDI-TOF-MS spectra of strains representative of the species or subspecies of the group of germs to which belongs the strain to be identified, said database containing for each representative strain, peaks having, compared to the peak with the highest intensity arbitrary set up to 1, a relative intensity higher than 0.1.

16. The method of claim 15, wherein said database contains for each representative strain, peaks having, compared to the peak with the highest intensity arbitrary set up to 1, a relative intensity higher than 0.05.

17. The method of claim 16, wherein said database contains for each representative strain, peaks having, compared to the peak with the highest intensity arbitrary set up to 1, a relative intensity higher than 0.1.

18. Databases consisting of peaks selected in the spectra obtained by MALDI-TOF-MS analysis of representative strains of species or subspecies, said databases being obtained
using a set of strains, each species or subspecies being represented by one or several strains, and
recording the spectrum of each strain of such group by using MALDI-TOF-MS and retaining the peaks with a relative intensity above 0.01 for fungi identification, and above 0.02 for other germs, which are present in at least 2 sub-culture.

19. The databases of claim 18, wherein, for each representative strain, the selected peaks have compared to the peak with the highest intensity arbitrary set up to 1, a relative intensity higher than 0.05.

20. The databases of claim 18, wherein, for each representative strain, the selected peaks have compared to the peak with the highest intensity arbitrary set up to 1, a relative intensity higher than 0.1.