Accuracy of conventional identification methods used for Enterobacteriaceae isolates in three Nigerian hospitals

BackgroundEnterobacteriaceae are ubiquitously present in nature and can be found in the intestinal tract of humans and animals as commensal flora. Multidrug-resistant Enterobacteriaceae are increasingly reported and are a threat to public health implicating a need for accurate identification of the isolates to species level. In developing countries, identification of bacteria basically depends on conventional methods: culture and phenotypic methods that hamper the accurate identification of bacteria. In this study, matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique was compared to conventional identification techniques.Materials and MethodsIn total, 147 Enterobacteriaceae isolates were collected from March to May 2015 from three medical microbiology laboratories of hospitals in Edo state, Nigeria, after being tested according to the individual laboratories standard operating procedures. All isolates were stored at −20°C until tested centrally by MALDI-TOF MS.ResultsOne hundred and forty five (98.6%) isolates had a MALDI Biotyper best score > or =2.0, indicating a secure genus and probable species identification; and 2(1.36%) isolates had a best score <2.0 indicating probable genus identification. Isolates with best scores of > or =2.0 comprised nine genera and 10 species, respectively. A total of 57.2% and 33.1% of isolates identified had agreement between MALDI-TOF MS and conventional techniques for identification at genus and species level, respectively, when analyzing bacteria with MALDI Biotyper best scores > or =2.0.ConclusionThe results of our study show that the applied conventional identification techniques for Enterobacteriaceae in the investigated Nigerian hospitals are not very accurate. Use of state-of-the-art identification technologies for microorganisms is necessary to guarantee comparability of bacteriological results.

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Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry in diagnosis of clinical Nocardia species

Journal of Laboratory Medicine ◽

10.1515/labmed-2018-0180 ◽

2019 ◽

Vol 43 (3) ◽

pp. 157-162

Author(s):

Gülşen Hasçelik ◽

Markus Kostrzewa ◽

Hamit Kaan Müştak ◽

Celalettin Uner ◽

Kadir Serdar Diker

Keyword(s):

Species Level ◽

Rrna Gene ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Maldi Biotyper ◽

Rrna Gene Sequencing ◽

Tof Ms

Abstract Background The routine identification to the species level of Nocardia genus by conventional methods is a fastidious and time-consuming process owing to the limited biochemical reactivity of these microorganisms, often requiring 1 or more days to complete identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a new technology for definitive and rapid species identification. Methods We evaluated the MALDI-TOF MS for the identification of 44 clinical isolates of Nocardia species in comparison to 16S ribosomal RNA (rRNA) gene sequencing. Nocardia isolates were identified by microbiological examination, phenotypical tests and MALDI-TOF MS and the results were compared by 16S rRNA gene sequencing. Results Of the 44 Nocardia strains, the identification of 28 isolates was determined with MALDI Biotyper database. According to this, 16 isolates (57.1%) of the strain log scores were ≥2. Two (7.1%) were identified to the species level (log scores of ≥2) as Nocardia otitidiscaviarum. The addition of a newly established Nocardia database (16 new Nocardia strains included to the original database) did significantly improve the scores. The results were 43 (97.7%) correct identification to the species level (log scores of ≥2). Conclusions This study showed that the identification of clinical Nocardia isolates by the Bruker MALDI Biotyper is highly reliable, whereas identification rates are generally lower than those for some Gram-negative bacteria and Gram-positive cocci. Based on our data, the identification rates can be improved by validated new database entries and the results can be confirmed with nucleic acid sequence analysis.

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Test Agreement among Biochemical Methods, Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry, and 16S rRNA Sequencing for Identification of Microorganisms Isolated from Bovine Milk

Journal of Clinical Microbiology ◽

10.1128/jcm.01381-18 ◽

2018 ◽

Vol 57 (3) ◽

Cited By ~ 2

Author(s):

David J. Wilson ◽

John R. Middleton ◽

Pamela R. F. Adkins ◽

Gregory M. Goodell

Keyword(s):

16S Rrna ◽

Species Level ◽

Content Type ◽

Maldi Tof Ms ◽

Milk Samples ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Identification Of Bacteria ◽

Biochemical Testing ◽

Tof Ms

ABSTRACT The objective of this prospective study was a blinded comparison of three methods for the identification of bacteria isolated on Columbia blood agar from milk samples of dairy cows. Basic biochemical testing, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and 16S rRNA partial genome sequence analysis were compared for bacterial identification to the genus or species level. Milk samples submitted from a commercial dairy farm from recently calved cows or clinical mastitis cases were cultured, and 181 isolates were identified by biochemical testing, MALDI-TOF MS, and 16S rRNA sequence analysis (179 isolates; 2 isolates could not be recovered from storage). For Staphylococcus aureus and Escherichia coli, agreement was determined at the species level. For other microbes, agreement was determined at the genus level or at the group level for streptococcus-like organisms. The positive agreement among all 3 diagnostic methods was 94%, with 95% to 98% between each pair of methods. The overall (including negative agreement) agreement among all 3 methods ranged from 97% to 100%. MALDI-TOF MS is becoming more commonplace for the genus- and/or species-level identification of bacteria isolated from milk samples and, in some laboratories, has replaced conventional biochemical methods. The results of the present study suggest that when identifying pathogens at the genus or group level, conventional culture followed up with either secondary biochemical testing or MALDI-TOF MS is of practical value. For purposes of milk quality and udder health monitoring or research, any of the 3 methods is a valuable tool for genus-level identification of bacteria isolated from dairy cow milk.

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Performance of Five Commercial Identification Platforms for Identification of Staphylococcus delphini

Journal of Clinical Microbiology ◽

10.1128/jcm.00721-19 ◽

2019 ◽

Vol 57 (11) ◽

Cited By ~ 2

Author(s):

Matthew C. Canver ◽

Tsigereda Tekle ◽

Samantha T. Compton ◽

Katrina Callan ◽

Eileen M. Burd ◽

...

Keyword(s):

Distinct Species ◽

Human Infection ◽

Species Level ◽

Accurate Identification ◽

Content Type ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Staphylococcus Intermedius ◽

Animal Pathogens ◽

Tof Ms

ABSTRACT The Staphylococcus intermedius group (SIG) is a collection of coagulase-positive staphylococci consisting of four distinct species, namely, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus intermedius, and Staphylococcus pseudintermedius. SIG members are animal pathogens and rare causes of human infection. Accurate identification of S. pseudintermedius has important implications for interpretation of antimicrobial susceptibility testing data and may be important for other members of the group. Therefore, we sought to evaluate the performance of five commercially available identification platforms with 21 S. delphini isolates obtained from a variety of animal and geographic sources. Here, we show that automated biochemical platforms were unable to identify S. delphini to the species level, a function of its omission from their databases, but could identify isolates to the SIG level with various degrees of success. However, all automated systems misidentified at least one isolate as Staphylococcus aureus. One matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system was able to identify S. delphini to the species level, suggesting that MALDI-TOF MS is the best option for distinguishing members of the SIG. With the exception of S. pseudintermedius, it is unclear if other SIG members should be routinely identified to the species level; however, as our understanding of their role in animal and human diseases increases, it may be necessary and important to do so.

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Identification of the ‘Streptococcus anginosus group’ by matrix-assisted laser desorption ionization – time-of-flight mass spectrometry

Journal of Medical Microbiology ◽

10.1099/jmm.0.076653-0 ◽

2014 ◽

Vol 63 (9) ◽

pp. 1143-1147 ◽

Cited By ~ 12

Author(s):

Katherine Woods ◽

David Beighton ◽

John L. Klein

Keyword(s):

Species Level ◽

Direct Transfer ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Streptococcus Anginosus ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Transfer Method ◽

Tof Ms

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid, accurate and cost-effective identification of a range of bacteria and is rapidly changing the face of routine diagnostic microbiology. However, certain groups of bacteria, for example streptococci (in particular viridans or non-haemolytic streptococci), are less reliably identified by this method. We studied the performance of MALDI-TOF MS for identification of the ‘Streptococcus anginosus group’ (SAG) to species level. In total, 116 stored bacteraemia isolates identified by conventional methods as belonging to the SAG were analysed by MALDI-TOF MS. Partial 16S rRNA gene sequencing, supplemented with sialidase activity testing, was performed on all isolates to provide ‘gold standard’ identification against which to compare MALDI-TOF MS performance. Overall, 100 % of isolates were correctly identified to the genus level and 93.1 % to the species level by MALDI-TOF MS. However, only 77.6 % were correctly identified to the genus level and 59.5 % to the species level by a MALDI-TOF MS direct transfer method alone. Use of a rapid in situ extraction method significantly improved identification rates when compared with the direct transfer method (P<0.001). We recommend routine use of this method to reduce the number of time-consuming full extractions required for identification of this group of bacteria by MALDI-TOF MS in the routine diagnostic laboratory. Only 22 % (1/9) of Streptococcus intermedius isolates were reliably identified by MALDI-TOF MS to the species level, even after full extraction. MALDI-TOF MS reliably identifies S. anginosus and Streptococcus constellatus to the species level but does not reliably identify S. intermedius.

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Matrix-assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a Reliable Tool to Identify Species of Catalase-negative Gram-positive Cocci not Belonging to the Streptococcus Genus

The Open Microbiology Journal ◽

10.2174/1874285801610010202 ◽

2016 ◽

Vol 10 (1) ◽

pp. 202-208 ◽

Cited By ~ 7

Author(s):

Marisa Almuzara ◽

Claudia Barberis ◽

Viviana Rojas Velázquez ◽

Maria Soledad Ramirez ◽

Angela Famiglietti ◽

...

Keyword(s):

Extraction Methods ◽

Species Level ◽

Ionization Time ◽

Maldi Tof Ms ◽

Genus Level ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Level Identification ◽

Gram Positive Cocci ◽

Tof Ms

Objective:To evaluate the performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) by using 190 Catalase-negative Gram-Positive Cocci (GPC) clinical isolates.Methods:All isolates were identified by conventional phenotypic tests following the proposed scheme by Ruoff and Christensen and MALDI-TOF MS (Bruker Daltonics, BD, Bremen, Germany). Two different extraction methods (direct transfer formic acid method on spot and ethanol formic acid extraction method) and different cut-offs for genus/specie level identification were used. The score cut-offs recommended by the manufacturer (≥ 2.000 for species-level, 1.700 to 1.999 for genus level and <1.700 no reliable identification) and lower cut-off scores (≥1.500 for genus level, ≥ 1.700 for species-level and score <1.500 no reliable identification) were considered for identification. A minimum difference of 10% between the top and next closest score was required for a different genus or species.MALDI-TOF MS identification was considered correct when the result obtained from MS database agreed with the phenotypic identification result.When both methods gave discordant results, the 16S rDNA orsodAgenes sequencing was considered as the gold standard identification method. The results obtained by MS concordant with genes sequencing, although discordant with conventional phenotyping, were considered correct. MS results discordant with 16S orsodA identification were considered incorrect.Results:Using the score cut-offs recommended by the manufacturer, 97.37% and 81.05% were correctly identified to genus and species level, respectively. On the other hand, using lower cut-off scores for identification, 97.89% and 94.21% isolates were correctly identified to genus and species level respectively by MALDI-TOF MS and no significant differences between the results obtained with two extraction methods were obtained.Conclusion:The results obtained suggest that MALDI-TOF MS has the potential of being an accurate tool for Catalase-negative GPC identification even for those species with difficult diagnosis asHelcococcus,Abiotrophia,Granulicatella, among others. Nevertheless, expansion of the library, especially including more strains with different spectra on the same species might overcome potential “intraspecies” variability problems. Moreover, a decrease of the identification scores for species and genus-level identification must be considered since it may improve the MALDI-TOF MS accuracy.

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Establishment and Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Detection of Shewanella Genus

Frontiers in Microbiology ◽

10.3389/fmicb.2021.625821 ◽

2021 ◽

Vol 12 ◽

Author(s):

Keyi Yu ◽

Zhenzhou Huang ◽

Ying Li ◽

Qingbo Fu ◽

Lirong Lin ◽

...

Keyword(s):

Laser Desorption ◽

Rapid Identification ◽

Species Level ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Type Strains ◽

Tof Ms

Shewanella species are widely distributed in the aquatic environment and aquatic organisms. They are opportunistic human pathogens with increasing clinical infections reported in recent years. However, there is a lack of a rapid and accurate method to identify Shewanella species. We evaluated here matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of Shewanella. A peptide mass reference spectra (PMRS) database was constructed for the type strains of 36 Shewanella species. The main spectrum projection (MSP) cluster dendrogram showed that the type strains of Shewanella species can be effectively distinguished according to the different MS fingerprinting. The PMRS database was validated using 125 Shewanella test strains isolated from various sources and periods; 92.8% (n = 116) of the strains were correctly identified at the species level, compared with the results of multilocus sequence analysis (MLSA), which was previously shown to be a method for identifying Shewanella at the species level. The misidentified strains (n = 9) by MALDI-TOF MS involved five species of two groups, i.e., Shewanella algae–Shewanella chilikensis–Shewanella indica and Shewanella seohaensis–Shewanella xiamenensis. We then identified and defined species-specific biomarker peaks of the 36 species using the type strains and validated these selected biomarkers using 125 test strains. Our study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid identification of Shewanella strains at the species level.

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Evaluation of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Rapid Identification of Bacteria in Processed Soybean Products

Journal of Food Research ◽

10.5539/jfr.v2n3p104 ◽

2013 ◽

Vol 2 (3) ◽

pp. 104 ◽

Cited By ~ 2

Author(s):

Yuko Furukawa ◽

Mitsuru Katase ◽

Kazunobu Tsumura

Keyword(s):

Laser Desorption ◽

Rapid Identification ◽

Bacterial Cells ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Identification Of Bacteria ◽

Tof Ms

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently been demonstrated as a rapid and reliable method for identifying bacteria in colonies grown on culture plates. Rapid identification of food spoilage bacteria is important for ensuring the quality and safety of food. To shorten the time of analysis, several researchers have proposed the direct MALDI-TOF MS tequnics for identification of bacteria in clinical samples such as urine and positive blood cultures. In this study, processed soybean products (total 26 test samples) were initially conducted a culture enrichiment step and bacterial cells were separated from interfering components. Harvested bacterial cells were determined by MALDI-TOF MS and 16S rRNA gene sequencing method. Six processed soybean products (23%) were increased bacterial cells after culture enrichiment step and they were sucessfully obtained the accurate identification results by MALDI-TOF MS-based method without colony formation.

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Identification of Nocardia species using Autof MS 1000 and Bruker MALDI-TOF MS systems: A comparison

10.1101/2021.01.11.426310 ◽

2021 ◽

Author(s):

Bing Ma ◽

Yunqi Tian ◽

Yungang Han ◽

Lifang Ban ◽

Junwen Yang ◽

...

Keyword(s):

Species Identification ◽

Protein Extraction ◽

Reference Method ◽

Invasive Disease ◽

Species Level ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Significant Difference ◽

Tof Ms

ABSTRACTNocardia is an important cause of clinically invasive disease, but for most clinical laboratories, identification of these isolates to the species level is challenging. Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for identification of most bacterial and fungal isolates. In this multicenter study, we evaluated the identification of Nocardia isolates using Autof MS1000 and Bruker Biotyper. A total of 86 non-duplicate Nocardia isolates from 7 hospital laboratories were evaluated. Further, we carried out sequence analysis of 16S rRNA, gyrB, secA1, hsp65, and rpoB genes as a reference method for Nocardia species identification. The 86 isolates were directly spotted on the target plate and plate protein extraction was performed. Data were analyzed by SPSS 19.0. In total, 72 (83.7%) strains (score ≥ 9.0) and 70 (81.4%) strains (score ≥ 2.0) were correctly identified by the Autof MS1000 and Bruker Biotyper systems, respectively, at the species level. There was no significant difference (P > 0.05) between the two systems using the same protein extraction method. In conclusion, the Autof MS 1000 and Bruker MALDI-TOF systems showed no difference in identification of Nocardia spp. to the species level and could meet the most important clinical requirement for species identification.

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Application of MALDI-TOF MS for the Identification of Food Borne Bacteria

The Open Microbiology Journal ◽

10.2174/1874285801307010135 ◽

2013 ◽

Vol 7 (1) ◽

pp. 135-141 ◽

Cited By ~ 63

Author(s):

Melanie Pavlovic ◽

Ingrid Huber ◽

Regina Konrad ◽

Ulrich Busch

Keyword(s):

Associated Bacteria ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Current State ◽

Food Borne ◽

Routine Identification ◽

Identification Of Bacteria ◽

Cost Efficient ◽

Tof Ms

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS based identification of bacteria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality. This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed.

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Multicenter evaluation of three different MALDI-TOF MS systems for identification of clinically relevant filamentous fungi

Medical Mycology ◽

10.1093/mmy/myaa037 ◽

2020 ◽

Vol 59 (1) ◽

pp. 81-86

Author(s):

Yue Sun ◽

Jian Guo ◽

Rong Chen ◽

Liang Hu ◽

Qihang Xia ◽

...

Keyword(s):

Sample Preparation ◽

Filamentous Fungi ◽

Reference Method ◽

Species Level ◽

Its Sequencing ◽

Maldi Tof Ms ◽

Genus Level ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Tof Ms

Abstract Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) holds promise as a potential tool for clinical identification of filamentous fungi. However, due to the lack of an appropriate extraction protocol and the difficulty of database building, the identification power of each system differs. In this study, we selected 126 clinical mould isolates comprising 28 species identified using internal transcribed spacer (ITS) sequencing as the reference method to evaluate three MALDI-TOF MS systems. When using cultures and sample preparation as recommended by the respective vendors, of the 126 strains tested, VITEK MS identified 121 (96.0%) to species-level and 124 (98.4%) to genus-level; Biotyper identified 53 (42.1%) to species-level and 54 (42.9%) to genus-level; Autof identified 74 (58.7%) to species-level and 76 (60.3%) to genus-level. For the Autof system, the tube extraction method recommended by the vendor performed better (59%) than the on-plate lysis (51%). Our study demonstrates that MALDI-TOF MS systems can successfully identify most clinically relevant fungi, while performance is still highly dependent on the database and sample preparation protocol.

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