Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

Lista, Florigio; Reubsaet, Frans A.G.; Santis, Riccardo De; Parchen, Rene R; Jong, A.L. de; Kieboom, J.; Laaken, Anton L van der; Voskamp-Visser, Ingrid A.I.; Fillo, Silvia; Jansen, Hugo-Jan; Plas, J. van der; Paauw, Armand

doi:10.1186/1471-2180-11-267

Cited by 85 publications

(80 citation statements)

References 33 publications

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“…This protocol is considerably faster than previously described protocols, which require several reagents, centrifugation steps, and long incubations (12)(13)(14)(15)(16) or expensive gamma ray sources (17). Sample preparation with the solvent mixture allowed correct MALDI-TOF MS identification of all Brucella strains tested and several other bacterial pathogens.…”

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confidence: 99%

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

et al. 2016

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dWe describe a simple protocol to inactivate the biosafety level 3 (BSL3) pathogens Brucella prior to their analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. This method is also effective for several other bacterial pathogens and allows storage, and eventually shipping, of inactivated samples; therefore, it might be routinely applied to unidentified bacteria, for the safety of laboratory workers. Brucellosis is a zoonosis caused by bacteria of the genus Brucella, which are transmitted to humans through direct contact, ingestion of contaminated animal products, or aerosolization (1, 2). Brucellosis is endemic in several regions of the world, with more than 500,000 new cases each year (2-5). To date, 10 recognized species of Brucella have been described, with the most pathogenic for humans being Brucella melitensis (2). Diagnoses of clinical brucellosis are made initially using serological tests and are confirmed by isolation of the agent (6, 7). The recent introduction of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of bacteria and yeasts (8, 9). However, the procedures recommended by the manufacturers for non-biosafety level 3 (BSL3) organisms are not adequate for the manipulation of Brucella strains, which are classified as BSL3 (potential bioterrorism pathogens) and represent potential health hazards for laboratory workers (1, 10). Any inactivation procedure must allow safe sample handling outside the BSL3 environment and must avoid destruction of the biomarkers used for identification. Therefore, we developed a simple, safe, and efficient sample preparation method for Brucella isolates that is compatible with their analysis by the current MALDI-TOF MS platforms.Solvent inactivation of Brucella. We first applied the direct transfer procedure recommended by the manufacturer to a panel of Brucella strains, working in class II microbiological safety cabinets in a BSL3 laboratory (Table 1). The target plate (Vitek MS DS slide; bioMérieux) was inoculated by picking a portion of a colony from a plate with a 1-l disposable loop (Sarsted), and the deposit was overlaid with 1 l of ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (saturated solution of CHCA in a solvent mixture composed of 33% acetonitrile, 33% absolute ethanol, 3% trifluoroacetic acid, and 31% water) and then air dried for 2 min at room temperature. The dried material was recovered with a sterile swab and spread on culture plates, which were then incubated for up to 3 weeks under the optimal growth conditions for the corresponding bacteria indicated in Table 1. Viable bacteria were recovered for most strains, showing that this protocol does not kill all Brucella strains, possibly because the bacterial deposit was not completely covered with the matrix solution. This risk is not acceptable for BSL3 pathogens. To overcome this problem, different liquid-phase inactivation protocols were tested in 1.5-ml Eppendorf tubes (see F...

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A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

et al. 2016

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“…MALDI-TOF MS has been used to identify bacterial species and subspecies (14,15), and in some outbreaks, MALDI-TOF MS has been reported to be able to identify the lineages of strains (16)(17)(18). Recently, MALDI-TOF MS has also been used to detect clinical pathogens previously misidentified or ambiguously identified (19)(20)(21)(22)(23)(24). Detection of antimicrobial resistance using MALDI-TOF MS has been reported for Staphylococcus aureus (25)(26)(27)(28)(29)(30)(31)(32), Acinetobacter baumannii (26), Escherichia coli, and other members of the family Enterobacteriaceae (33)(34)(35).…”

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Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2013

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bDuring the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

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“…It should be pointed out that because of the high transmissibility of Brucella organisms, an initial bacterial inactivation step with absolute ethanol was added as a measure of caution, to be followed by extraction with formic acid and acetonitrile [69,70]. Although in some studies, the method also enabled discrimination at the species level and even at the biovar level for B. suis [66,67], other investigators reported unreliable discrimination between the different Brucella species [68].…”

Section: Rapid Phenotypic Identification Methodsmentioning

confidence: 99%

“…The method obviates the need for biochemical testing and, thus, is suitable for high-throughput by less skilled laboratory personnel [66,67]. This novel approach can be employed on bacterial colonies growing on agar plates, as well as from positive culture broths and, therefore, it enables direct identification of organisms from blood culture vials [68].…”

Section: Rapid Phenotypic Identification Methodsmentioning

confidence: 99%

Blood Cultures for the Diagnosis of Human Brucellosis

Yagupsky¹

2015

Updates on Brucellosis

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Brucellosis represents a serious health threat to human populations living in areas endemic for the disease. The clinical manifestations of brucellosis are protean and nonspecific, and laboratory confirmation of the diagnosis is crucial for an adequate management of the patient and implementation of infection control measures aimed to control the disease in affected herds. Although brucellosis can be confirmed by serologic tests and nucleic acid amplification assays, culture detection of circulating Brucella organisms remains a diagnostic cornerstone. Traditionally, prolonged incubation of media and performance of blind subcultures of negative blood culture vials have been recommended to maximize isolation of the organism. In recent years, modern automated blood culture systems have revolutionized the diagnosis of human brucellosis by improving sensitivity and enabling detection of brucellae within the routine one-week incubation protocol followed in most Clinical Microbiology laboratories. Development of molecular techniques and mass-spectrometry technology have also shortened the time needed to identify members of the genus, whereas use of biological safety cabinets considerably reduce the risks of contagion to laboratory personnel.

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Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

Cited by 85 publications

References 33 publications

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Blood Cultures for the Diagnosis of Human Brucellosis

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