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RSC Advances View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. REVIEW View Journal | View Issue Bacterial identification: from the agar plate to the mass spectrometer Cite this: RSC Advances, 2013, 3, 994 Patricia Aparecida Campos Braga,a Alessandra Tata,a Vanessa Gonçalves dos Santos,a Juliana Regina Barreiro,b Nicolas Vilczaki Schwab,a Marcos Veiga dos Santos,b Marcos Nogueira Eberlina and Christina Ramires Ferreira*a For more than a century, bacteria and fungi have been identified by isolation in culture followed by enzymatic reactions and morphological analyses. The identification of environmental microorganisms, however, remains a challenge because biochemical and staining protocols for bacteria identification are tedious, usually stepwise, can be long (days) and are prone to errors. Molecular techniques based on DNA amplification and/or sequencing provide more secure molecular identification of specific bacteria, but identification based on mass spectrometry (MS), mainly on MALDI-MS, has been shown to be an alternative accurate and fast method able to identify unknown bacteria on the genus, species and even subspecies level based profiles of proteins and peptides derived from whole bacterial cells. Breakthroughs such as non-culture-based identification of bacteria from biological fluids and MS detection of antibiotic Received 6th September 2012, Accepted 24th October 2012 resistance have recently been reported. This review provides an overview of the traditional bacterial and fungal identification workflow and discusses the recent introduction of MS as a powerful tool for the identification of microorganisms. Principles and applications of MS, followed by the use of high-quality DOI: 10.1039/c2ra22063f databases with dedicated algorithms, are discussed for routine microbial diagnostics, mainly in human www.rsc.org/advances clinical settings and in veterinary medicine. Introduction If a microbiologist working in the first decades of the 20th century stepped into a current microbiology laboratory, it is likely that he would not have a hard time feeling at home. In fact, most methods for bacterial isolation and identification have remained unchanged and are still based on the use of specific culture media for isolation and on classical morphological, staining and biochemical enzymatic assays for microorganism identification.1 Improved assays, high-throughput microbiological analysis with automated systems, molecular technologies based on DNA amplification/sequencing for microorganism identification and the detection of antimicrobial resistance are available.2,3 However, challenges such as the urgent identification of microorganisms and their antibiotic resistances in septicemic patients and in infants, the occurrence of genetic rearrangements in microorganisms that alter their behavior in enzymatic assays, and the need to identify less frequent or rare microorganisms are difficult to tackle with classical microa ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas, Campinas, 13083-970, SP, Brazil. E-mail: [email protected]; Fax: +55 19-3521 3073; Tel: +55 19-35213049 b University of São Paulo, School of Veterinary Medicine and Animal Science, Pirassununga, 13635-900, São Paulo, Brazil. E-mail: [email protected]; Fax: +55 19-5616215; Tel: +55 19-3565 4240 994 | RSC Adv., 2013, 3, 994–1008 biological approaches and may lead to incorrect identifications. In response to these challenges, a paradigm break for microbiology has been the introduction of mass spectrometry (MS)-based microorganism identification. This strategy emerged after the development of electrospray ionization (ESI)4 and matrix-assisted laser desorption/ionization (MALDI)5 at the end of the 1980s. Since the early 1990s, more than 13 000 Pubmed indexed manuscripts on microbiology associated with MS have been published. However, microorganism identification by MS is not only performed for research purposes. Dedicated instruments equipped with automated database search functions for almost real-time microorganism identification are being installed in hospitals, clinical institutes and commercial settings, mainly in Europe. In the United States, the Food and Drug Administration has still not approved any MALDI-MS system for organism identification, which limits the widespread implementation of this approach in this country.6 This review provides a general overview of classical microbiological approaches and the MS ionization strategies that have been mostly applied in microbiology. The most recent achievements in MS-based microorganism identification, such as the identification of uncommon pathogens and non-fermenting bacteria, non-culture identification of bacteria in biological fluids, identification of antibiotic resistance and This journal is ß The Royal Society of Chemistry 2013 View Article Online RSC Advances mixed culture analysis, are also described and discussed. Finally, real-world perspectives on moving bacterial identification from the agar plate to the mass spectrometer are presented. Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. Traditional bacterial and fungal identification based on bacterial isolation by culture and enzymatic assays There are several methods for routine microbial identification in clinical and research microbiology laboratories, but microbiologists have continuously pursued efficient systems for microorganism identification. These methods are mainly based on morphological and biochemical characteristics of bacterial colonies. For example, bacterial colony morphology can be evaluated under defined growth conditions to show hemolytic capacity, by Gram staining (Fig. 1a–b), and by examining growth performance on selective media that allow only specific bacteria to multiply, the potential to ferment sugars, specific biochemical reactions (Fig. 1c), metabolic characteristics, antigenic and pathogenic capacity, and antibiotic susceptibility.7 When determining the characteristics of a microorganism to provide its identification, a pure culture population of identical cells is needed; this means that these cells originate from the same parental cell. However, microorganisms in nature are typically found in mixed cultures with many different species occupying the same environment. Therefore, in a microbiological laboratory, the first step in the microorganism identification workflow is the isolation of the various species contained in a specimen. For this purpose, there is an extensive list of available commercial tools, and the chosen strategy depends on numerous factors. The main considerations are the source of the sample, the species that are expected to be present and the nutritional needs of these microorganisms. For example, the isolation medium may contain specific compounds that inhibit or prevent general Fig. 1 (a) Staphylococcus aureus (left) and Staphylococcus spp. (right) on blood agar. Note the hemolysis caused by S. aureus; (b) Bright light microscopy analysis: Gram-positive stained cocci; (c) Biochemical tests used to identify bacteria usually include color codes. This journal is ß The Royal Society of Chemistry 2013 Review microbial growth yet simultaneously are appropriate for growing the species that are determined to be present (Fig. 1a).8 After isolation of the microorganism, phenotypic characteristics such as enzymatic profiles, sensitivity to antibiotics and chromatographic analysis of fatty acids can be performed to characterize the strain.9 Microscopy is frequently used to characterize microorganisms (Fig. 1b), and there are two main microscopic techniques: light microscopy and electron microscopy. Light microscopy is routine in the microbiology setting and can be performed with bright field, dark field, fluorescence, and phase control. Constant development and refinement of the techniques used for optical microscopy allow one to perform additional specialized functions, such as evaluating biochemical processes occurring within living cells.10 For close to a century, bacterial identification relied on the interpretation of a skilled microbiologist using a microscope, specific media and antibiotics, but technological improvements over the past 50 years have allowed for both automation and simplification of analysis.2 Automated testing for bacterial identification in laboratories has become necessary as microbiologists attempt to answer higher demands and the need for fast diagnoses, such as in cases of patients with septicemia or neonatal infections. Automation also allows increasing numbers of specimens to be studied.11 Usually, the microorganism cell count can be determined in real time by incubators equipped with a system to measure the light absorption of liquid cultures because turbidity can be related to the number of cells. Another strategy of an automated laboratory system is based on testing the susceptibility of bacteria to a large number of antibiotics.10 Advances in the molecular biology field in the early 1980s resulted in novel approaches for microbial identification and characterization based on polymerase chain reaction (PCR) to amplify specific gene sequences of bacteria. PCR allows in vitro amplification of specific DNA or RNA sequences, the latter being performed following the synthesis of complementary deoxyribonucleic acid (cDNA). PCR is a technique with high specificity and applicability, and hundreds of methods have been described.12 The most important feature of PCR is its ability to exponentially amplify copies of DNA from small amounts of material. For PCR analysis, nucleic acids must be extracted, and several protocols using specific reagents and different strategies have been described for this purpose.10 Though time-consuming, costly and difficult in the case of multiplex assays, PCR-based bacterial identification is now a common and often indispensable technique used in medical and biological research for a variety of other applications, including forensic DNA typing, clinical diagnosis, DNA amplification for cloning or sequencing, paternity testing, construction of DNA libraries and detection of mutations.12–14 For example, Campylobacter, which is the most common cause of acute bacterial gastroenteritis in the world, may be detected in pork samples using this approach. Sixty Campylobacter strains isolated from porcine rectal swabs and from different areas in a pork processing plant were shown by PCR analysis to be mostly C. coli (86.9%) and C. jejuni.(13.1%).15 PCR is less prone to errors compared to traditional methods of identifying microbes that rely exclusively on phenotypic RSC Adv., 2013, 3, 994–1008 | 995 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. Review features and some the morphological characteristics of the organism to identify the strain, such as enzyme profiles, antibiotic sensitivity profiles, and chromatographic analyses of fatty acids.9 However, the expanding and impressive capabilities of MS-based microorganism identification are causing a revolution in the field of microbiology. Even though bacterial identification represents the largest interest in routine microbiology, invasive fungal disease plays an important role in the morbidity and mortality of immunocompromised patients.16 Approximately 60 years ago, Wickerham described a broth method that is specific for metabolic assimilation and fermentation testing of yeasts.17,18 Assimilation tests determine the ability to use various substrates as the sole source of carbon (e.g., sucrose) or nitrogen (e.g., KNO3). This method is still considered a powerful tool and was used to characterize and determine the taxonomy of yeasts. Most Wickerham media are not commercially available and are produced in the laboratory. This process is usually long and arduous and is employed only by a few laboratories because basal media and various substrates need to be prepared, sterilized, and dispensed prior to inoculation. Since many types of yeast can ‘‘carry-over’’ nutrients from the isolation medium, one must run negative controls for each test type and organism. Metabolic assimilation tests are read for turbidity, fermentation and gas production for up to four weeks. Due to their time-consuming nature, these ‘‘gold standard’’ assays have been replaced by more practical methods that are currently available. The first technical progress to improve the speed and sensitivity of yeast identification from fungal blood cultures and histological practices was the development of highly sensitive and specific molecular techniques, including PCR. At the molecular level, genetic sequence variation offers an alternative to culturing for the detection and identification of fungi. For example, ribosomal genes have conserved sequence regions that are ideal for primer targeting as well as regions of variability that are useful for species identification. DNA amplification techniques, with subsequent species-specific probing of the amplicons or PCR-enzyme immunoassay, have also been introduced to overcome the problems of sensitivity, specificity, and delay that are encountered with conventional methodology. These methods have already shown great promise in the field of diagnostics. The use of species-specific probes, however, is not always an efficient approach in mycology, given the large number of potentially pathogenic fungi.19,20 In parallel with the development of molecular methods, there has also been an emphasis on improving commercial kits based on substrate utilization or hydrolysis. The results are determined by increased turbidity, the generation of colored products, or the detection of fluorescent products. Some colored products require the addition of reagents to reveal the color, while others are self-revealing. These kits enable presumptive identification of the most important etiologic agents of yeast infections. Another focus of these rapid tests has been to screen for species that are commonly associated with resistance to antifungal compounds.21 Some kits are read manually, while others are read automatically. 996 | RSC Adv., 2013, 3, 994–1008 RSC Advances Many systems employ the aid of computer algorithms for rapid and reproducible data analysis.21 Merits of mass spectrometry in microorganism identification: time-saving and reliablity ELECTROSPRAY-BASED BACTERIAL IDENTIFICATION (PCR-ESI-QTOF). Scientific advances in MS, such as the development of the ‘‘soft’’ ionization techniques MALDI5 and ESI,4 have allowed the ionization, detection and characterization of large intact biomolecules. An improved understanding of the limitations associated with MS analysis of nucleic acids led to the ionization of intact PCR products by ESI.4,22 This capability resulted in the development of MS analysis of nucleic acids for microorganism identification, which was first described in 2005 by Hofstadler and co-workers.23–26 This approach, previously termed TIGER (Triangulation Identification for Genetic Evaluation of Risks) or PCR-ESIQTOF-MS, uses broad-range primers for PCR analysis to amplify products from diverse organisms, such as viruses, bacteria, fungi and protozoa within a taxonomic group, that are present in samples combined with PCR product calculations using MS (Fig. 2). The Ibis T5000 Universal Biosensor is an automated platform for pathogen identification that is based on TIGER technology. In its commercial form, Ibis T5000 is capable of identifying and strain typing a broad range of pathogens in a blinded panel from human or animal samples.28–30 Because the Ibis T5000 provides digital signatures of identified microorganisms, this technology allows the collection and dissemination of epidemiological information in real-time.31–33 A major advantage of this methodology is the ability to characterize an organism without prior knowledge by the instrument operator as well as rapid sample preparation. Since rapid pathogen identification significantly reduces rates of patient mortality, technologies for the correct and timely diagnosis of bloodstream infections are urgently needed. PCR-ESI-MS has been used as a new strategy for detecting bloodstream infections and has provided high concordance with results from standard methods, particularly at the genus level. The results from this technique can also be obtained in five to six hours, whereas culture and biochemical characterization techniques typically require one to five days for confirmation of microbial identification.34,35 Rapid detection and identification of Ehrlichia species, a tick-borne pathogen responsible for causing Ehrlichiosis disease, was performed by PCR-ESI-MS directly from crude blood samples without microorganism culture. The results from an enzyme immunoassay that was also performed showed 100% agreement with the PCR-ESI-MS results.36 The detection of bloodstream infections can be biased, as blood cultures are reported to be negative in more than 50% of the cases where bacteria are believed to exist. This approach allows the detection and identification of both culturable and unculturable organisms by the same method, in addition to the identification of mixed populations of bacteria. The PCRESI-MS platform not only identifies organisms that are present in a clinical sample but is also capable of providing information about the strain type.37 This approach has been applied for the genotypic characterization of S. aureus isolates and to detect the presence or absence of genetic elements that This journal is ß The Royal Society of Chemistry 2013 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. RSC Advances Review Fig. 2 An overview schematic of the traditional bacterial identification workflow and the recent integration of mass spectrometry techniques. Figure adapted from Drake et al., 201127 with permission from John Wiley and Sons. encode potential virulence factors and antibiotic resistance elements. PCR-ESI-MS can also distinguish S. aureus from other coagulase-negative staphylococci (CoNS) isolates.38,39 Members of the genus Acinetobacter, which are aerobic Gram-negative organisms that are widely distributed in soil and water in the natural environment and are important nosocomial (hospital-acquired) pathogens, were isolated from infected soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq. Through the PCR-ESI-MS technique, it was possible to distinguish at least 16 Acinetobacter species, and the genotyping of A. baumannii showed a genetic relationship between endemic European isolates and many of the isolates found in patients and in military hospitals, indicating that this approach provides a better understanding of the origins of these infections and will improve infection control and prevention measures.40 The application of this technique has also been described for the genotyping of pathogens related to food-borne illnesses, and it proved to be highly effective in differentiating C. jejuni isolates in a panel of 50 Campylobacter isolates as well as determining the correct classification of C. coli instead of C. jejuni (Fig. 3).41 This journal is ß The Royal Society of Chemistry 2013 The Ibis 5000 platform has also been applied in aquatic environmental analysis to identify different Vibrio species directly from natural aquatic samples. From 278 total water samples that were screened, nine different Vibrio species were detected, and 41% of samples were positive for V. cholerae, a pathogen responsible for cholera disease. The results also indicated that V. mimicus could be correctly identified and distinguished from the close species V. Cholerae.42 PCR-ESIMS has been described as a high-throughput method to simultaneously identify, based on genotype, a number of bacterial species from complex mixtures in respiratory samples taken from military recruits during respiratory disease outbreaks and follow up surveillance at several military training facilities.43 PCR coupled to ESI-MS has also been described as a powerful tool for detecting other microorganisms such as viruses.44–50 Use of ambient desorption/ionization techniques for direct bacterial identification Ambient desorption/ionization describes a new set of MS techniques that are performed in an open atmosphere directly on samples in their natural environments or matrices or by using auxiliary surfaces. Ambient MS has greatly simplified RSC Adv., 2013, 3, 994–1008 | 997 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. Review RSC Advances Fig. 3 Deconvoluted ESI-TOF mass spectra of PCR amplicons of the tkt housekeeping genes from six different C. jejuni strains. Both the forward (e) and the reverse (#) strands of the PCR amplicons from each strain are clearly evident in the spectra (e.g., for strain RM4197, the forward strand is A49, G22, C26 and T45 and the reverse strand is A45, G26, C22 and T49). As can be observed in the stacked spectra, differences due to variations in the sequence (and thus the base composition) are readily discernible. Note that any mass differences resulting from changes in the number of guanosines are enhanced by the use of 13C guanosine (G*). The T5000 software automatically determines the base composition of each strain and provides a strain association by using a set of eight primer pairs. Reproduced from ref. 41 with permission from the American Society for Microbiology. and increased the speed of MS analysis, and especially after 2004, this approach has experienced a large trend towards realworld rapid chemical analysis of untreated samples in ambient conditions.51,52 Recently, several new high-throughput ambient desorption/ionization methods have been reported. One of the most studied ambient ionization methods is desorption electrospray ionization (DESI), which was introduced by Cooks and co-workers in 2004.53 DESI involves spraying untreated samples with ionized solvent droplets from a pneumatically-assisted electrospray. Desorption and ionization of analytes occurs through interactions of the charged droplets with the surface from which they pick up organic molecules, and they are delivered as desolvated ions into the mass spectrometer (Fig. 4a). Another well-explored ambient ionization technique is the direct analysis in real time (DART) method, first described by Cody and co-workers in 200554 (Fig. 4b). Due to the increasing importance of rapid identification of bacteria for food, biosafety and medical analysis, ambient desorption/ionization methods are of substantial interest.55 Both the DESI and DART techniques allow direct and rapid analysis of condensed phase samples without any sample preparation or the need to introduce the samples into the vacuum system of the mass spectrometer. DESI and DART have been widely utilized in many different applications, including bacterial analysis and identification.51 The most important characteristic of DESI and DART-MS approaches is the absence of sample preparation, so the real-time identification of microorganisms by these approaches appears to be feasible.56,57 Cooks and co-workers were the first to recognize the potential of these ambient desorption/ionization methods for microorganism identification when they performed DESI-MS 998 | RSC Adv., 2013, 3, 994–1008 Fig. 4 Schematic of ambient MS techniques used for direct microorganism analysis. These approaches usually make use of untreated samples, which are desorbed and ionized from surfaces or solutions under normal atmospheric conditions. (a) For DESI, an ionized solvent is pneumatically sprayed onto the sample, forming a thin film in which the sample molecules are dissolved. Secondary droplets containing ionized analytes are then delivered in the direction of the mass spectrometer inlet. (b) DART uses an electrical potential applied to a gas with a high ionization potential (typically nitrogen or helium) to form a plasma of excited-state atoms and ions, which desorbs low-molecular weight molecules from the surface of a sample. (c) In LTP-MS, there is no need for any solvent. The ion source consists of a glass tube with an internal grounded electrode centered axially and an outer electrode of copper tape surrounding the outside of the glass tube. An alternating voltage is applied to the outer electrode with the center electrode grounded to generate the dielectric barrier discharge. The discharge AC voltage is provided by a custombuilt power supply with total power consumption below 3W. Helium is used as the discharge gas, and it is fed through the glass tube to facilitate the discharge to direct the plasma onto the sample surface and to transport analyte ions to the mass spectrometer. on freshly harvested cells from untreated E. coli and Pseudomonas aeruginosa samples deposited on a polytetrafluoroethylene (PTFE) target. The characteristic DESI mass spectra for each microorganism that was analyzed demon- This journal is ß The Royal Society of Chemistry 2013 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. RSC Advances strated the potential of the technique for microbiological application.58 The similarity of the spectra for different samples of the same culture or for E. coli different cultures was evaluated and indicated that DESI-MS for microorganisms was reproducible. The characteristic constituents of bacteria were observed without chemical derivatization; in particular, acylium ions of fatty acids were observed directly, not as the usual methyl esters. Principal component analysis (PCA) was performed on the DESI-MS data to differentiate the bacteria studied into two well-separated groups that could be identified based on the first principal component (PC1), which corresponds to the differentiation between E. coli and S. typhimurium. Because the mass spectra were recorded directly from freshly harvested microorganisms, and no chemical reagent or other processing step was used to disrupt the cells before MS analysis, any variations in the final spectra associated with sample pretreatment were eliminated in collaboration with the separation. Although only two different species were evaluated, this study demonstrated the possibility of performing in situ identification using DESI-MS, including sub-species differentiation of microbiological agents.56 Meetani et al. (2007) also applied DESI-MS to bacterial identification for a larger number of different samples. In this study, seven different bacterial species were evaluated on the basis of their spectra profiles. Incubated bacterial cells were transferred from agar plates, washed to remove media components, applied on glass slides and directly subjected to DESI-MS analysis. The mass range from 50–500 was used, and the observed ions in the mass spectra revealed the presence of free fatty acids, such as palmitic acid (16:0) at m/z 257 for the protonated molecule and m/z 279 for the sodium adduct. Further comparisons of the mass spectra with lowmass matrix-assisted laser/desorption ionization (MALDI) mass spectra of bacteria did not show common ion signals, indicating the likely complementary nature of DESI-MS and MALDI-MS for whole-bacteria identification. The mass spectra of negative ions were also evaluated, showing a greater number of detected ions than their counterpart positive ion spectra throughout the measured m/z range.57 In vivo recognition of bacteria was evaluated in another study from the Cooks group that examined direct profiles of intact biofilms of Bacillus subtilis by DESI-MS, noting that the biofilms were still viable after the experiment. The authors reported that the DESI plume primarily desorbs materials from the bacterial cell envelopes outer layers together with excreted metabolites. Bacteria with a rigid cell wall can withstand the impinging sprayed droplets. This assumption was corroborated by experimental results from Gram-negative and Gram-positive bacteria. The outermost layer of Gramnegative bacteria is the cell outer membrane, while the outermost layer of Gram-positive bacteria is a thicker cell wall. For Gram-negative species, the outer membrane is relatively easy to break, and its major phospholipids (PL) can be readily ionized, which leads to PL dominance in the resulting DESI mass spectra. However, the thicker cell walls of Gram-positive species are more difficult to break, and their major components, which correspond to 90% of glycans, are much more easily ionized than PL. Consequently, the excreted This journal is ß The Royal Society of Chemistry 2013 Review metabolites are observed as the dominant species in the resulting DESI mass spectra, especially those lipopeptides that are produced in large quantities, are surface-active and ionize with high efficiency, such as the surfactins.59 Fernandez and co-workers have also applied DART-MS to two different bacterial samples.60 They describe the detection of fatty acid methyl ester (FAME) ions from whole bacterial cell suspensions and their identification by accurate-mass orthogonal TOF-MS. This study is interesting because the ‘‘gold standard’’ methods routinely used in bacterial taxonomy and classification are based on the determination of microbial FAME composition after culturing, a process that forms the basis of the commercial Sherlock microbial identification system (MIDI Inc., Newark, Delaware, US). Routine FAME analysis involves lengthy sample preparation, starting with the hydrolysis of bacteria cells followed by fatty acid methylation. Gas chromatography coupled to mass spectrometry (GC-MS) is then used for separation and the detection of FAME composition. Each GC-MS run generally takes 20 to 30 min, whereas the total DART-MS analysis takes less than 10 min.55,61 FAME were generated from approximately 107 cell mL21 Streptococcus pyogenes and E. coli. After incubation, cells were washed with TRIS-sucrose buffer, suspended in water, and diluted with a solution of tetramethylammonium hydroxide (TMAH) to produce thermal hydrolysis and methylation of bacterial lipids. An aliquot of the whole bacterial cell suspension mixed with TMAH was deposited in the bottom of the capillary tube. The capillary was positioned so that the bottom of the tube came in contact with the DART He stream directly in front of the mass spectrometer inlet orifice after sliding the sample holder arm. The protonated FAME C9:0, C10:0, C11:0, C12:0, C14:0, C15:0, C17:1/cycloC17:0, and C19:1/cycloC19:0 were found to be present only in E. coli, while C11:1 was uniquely detected in S. pyogenes. C17:1/cycloC17:0 and C19:1/cycloC19:0 were found in E. coli at relatively high abundances but were not detected in the S. pyogenes spectrum, which is in accordance with the membrane characteristics of Gramnegative bacteria. Some FAME ions were common to E. coli and S. pyogenes; however, clear differences existed in the relative abundances of these ions in the mass spectra. Differences among samples were thus observed in the spectral temporal and intensity domains.60 Recently, a new ambient ionization technique termed low temperature plasma mass spectrometry (LTP-MS) was applied for bacterial identification.60 This ambient ionization method was introduced in 2008 by Cooks and co-workers, and the absence of any solvent is the distinguishing feature of this plasma-based method (Fig. 4c).55 LTP-MS was employed to detect fatty acid ethyl esters (FAEE) from bacterial samples in a direct way. Positive ion mode FAEE mass spectrometric profiles for 16 different bacterial samples were obtained without extraction or other sample preparation. Data were examined by PCA to determine the degree of possible differentiation among the bacterial species. Growth media effects were observed, but in this case, they did not interfere with species recognition based on the PCA results.55 Ambient desorption/ionization techniques can therefore be applied to bacterial identification, but in some cases, such as for DESI that uses a high velocity nebulizing gas, it is necessary RSC Adv., 2013, 3, 994–1008 | 999 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. Review to secure or fix bacterial cells onto the DESI probe surface to prevent sample dispersion and/or aerosolization during analysis. This procedure is required for the analysis of pathogenic bacteria, and heating (for example, at 220 uC for 30 s) or long-term drying (.1 h at room temperature) of bacterial cells are effective means of fixing the sample onto a glass slide surface.57 Different groups using the same ambient ionization technique have presented different results due to the dependence of mass spectral profiles of intact bacteria on the experimental design and the way in which growth media was used and/or prepared. Future investigations in this area appear to require detailed evaluation of the experimental design and its influence on the data output.57 However, the successful demonstration of identification by ion composition from whole bacterial cells via ambient MS analysis, mainly for segregating bacterial strains according to Gram status, constitutes the first step that could in the future lead to the successful development of new approaches for high-throughput microbial identification from a variety of biological, food, and water samples in the open air with minimum sample preparation.57 MALDI-MS based platforms: real-world breakthroughs in microbiology PROTEOMIC STUDIES FOR THE IDENTIFICATION OF BIOMARKERS. Approaches that use proteomics as a tool for studying expressed proteins are increasingly being utilized to address diverse biomedical questions. Via the identification of specific and conserved biomarkers, such as ribosomal proteins, peptides and lipids, it is possible to provide cancer diagnoses, study inflammatory and degenerative diseases and to determinate pathogens responsible for a broad range of diseases.62–66 Protein profiles obtained from direct MALDI-MS analysis of intact microorganisms or protein extracts have revealed robust biomarkers that are mostly related to conserved and specific ribosomal proteins. Currently, bacterial species identification by MALDI-MS is the MS approach that has the highest impact in the field of microbiology.67,68 This approach is based on the acquisition of ribosomal protein fingerprints directly from protein extracts from intact organisms. Interestingly, these protein profiles, which primarily contain ribosomal proteins, have been found to vary considerably and allow the proper characterization of different microorganisms. These protein markers are rapidly being incorporated into human clinical microbiology routines due to the availability of bioinformatics tools for databank searches, allowing secure identification and high laboratory reproducibility.67,68 MALDI-MS has been proved by many reports to be easier, faster and sometimes more reliable than classical protocols even when compared to more sophisticated DNA analysis-based technologies.69,70 MALDI-MS-based microorganism identification has rapidly been introduced in laboratory and clinical settings and delivers fast and reliable diagnostic results, not only for genus level identification but also at the species level for bacteria,63,70–81 fungi,16,82–91 algae,92 viruses93–96 and protozoa.97 An essential step in the identification of microorganisms at the species level by MALDI-MS has been the use of dedicated databases with rigorous data quality control and powerful 1000 | RSC Adv., 2013, 3, 994–1008 RSC Advances algorithms for comparison with mass fingerprinting. These databases can be run in parallel with MS acquisition data, giving almost real-time bacterial identification results.33,98,99 Because much effort has focused on MALDI-TOF-MS, this approach is already in use in clinical diagnostic laboratories.68,100–107 The observed biomarkers in the mass spectrum enable not only the detection of pathogenic bacteria but also the ability to distinguish them from corresponding nonpathogenic species.108 Many Campylobacter species and Helicobacter strains cause gastrointestinal diseases and can be discriminated via protein profiles observed by MALDI-MS.109–113 Biomarker assignment also makes it possible to distinguish subspecies of members of the Enterobacteriaceae family so that their fingerprints can be used as family-specific biomarkers for accelerated bacterial identification via database searches.114 Biomarker monitoring by MALDI-MS can also be used to identify environmental toxin producers. MS analyses of peptides and polyketides from intact cyanobacteria were used to identify toxic and nontoxic water blooms115–117 and pathogens isolated from seafood, which are associated with food-borne diseases. This approach was also used to further study the different protein profiles of azaspiracid toxin biomarkers in contaminated and non-contaminated blue mussels (Mytilus edulis).118 Burkholderia cepacia, which are important agents of chronic pulmonary disease in cystic fibrosis patients and are problematic to accurately identify due to their complex taxonomy, have been successfully discriminated by MALDI-MS.119–122 Mycobacterial species, which are responsible for causing significant morbidity in humans by diseases such as tuberculosis, and Haemophilus spp., which are well known etiological agents of pneumonia, meningitis and conjunctivitis, have been identified by MALDI-MS via their protein profile spectra.123–125 In the veterinary field, bacteria isolated from cows presenting subclinical mastitis, a common and easily disseminated disease in dairy herds, were diagnosed in a few minutes through the analysis of ribosomal protein biomarkers isolated from microorganisms present in milk samples by MALDI-MS with the use of the Biotyper database, a commercial software for MALDI-MS-based microorganism identification that allows earlier treatment with appropriate antibiotics.126 Immunoproteomic analyses of Mannheimia haemolytica, the most important bacterial pathogen associated with bovine pneumonia, have been performed by MALDI-MS to search for and identify biomarkers from outer membrane proteins that may hold potential as candidate vaccine antigens.127 Pigments and proteins from chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum, were characterized directly from organelles and bacteriochlorophyll, and homologs were detected to provide fingerprints for these biomarkers.128 MALDI-MS analysis was used to detect the increased expression of cold shock proteins in bacteria collected from the Siberian permafrost, and distinct proteins and peptide profiles were observed as a function of temperature. The recent capability of MALDI-MS imaging has been used to study synergism and antagonism in microbial communities in the This journal is ß The Royal Society of Chemistry 2013 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. RSC Advances nests of leaf-cutting ants through the identification of antimicrobial and antifungal compounds that are produced to defend a symbiotic fungus, which is a major food source for ant species.129 These numerous examples of MALDI-MS applications are supported by the availability of dedicated instrumentation and software containing fingerprinting databases for bacterial identification. Such instruments are operator-friendly, and technicians can operate them in microbiological settings and hospitals with diagnostic purposes with minimal specialization in MS or microbiology and with high-throughput, efficient and trustworthy results. As a result, diverse human clinical settings start by comparing the results of traditional methods versus MALDI-MS-based bacterial identification, and, after some time, they move to a solely MALDI-MS-based approach because of the many advantages and the desire to avoid numerous time-consuming and tedious assays. Identification of uncommon bacterial pathogens, fungi and yeast by MALDI-MS Microorganisms still represent the largest reservoir of biodiversity yet to be studied. It has been estimated that only between 1 and 10% of bacteria have been properly described. Correct microorganism identification is essential for appropriate classification, and the criteria for the identification of diverse microorganism species are still equivocal. Some strains can be misidentified as closely related species. This is the case, for example, for Cronobacter spp.130 that can easily be misidentified as apathogenic Enterobacter turices, E. helveticus and E. pulveris. Cronobacter spp. are Gram-negative opportunistic food-borne pathogens and are known as rare but important causes of neonatal infections. To overcome this problem, MALDI-MS was successfully used for rapid genus and species-specific identification. Moreover, multi-isotope imaging mass spectrometry is contributing to understanding the bacterial diversity of bacterioplankton and helping to link microbial diversity to the biogeochemistry of the pelagic zone of the aquatic system.131 The marine environment has also been proven to be a source of diverse arrays of bioactive metabolites with great potential for pharmaceutical and other applications. In fact, MALDI-MS was applied to classify environmental Sphingomonadaceae using ribosomal subunit proteins coded in the S10-spc-alpha operon as biomarkers.132 In particular, sponges have a largely unexplored biosynthetic potential. Sets of bacteria were cultured from marine sponges (Isops phlegraei, Haliclona sp., Phakellia ventilabrum and Plakortis sp. growing on the Norwegian coast). Intact cell MALDI-MS was used for the rapid screening and proteometric clustering of a subset of the strain collection comprising 456 isolates. The 11 resolved groups were also verified by 16S rDNA analyses. The results indicated that MALDI-MS is effective for the rapid identification of isolates, for the selection of strains representing rare species and for their dereplication, i.e., rapid grouping of bacterial isolates for subsequent characterization.133 Screening for microbial population complexity and diversity in the sediment of contaminated environments has also received increased interest, particularly due to the significance of these microbes for environmental protection. The taxono- This journal is ß The Royal Society of Chemistry 2013 Review mical identification of microbial isolates obtained from sediment samples contaminated with polychlorinated biphenyls was successfully performed with MALDI-MS with minimal time demand and reduced costs.134 The contribution of MS to worldwide biodefence has also been substantial.135,136 In fact, some potential agents for biological attacks are microorganisms and biotoxins. MS was demonstrated to be a valid tool for the rapid identification of potential bioagents. Confident identification of an organism can be achieved by top-down proteomics following identification of individual protein biomarkers from their tandem mass spectra. In bottom-up proteomics, the rapid digestion of intact protein biomarkers is again followed by MS to provide unambiguous bioagent identification and characterization.133,134 Accurate discrimination between species of filamentous fungi is also essential because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. Direct surface analysis of fungal cultures90,137,138 and yeasts139 by MALDI-MS has been evaluated for species identification. The protein profiles of intact fungal spores83,140 such as Aspergillus, Fusarium and Mucorales demonstrated that MALDI-MS is appropriate for the routine identification of filamentous fungi in medical microbiology laboratories.73,138 Culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales were used to establish one reference database for MALDI-MS-based species identification with the MALDI BioTyper 2.0 software. To evaluate the database, 103 blind-coded fungal isolates collected in a routine clinical microbiology laboratory were tested, and 96.8% of the isolates were correctly identified to the species level in agreement with reference methods. Eight technical replicates of 15 strains were also obtained to study the variation of mass spectra. Little variation was observed for each spectrum, whereas enough MS variation could be observed to separate each strain (Fig. 5).90 Yeast infections cause significant mortality in critically ill and immunocompromised patients. In particular, Candida spp. are the 4th most common cause of nosocomial bloodstream infections in the United States, and Cryptococcus neoformans is the most common cause of fungal meningitis worldwide.141,142 Candida species were reliably identified by MALDI-MS, which had superior performance over conventional methods, and it was possible to discriminate between different molecular types of Cryptococcus neoformans and Cryptococcus gattii with the same technique.143,144 Non-fermenting bacteria Non-fermenting bacteria are a taxonomically heterogeneous group of bacteria of the Proteobacteria division, which cannot catabolize glucose. The genera Pseudomonas, Burkholderia, Stenotrophomonas and others belong to this large group. They are ubiquitous environmental opportunists, and some species can cause severe infections,145 particularly in immunocompromised or cystic fibrosis (CF) patients.146 It has been demonstrated that classical phenotypic methods can frequently misidentify non-fermenters.147,148 RSC Adv., 2013, 3, 994–1008 | 1001 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. Review RSC Advances Fig. 5 Three-dimensional principal component analysis (PCA) plot of the technical replicates of selected reference strains of (a) Aspergillus (seven strains), (b) Fusarium (four strains) and (c) Mucorales (four strains). Reproduced from ref. 90 with permission from John Wiley and Sons. For this class of bacteria, molecular tools such as 16S rDNA gene sequencing provide reliable results, but less accurate results have been obtained at the species level. Therefore, a reference database for MALDI-MS based on the identification of non-fermenters was established by Mellmann et al.149,150 and 16S rRNA gene sequencing was used for comparison. Different cultivation conditions and mass spectrometer instruments were used, and the methodology was evaluated with 80 blind-coded clinical non-fermenter strains. The study demonstrated that the MALDI-MS method provides fast and thorough identification of non-fermenting bacteria, even more accurately than partial 16S rRNA gene sequencing for species identification of members of the Burkholderia cepacia complex. A large international multicenter study has also demonstrated the high reproducibility of the identification of non-fermenting bacteria by MALDI-MS, with 98.75% correct species identification. This study demonstrated the suitability of the technique as an alternative to partial 16S rRNA platforms. A very recent study also evaluated the ability of the technique to identify non-fermenting bacteria from among a total of 182 isolates from 70 CF patients because non-fermenters are the main cause of mortality in this type of patient.151 MALDI- MS was found to improve routine identification, particularly by enlarging the Biotyper 2.0 database to include the rare and infrequent microorganisms recovered from CF patients.152 The routine identification of microorganisms that contaminate milk is mostly based on phenotypic characteristics such as colony morphology, hemolytic potential and several biochemical reactions, which are time-consuming and costly.157 Additionally, these tests may fail to correctly identify all agents, and even though methods based on PCR are developing rapidly, there may be no agreement among the techniques that phenotypically and genotypically differentiate bacterial species, resulting in the false identification of agents. Non-culture MALDI-MS identification based on protein fingerprinting from bacteria (E. coli, S. aureus and E. faecalis) inoculated and recovered directly from milk samples has been successful (Fig. 6). Although relatively high bacterial loads (106 to 107 bacteria mL21 of milk) must be present, the simple incubation of an initial load of 104 bacteria mL21 of milk can be used to facilitate bacterial replication and successful Non-culture-based identification of bacteria from blood and milk In addition to being increasingly used for the rapid identification of bacteria and fungi, MALDI-MS also holds promise for bacterial identification from blood culture (BC) broths in hospital laboratories153–155 and bacterial identification directly from milk samples.156 A MALDI-MS-based approach has been shown to perform rapid (,20 min) bacterial identification directly from positive BCs with high accuracy. Positive predictive values for the direct identification of both Gram-positive and Gram-negative bacteria from monomicrobial blood culture broths were 100% to the genus level. A diagnostic algorithm for positive blood culture broths that incorporates Gram staining and MALDI-MS should be able to identify the majority of pathogens, particularly at the genus level.153,154 1002 | RSC Adv., 2013, 3, 994–1008 Fig. 6 MALDI-MS ribosomal protein fingerprints for the identification of bacteria in whole milk. Data were collected in the m/z 4000–22 000 range after processing 900 mL of whole milk that had been experimentally contaminated with E. coli at (a) 103, (b) 104, (c) 105, (d) 106, (e) 107, (f) 108, or (g) 109 cfu ml21. Reproduced from Proteomics ref. 156 with permission from John Wiley and Sons. This journal is ß The Royal Society of Chemistry 2013 View Article Online RSC Advances identification. Fast, reliable and sensitive protocols for the analysis of relatively low concentrations of bacteria present in milk could be of great value for the dairy industry. Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. Testing for antibiotic resistance with MS and polymicrobial culture analysis Antibiotic resistance is the ability of a microorganism to withstand the effects of an antibiotic drug, and this ability represents a huge health concern in the medical and veterinary fields. Recent studies have shown that individuals are at risk of carrying antibiotic-resistant bacteria after a series of antibiotic treatments due to the resulting selection for antibiotic-resistant microorganisms. The most common mechanisms of antibiotic resistance can be divided in three classes: alteration of the antibiotic target, restriction of antibiotic access to the target and inactivation of the antibiotic.158–160 Recently, the ability of MALDI MS to effectively discriminate bacteria strains which have acquired resistance to a variety of antibiotics has been demonstrated, indicating that this technique has the potential to differentiate bacterial strains with varying degrees of antibiotic resistance.161 The rapid detection of resistance type is necessary to select the best antibiotic therapy. b-Lactam antibiotics represent a broad class of antibiotics that contain a b-lactam nucleus in their molecular structure. These antibiotics include penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. b-Lactam antibiotics act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls, Fig. 7 (A and B) MALDI-MS of ampicillin after incubation with a b-lactamaseproducing strain (B). (C) Inhibition of hydrolysis by a b-lactamase-producing strain was performed in the presence of clavulanic acid. Peaks corresponding to the non-hydrolyzed form of ampicillin are highlighted in gray. Peaks corresponding to the hydrolyzed form of ampicillin are indicated with an arrow. Reproduced from ref. 164 with permission from the American Society for Microbiology. This journal is ß The Royal Society of Chemistry 2013 Review which is important for cell wall structural integrity. b-Lactam antibiotics mainly affect Gram-positive organisms because peptidoglycan is the outermost and primary component of their cell wall. This antibiotic class binds in the active site of penicillin-binding proteins (PBPs), preventing the final crosslinking (transpeptidation) of the nascent peptidoglycan layer and disrupting cell wall synthesis.162 Since these antibiotics are widely administered to treat infections in human and domestic animals, many Gram-positive bacteria have already developed resistance mechanisms. Antimicrobial susceptibility has classically been determined using a variety of in vitro methods such as disk diffusion and broth microdilution as well as automated instrument-based methods. These methods may require from a few hours, such as for the antimicrobial susceptibility test (AST), to 24–96 h for a pure culture of the suspected pathogen to be obtained and subjected to disk diffusion assays.3,163 A novel MALDI-MS method for the detection of b-lactamase resistance has recently been reported. Resistance to b-lactam antibiotics can be easily monitored by MS because hydrolysis of the central b-lactam ring by b-lactamases results in the disappearance of the original ion, which is shifted 18 m/z units higher in the spectrum of the antibiotic. In many cases, hydrolysis is directly followed by a decarboxylation of the hydrolyzed product, resulting in a further shift of 244 m/z units due to detection of the hydrolyzed form. Because MS easily monitors such m/z shifts, a MALDI-MS assay was set up to analyze the hydrolysis reactions of different b-lactam antibiotics (Fig. 7).164 MALDI-MS can also be applied to study bacterial resistance to antibiotics or antimicrobial compounds secreted by other bacterial species.68,165,166 Reportedly, antibiotic-resistant and non-resistant strains of an important human pathogen, S. aureus, can be differentiated by MALDI-MS by rapid and accurate discrimination between methicillin-sensitive and methicillin-resistant strains of this organism, which can could lead to major improvements in the treatment strategies for infected patients.167 The same microorganism has been identified via biomarker analysis as one of the main pathogens responsible for prosthetic joint infections, indicating reliable differentiation between S. aureus and coagulase-negative staphylococci.168 The resistance mechanism of colistin-resistant variants of Acinetobacter baumannii was elucidated by MALDI-MS169 by determining the phosphoethanolamine modification of lipid A. For Bacteroides fragilis, it was recently shown that the differentiation of cfiA gene-encoded class B metallo-b-lactamase was possible by direct MALDI-MS.170,171 The carbapenem resistance of B. fragilis is due to a species-specific metallob-lactamase, which is encoded by the cfiA (ccrA) gene of the organism. Almost 100% of the carbapenem-resistant bacteroid strains were cfiA-positive B. fragilis isolates. However, such clonal differentiation for resistant and susceptible clones cannot be expected for the majority of bacteria. Antibiotic resistance in Gram-negative rods, particularly Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., has been an increasing problem worldwide. Infections by multidrug-resistant Gram-negative bacteria are usually treated with carbapenems. This resistance is caused by an alteration RSC Adv., 2013, 3, 994–1008 | 1003 View Article Online Published on 24 October 2012. Downloaded by UNIVERSIDAD SAO PAULO on 01/12/2013 20:51:28. Review in the outer membrane of the cell wall and by the production of carbapenemases.172 Carbapenamase activity was detected in 124 strains following comparison between well-typed bacterial carbapenem non-susceptible isolates and clinical isolates susceptible to carbapenems. Antibiotic resistance was demonstrated by detecting the disrupted amide bond and its cationized forms.173 Although the microbiological methods of microorganism culture and isolation are successful, mixed cultures or polymicrobial cultures may occur. For example, one bacterial isolate from subclinical bovine mastitis was identified as Staphylococcus aureus by an enzymatic assay, and yet the same sample, when analyzed by Biotyper software after MALDI-MS, displayed a signature of Enterococcus faecalis mixed with S. aureus, which was confirmed by 16S RNA sequencing.174 When bacterial identification of human clinical isolates was performed by MALDI-MS directly on 500 blood broth cultures, there were 27 polymicrobial cultures, and 25 (92.6%) had at least one species correctly identified by the instrument database. Using the same instrument and software platform, similar results have been observed by Moussaoui et al.175 and Christner et al.,176 who reported that 80.9% and 81.2%, respectively, of at least one of the species present were identified in polymicrobial cultures obtained directly from blood culture vials.175,176 RSC Advances around U$ 2–3 per isolate, depending on the cost of the instrument. Therefore, microbiology is ready to enter into a new era in which molecular rather than morphological or biochemical characteristics of microorganisms will be rapidly assessed (directly from biological fluids) by MS for identification. The use of this technique is not only limited to clinical diagnosis, but it has been shown to have successful application for detecting antibiotic resistance, characterizing microorganisms that are difficult to isolate and culture and exploring the biodiversity of microorganisms present in the environment and in the digestive tracts of animals and humans. Mass spectrometry-based proteomics approaches have also been applied to gain a greater understanding of the pathophysiology and virulence of microorganisms. This approach is providing key insights to better understand the molecular processes involved in protein secretion, modification, synthesis and degradation.177–179 A new era has indeed emerged in which bacterial identification seems fully prepared to make the transition from the agar plate and visual inspection to the mass spectrometer for characterization at the more accurate molecular level. References Conclusions and real-world perspectives Diverse ionization methods and MS techniques can be successfully used for microorganism identification and research. MALDI-MS is the leading MS technique for clinical and commercial microbiological use, and there are consistent reports demonstrating the confidence of this approach in comparison to other non-MS based gold-standard approaches to identify a large range of microorganisms. The broad use of this technique now appears to only be dependent on regulatory issues in various countries, including the United States. Limitations of MS-based microorganism identification are dependent on the initial cost of the technology, which is related to the instrument configurations. Also, most laboratories will need an internal validation period in which the transition between the traditional methods to MS-based microorganism identification and staff training is performed. Since one instrument is sufficient for a high number of samples/day, technical problems can interrupt the routine and impact clinical decisions, especially in septicemic conditions. These issues should be managed with appropriate planning such as the long-term benefits related to the cost/sample. The identification of bacteria isolated from 928 human clinical samples in a routine microbiology setting has been recently compared using the BD Phoenix, API panels and other recommended procedures and MALDI-MS using a TOF analyzer and the Biotyper software. 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