MALDIToF Mass Spectrometric Analysis for the Identification of Plague, Cholera, and Tularemia Causative Agents M. V. Afanas’ev, L. V. Mironova, and S. V. Balakhonov Irkutsk Antiplague Research Institute of Siberia and the Far East, Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, ul. Trilissera 78, Irkutsk, 664047 Russia email: [email protected], mironova[email protected], [email protected] Received December 17, 2013
Abstract—General questions of the methodology of MatrixAssisted Laser Desorption/Ionization Timeof Flight Mass Spectrometry (MALDIToF MSanalysis, a new technology in laboratory diagnostics of infec tions), as well as a number of special questions concerning the use of this technology in the identification and typing of causative agents of extremely dangerous infections (plague, cholera, and tularemia), are considered in the present review. The problems of sample preparation for study and the ensurance of biological safety are discussed. Keywords: MALDIToF MSanalysis, MALDIToF MSidentification, Yersinia pestis, Vibrio cholera, Fran cisella tularensis DOI: 10.3103/S0891416815020020
GENERAL INFORMATION ON MALDIToF MASS SPECTROMETRY
the time of this movement, it is possible to calculate the rate of ion movement and, based on its value, to calculate the mass of particles presented in the ana lyte, as well as to generate a spectrum that character izes the qualitative composition of the studied object. The clinical and biological chemistry traditionally considered to be one of the main areas of MALDI ToF MSanalysis application in biology and medicine; this method was used for the qualitative and quantita tive detection of biomolecules of different nature in clinical material, including blood serum, urine, saliva, cerebrospinal fluid, tears, and tissue fragments . MALDIToF MS was for the first time used for the study of microorganisms quite a long time ago, in the middle of the 70s of the last century ; however, active introduction of this method in the practice of laboratory diagnostics started in the last decade. It was largely associated with the improvement of the instru ment base, the accumulation of factual material on the opportunities of the technology of mass spectrometric analysis for identification, and a deepened character ization of microorganisms. The identification of microorganisms using MALDIToF MS is performed by comparison of the protein spectrum of the studied strain with a basic spectrum collection of reference microorganisms of known species. Based on the degree of matching of spectra, the belonging of the studied microorganism to a certain species (genus) is determined . In addi tion, there is an algorithm of identification, in which the values of ion masses in the obtained spectrum of unknown microorganisms are compared with masses
In recent years, MatrixAssisted Laser Desorp tion/Ionization TimeofFlight Mass Spectrometry (MALDIToF MS) technology is one of the most actively developing directions in laboratory diagnos tics of infectious diseases . The MALDIToF method is based on the procedure of the soft ioniza tion of the studied material (analyte) allowing the ion ization of biological macromolecules (peptides, pro teins, DNA, oligonucleotides, lipopolysaccharides, and sugars) without their fragmentation and destruc tion under the effect of a laser in the presence of a spe cial substance (the socalled matrix). The matrix is a substance of an acidic nature, which (being cocrystal lized with analyte) provides the transfer of laser energy to the studied object molecules (by ionizing them and transferring into a gas phase) when exposed to the laser . Substances of both a complex organic and inor ganic nature are used as matrixes [3, 4]. αcyano4 hydroxycinnamic acid (CHCA), sinapinic acid (SA), ferulic acid (FA), and 2,5dihydroxybenzoic acid (DHB) are most widely used. After desorption, mainly singlecharged ionized molecules are accelerated in the electric field, put into the distributive part of the device (a tube, in which cavity vacuum is maintained), after the passage of which ions reach a detector. The rate of motion and, correspondingly, time of passing the distance from the point of ionization to the detector is inversely propor tional to the mass of ions. Knowing the length of the ionmovement path from ionizer to detector, as well as 57
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of proteins annotated in proteome databases and/or pre dicted based on genome nucleotide sequences [10, 11]. In the process of experimental studies, it was estab lished that most spectrum peaks (especially in the range from 4 to 15 kDa) correspond to proteins. Intact and passed posttranslational modification ribosomal proteins prevail among them (up to half of the peaks presented in the spectrum). Coldshock proteins and DNAbinding proteins are also rather stably detected in the spectrum [12–14]. The MALDIToF MSanalysis procedure is rather simple and does not require large amount of time or specialized skill. A pure microorganism culture (single isolated colony) collected in the exponential growth phase is required for the study. Then, application on a special metal substrate (target of either pure culture without additional treatment or extract obtained after preliminary treatment of the studied culture suspen sion by physical or chemical methods) is possible [15– 17]. In recent years, a number of approaches allowing a direct identification of the causative agent in some types of clinical material (such as urine, cerebrospinal fluid, and blood) are developed [18–20]. As regards to the instrumental base, it should be noted that to date there are 3 commercial MALDI ToF MS platforms for microorganism identification, including Andromas (Andromas SAS, Paris, France), VitekMS (bioMérieux, France), and BioTyper (Bruker Daltonics, Germany), for each of which an identification base was created and an algorithm of identification and sample preparation was developed [6, 21]. Multiple studies demonstrate the efficiency of the MALDIToF MSidentification method for the determination of taxonomic belonging of microorgan isms from different species and groups [6, 22]. The disadvantages and limitations of the MALDI ToF MSidentification method include the high cost of the equipment, the impossibility of carrying out the identification and interspecific differentiation of some groups of microorganisms associated with similarity of their mass spectrometric profiles, the low quality of spectra due to the resistance of some objects to the components of standard sample preparation proto cols, the absence of reference spectra available in the database, the insufficient resolution of the method, as well as the necessity of working with pure cultures and isolated colonies . Taking into account the high productivity, rate of analysis, simplicity, and low cost of sample prepara tion, the MALDIToF MS identification method fits well with the algorithm of functioning of microbiolog ical laboratories, especially if largescale screening studies of primary seedings are necessary . The uni versality of the MALDIToF MS platform allows us not only to identify the microorganism, but also to deter mine its properties (for example, such as resistance to antibacterial drugs  and the genetic profile ).
Of course, the aboveindicated peculiarities and advantages of mass spectrometric identification deter mine the topicality of its use in laboratory diagnostics of extremely dangerous and natural focal infections, since making a decision about the requirement and volume of emergency antiepidemic and preventive measures depends on the rate and accuracy of species identification. The peculiarities of using MALDIToF MS tech nology in the identification of causative agents of extremely dangerous bacterial infections on the exam ple of plague, cholera, and tularemia are considered in this review based on the literature data and our own experimental studies. MASS SPECTROMETRIC ANALYSIS OF PLAGUE CAUSATIVE AGENT AND OTHER MICROORGANISMS FROM THE Yersinia GENUS Initially, MALDIToF MS was used as a traditional method of the physical and chemical analysis of bio logical macromolecules for the study of members of the Yersinia genus. Using this technology, the studies of peculiarities of chemical structure and lipopolysac charide modifications in causative agents of plague (Y. pestis)  and intestinal yersiniosis (Y. entero colitica) , pseudotuberculosis causative agent (Y. pseudotuberculosis) proteins  were conducted. The MALDIToF mass spectrometry became applicable for interspecific identification of the Yers inia genus members relatively recently. A collection consisting of 146 Yersinia spp. strains, which includes all known at the moment Yersinia species (13 species), as well 35 strains of the Enterobacteriaceae family members of other species, was studied in one of the first work, which considered the possibility of mass spectrometric analysis application for determination of taxonomic position of the Yersinia spp. members . The authors created a base of speciesspecific spectra and conducted a comparative analysis. Based on the data obtained, groups of biological markers (spectral peaks with certain m/z value) were isolated; they correspond to the Enterobacteriaceae family (m/z values 4185 and 8370), the Yersinia genus (m/z values 4350, 5427, 6046, and 6241), and certain species, including the Y. enterocolitica (m/z values 7149, 7262, 7318, 9238, 9608, and 9651), Y. pseudotuberculosis/ Y. pestis (m/z 6637, 7274, 7783, 9268, and 9659), Y. pseudotuberculosis (m/z 6474), Y. pestis (m/z 3065). Using proteome databases, we succeeded to identify a number of biomarkers, comparing m/z values with specific cellular protein/peptide. Additional proteome analysis of protein Y. pestis extracts using highefficiency liquid chromatography and tandem MALDIToF mass spectrometry allowed us to establish that the spectral peak with m/z 3065 (specific for the Y. pestis) corre sponds to a peptide consisting of 30 amino acid resi dues, which is generated in the process of posttrans lational modification of plasminogen activator (Pla)
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molecule as a result of Nterminal fragment detach ment. Cluster analysis based on the spectral characteris tics of the studied strains provided an opportunity to specify the Y. ruckerii taxonomic position as an iso lated species relative to other members of the Yersinia genus. It should be noted that based on mass spectro metric profiles we could not differentiate the Y. similis, which was in a single cluster with the Y. pseudotuber culosis and Y. pestis. In general (according to the results of this work), the authors concluded that using the MALDIToF method allows (especially in combination with addi tional methods of bioinformatic analysis) to rather efficiently identify members of the Yersinia genus, including those with a clinical significance. Efficient species determination by means of mass spectrometric analysis of the plaguecausative agent and its differentiation from Y. pseudotuberculosis (a closely related species), as well as the correct taxo nomic identification of nonpathogenic Yersinia spe cies, were demonstrated in the work by S. Ayyadurai et al. . In addition to microorganism determina tion at the species level, the authors were able to carry out intraspecific Y. pestis clusterization (based on statis tical analysis of markerpeak frequency distribution), to assign strains according to their belonging to three main biovars (Antiqua, Medievalis, and Orientalis). The differentiation of plague and pseudotubercu losis causative agents by MALDIToF MS method is of special interest and a problem for research, since contradictory results were registered both in the abovementioned works [28, 29] and in subsequent observations (including on mass spectrometric plat forms using other principles of data analysis ), when it was possible to distinguish one species from another only using additional algorithms of the treat ment and analysis of spectra. In addition to identification, MALDIToF MS can be used for the determination of the degree of similarity of the studied plague microbe strains in order to estab lish sources and probable pathways of infection distri bution. The potential possibility of such analysis was demonstrated when it become possible to establish a potential source of the penetration of the highly virulent Y. pestis strain (which has a large epidemic significance) into territories where such causative agent variants were not previously registered, by the mass spectrometric method in combination with classical microbiological and molecular genetic approaches . In general, based on the above, about the high effi ciency and prospects of application of MALDIToF MS analysis in identification and deepened character ization of causative agents of plague and other Yersinia species (primarily enteropathogenic) can be con cluded. However, the question of the differentiation of closely related species of plague and pseudotuberculo sis causative agents (especially taking into account the Y. pestis subspecies classification) remains incom
pletely resolved. It is obvious that accumulation of fac tual material and testing of alternative algorithms of the comparative analysis are required at this stage of the work. MASS SPECTROMETRIC ANALYSIS OF CHOLERA CAUSATIVE AGENT AND OTHER MICROORGANISMS FROM Vibrio GENUS Different vectors of the MALDIToF MS applica tion were also reflected during the study of the cholera causative agent. Using this approach, fundamentally oriented studies of V. cholerae are conducted, includ ing construction of a proteome vibrio map, determi nation of differentiated levels of expression and differ ent isoforms of protein products, analysis of exopro teins of cholera vibrio strains of different epidemic significance, terminal epitopes of O1antigen, etc. [32–34]. The first studies on the rapid identification and characteristics of the Vibrio genus microorganisms and their differentiation from closely related taxonomic groups based on determination of the microbe cell protein profile were carried out in 2009–2010 [35, 36]. R. Dieckmann et al.  performed MALDIToF mass spectrometric analysis of a representative sample out of 83 strains, including strains of 17 Vibrio genus species and 7 Aeromonas species, as well as Grimontia hollisae and Photobacterium damselae strains. In the process of analysis, they were able to carry out differ entiation of closely related species from the Vibrio genus, such as V. parahaemolyticus–V. alginolyticus and V. cholerae–V. mimicus, which allowed the authors to make a conclusion on the high efficiency of this method. At the same time, the species identified as V. fluvialis and V. furnissii according to biochemical tests were mass spectrometrically indistinguishable, which was in agreement with data on one of reference methods of microorganism genetic identification (the rpoB gene sequencing). The authors carried out the determination of speciesidentifying biomarker ions (SIBI) for the deepened differentiation at generic, specific, and intraspecific levels. The high diagnostic value of MALDIToF mass spectrometric profiling for rapid and accurate identi fication and differentiation of Vibrio genus microor ganisms was confirmed during the study of the V. cholerae not O1/O139, V. alginolyticus, V. fluvualis, and V. metschnikovii strains isolated from waste water in Morocco . Analysis of spectral patterns demon strated that those typical for the Vibrio genus spectra contain approximately 150 peaks in the range 2– 20 kDa with the largest intensity in the area of mass 2– 11 kDa. Along with significant interspecific similarity, spectra are characterized by the presence of species specific biomarker peaks (m/z 2400, 2800, 3800), and the differentiation of separate groups of strains depending on their specific belonging is observed dur ing the cluster analysis.
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Mass spectrometric analysis was used for acceler ated microorganism identification in the ballast waters of sea vessels . Eight strains among the microor ganisms isolated from water samples were identified as belonging to the Vibrio genus by their protein spec trum profile. The similarity both at protein and nucle otide (determination of 16S rRNA gene structure) lev els has been determined only for 50% of these isolates, while others demonstrated the discrepancy on the results of two tests. Previously, such results were observed during the study of possibility of the Vibrio genus microorganism identification using a complex of methods (MALDIToF mass spectrometry, 16S rRNA and rpoB gene sequencing). It was found that analysis of 16S rRNA gene sequencing did not provide the pos sibility to differentiate V. alginolyticus and V. para haemolyticus isolates for a high comparability of the results of identification based on protein spectra and rpoB gene structure . Nevertheless, a significant difference of V. para haemolyticus spectral patterns from the spectra of other closely related Vibrio genus microorganisms was also established with MALDIToF mass spectrometric study of the V. parahaemolyticus strain collection iso lated from patients and from environmental objects in different regions of United States, as well as reference strains . Using TaqIdent instruments, the authors identified 30 biomarker peaks present only in V. para haemolyticus mass spectra. At the same time, the fact of intraspecific variability of the protein profile of the V. parahaemolyticus (isolated in different geographical points at different times and characterizing by differ ent epidemic potential), as well as in induced mutants with deletion of “quorum sensing” genes involved in the system, is interesting; this determines the suitabil ity of MALDIToF mass spectrometry not only for reliable and significant identification of strains, but also as an instrument for the monitoring of the appear ance of new pathogen clones and identification of kin ship of certain isolates. S.M. Malainine et al.  identified more than 20 variable peaks in the structure of spectral patterns obtained during MALDIToF mass spectrometric analysis of 22 V. parahaemolyticus strains. Variable biomarker peaks were characterized by smaller inten sity as compared with common for all strains peaks and were mainly localized in 6–11 kDa region. Using of MALDIToF mass spectrometric analysis for identification of cholera causative agent in Russia was limited until recently by the absence of specific for the V. cholerae spectra in supplied databases of MALDI Biotyper software. The studies conducted on the creation of one’s own reference libraries of the V. cholerae spectra with subsequent import to MALDI Biotyper demonstrated the efficiency of cholera vibrio identification using broadened base, including at an intraspecific level [41–43]. Thus, the MALDIToF mass spectrometry method has a number of advantages and is widely used, not
only in proteome analysis for protein mapping, but also in clinical laboratory diagnostics and monitoring studies for the identification of the cholera vibrio and other Vibrio genus microorganisms based on the deter mination of the reference protein profile of the micro bial cell. The possibility of obtaining individual spe cific for each strain of protein patterns by a “finger print” type determines in a number of cases the efficiency of using direct protein profiling for intraspe cific typing, identification of kinship of certain iso lates, and epidemiological monitoring. MASS SPECTROMETRIC ANALYSIS OF TULAREMIA CAUSATIVE AGENT Very few studies have been devoted to identification of members of the Francisella genus (and particularly the F. tularensis tularemia causative agent) using MALDIToF MS analysis. This may be due to the peculiarities of causativeagent cultivation (which requires special nutrient media and continuous incu bation) and the frequent impossibility of obtaining the culture by direct seeding of the studied material, espe cially during the study of environmental objects. Nevertheless, the experience of successfully using mass spectrometric analysis in complex characteristics of lipopolysaccharide (LPS) (obtained from vaccine F. tularensis SLV strain) was described. The study of crustal LPS structure (A lipid), as well as the chemical structure of fatty acids and sugars (which is a part of LPS), allowed us to more completely characterize this molecule (demonstrating its complexity and variabil ity) . The possibility of mass spectrometric identification of tularemia causative agent till specific and subspe cific level was rather convincingly demonstrated [45, 46]. Marker peaks were isolated during the com parative analysis of spectra, including peaks with m/z value 6153 D and 7757 D for the F. philomiragia spe cies, 6730 D for the F. tularensis subsp. tularensis, and 7800 D for the F. tularensis subsp. holarctica. The results of mass spectrometric identification were con firmed by the method of nucleotide sequence determi nation for the 23S rRNA gene. The reproducibility of the data was established by using different nutrient media, cultivation conditions, after conducting bio logical samplings, as well as on different models of mass spectrometric equipment. The results obtained allowed us to make conclusions about the efficiency of the use of mass spectrometric identification in the diagnostics of tularemia, especially for the rapid deter mination of the subspecific belonging of the causative agent.
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QUESTIONS OF BIOLOGICAL SAFETY DURING MASS SPECTROMETRIC STUDY OF MICROORGANISMS OF I–II GROUPS OF PATHOGENICITY The samplepreparation procedure is an important problem in the study of causative agents of I–II groups of pathogenicity/danger by the MALDIToF MS analysis method. According to the classification of infectious disease causative agents accepted in Russian Federation, the Y. pestis belongs to pathogenic biolog ical agent group I, while toxigenic V. cholerae and F. tularensis are categorized as group II; therefore, all manipulations with them should ensure the possibility of decontamination of working surfaces, instruments, and equipment. Being a rather complex analytical instrument, the mass spectrometer excludes the possi bility of regular current and final disinfection of inter nal working surfaces. Obviously, it is necessary to use such protocols that provide for the disinfection of the studied sample before its introduction in the equipment. On the other hand, the samplepreparation method should provide the obtaining of qualitative, reproducible spectra containing a sufficient amount of peaks in the studied range of masses (from 2 to 15–20 kDa). In rela tion to the plaguecausative agent, a comparative anal ysis of several methods of protein isolation was con ducted. Thus, C. Couderc et al.  compared two methods of a plaguecausative agent sample prepara tion for mass spectrometric analysis (method of extraction using 80% trifluoroacetic acid and method of treatment of the causative agent water suspension by 70% ethanol for 60 min). Both methods demonstrated a similar efficiency by inactivation capacity and repro ducibility. However, better and more informative spec tra were obtained when using alcohol extraction, which was reflected in the amount of peaks, as well as relative to the signal/noise parameter for major peak with m/z value 6049 and value of identification com pliance index. The duration of sample preparation can be attributed to the shortcomings of alcohol treat ment. M. Drevinek et al.  conducted a wider study and compared the applicability and biological safety of 4 different methods of sample preparation (extraction by trifluoroacetic acid, alcohol treatment with subse quent extraction by formic acid and acetonitrile, extraction using chloroform, and extraction using acetonitrile) for a wide spectrum of causative agents of I–II groups of biological safety/pathogenicity, including the B. anthracis, C. botulinum, B. melitensis, B. mallei, Y. pestis, V. cholerae, and F. tularensis. The best results were demonstrated by the method of alco hol treatment with subsequent extraction by formic acid and acetonitrile, in which it became possible to achieve the sterility of the studied samples (except spore formers), to obtain a maximal yield of proteins after extraction, as well as the most saturated spectra with the best ratio of signal/noise levels. In addition, this method allowed us to save samples for 26 days after extraction without a significant decrease in the effi ciency of identification.
It should be noted that manufactures of the Bio Typer identification platform use a similar method of extraction for the generation of reference spectrum libraries and recommend as the main method of sam ple preparation during work with grampositive and gramnegative asporous microorganisms . CONCLUSIONS In general, the MALDIToF method of mass spec trometric identification (considered in the review) demonstrates good results and is a rather efficient and promising instrument for laboratory diagnostics of causative agents of extremely dangerous infections. In our opinion, the introduction of MALDIToF MS analysis technology to the system of microbiolog ical monitoring of cholera (which is traditionally asso ciated with large volumes of the studied material and necessity of wide spectrum of screening studies) is one of the most promising in this direction [41, 42]. The creation and mass spectrometric analysis of strain collections for causative agents of infections of the I–II groups of biological danger/pathogenicity (relating to different bio, serum, genovariants iso lated in different territories and having different clini cally and epidemically significant properties) is an important direction for further work. Based on these data, the solution of the question on the perspectives of mass spectrometric intraspecific differentiation of the studied microorganisms will become possible . Certain efforts should be directed to the overcom ing of a serious limitation faced by researchers that exploit mass spectrometric BioTyper platform (Bruker Daltonics, Germany), the absence of reference spec tra of microorganisms of groups I–II of biological danger/pathogenicity (including causative agents of plague, cholera, tularemia, brucellosis, anthrax) in the version of the identification database supplied in the Russian Federation. The existing possibility of addi tion by the user of an available reference spectrum database raises the problem for researchers of the cre ation of collections of reference strains of microorgan isms of I–II groups of biological danger with subse quent generation of reference spectra and on the con duction of interlaboratory estimation of the efficiency of identification of extremely dangerous infection causative agents using updated database variants. REFERENCES 1. Fournier, P.E., Drancourt, M., Colson, P., Rolain, J.M., La Scola, B., and Raoult, D., Modern clinical microbi ology: new challenges and solutions, Nat. Rev. Micro biol., 2013, vol. 11, pp. 574–585. 2. Knochenmuss, R., Dubois, F., Dale, M.J., and Zenobi, R., The matrix suppression effect and ioniza tion mechanisms in matrixassisted laser desorp tion/ionization, Rapid Commun. Mass Spectrom., 1997, vol. 10, pp. 871–877.
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