MALDI-TOF MS Method Research Articles

Investigation of high-risk ST131 clone in ESBL-producing Escherichia coli isolates isolated from urine and non-urinary clinical specimens with MALDI-TOF MS and real time PCR

In recent years, the ST131 clone was identified as a high risk pandemic clone among Escherichia coli isolates by multilocus sequence typing (MLST) studies and has been associated with extended spectrum beta-lactamase (ESBL) production (often with CTX-M-15) and antibiotic resistance especially against fluoroquinolones. The aim of this study was to determine the rate of high risk ST131 clone in ESBL producing E.coli isolates in our region, to investigate the sensitivity of MALDI-TOF MS in the detection of ST131 clone, and to compare the frequency of antimicrobial resistance among ST131 and non-ST131 isolates. A total of 251 urinary and 50 non-urinary E.coli isolates identified in our hospital central laboratory between February 2016-February 2017 were included in the study. Real-time PCR and MALDI-TOF MS methods were used for the detection of E.coli ST131 clone. For the statistical evaluation of the rate of antibiotic resistance among isolates of ST131 and non-ST131 clones, chi-square test was used. p value under 0.05 was considered as significant. Of the 301 isolates, 110 (36.6%) and 92 (30.6%) isolates were identified as ST131 clone by real-time PCR and MALDI-TOF MS, respectively. According to real-time PCR results, 91 (36.3%) of 251 urinary isolates and 19 (38%) of 50 non-urinary isolates were found as ST131 clone; there was no statistically significant difference between the groups. Ciprofloxacin resistance was found to be significantly higher in ST131 isolates than the non-ST131 isolates (78.2%, n= 86 vs. 53.4%, n= 102). No statistically significant difference was determined for the other antibiotics tested. For the detection of E.coli ST131 clone; sensitivity of MALDI-TOF MS was 84%, specificity was 100% while positive predictive value was 100% and negative predictive value was 92%. In conclusion, further investigation of the high risk E.coli ST131 clone in our country, in which ESBL ratios and antibiotic resistance rates, especially in fluoroquinolones, are high, is important for the development of new strategies to control antibiotic resistance. MALDI-TOF MS method is particularly useful for easy and fast detection of the high risk E.coli ST131 clone.

A novel method to rapidly distinguish the geographical origin of traditional fermented-salted vegetables by mass fingerprinting.

The geographical origin of kimchi is of interest to consumers and producers because the prices of commercial kimchi products can vary significantly according to the geographical origin. Hence, social issues related to the geographical origin of kimchi in Korea have emerged as a major problem. In this study, the geographical origin of kimchi was determined by comparing the mass fingerprints obtained for Korean and Chinese kimchi samples by MALDI-TOF MS with multivariate analysis. The results obtained herein provide an accurate, powerful tool to clearly discriminate kimchi samples based on their geographical origin within a short time and to ensure food authenticity, which is of significance in the kimchi industry. Furthermore, our MALDI-TOF MS method could be applied to determining the geographical origin of other fermented-salted vegetables at a reduced cost in shorter times.

Evaluation of repetitive-PCR and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid strain typing of Bacillus coagulans.

In order to establish rapid and accurate typing method for Bacillus coagulans strains which is important for controlling in some canned foods and tea-based beverages manufacturing because of the high-heat resistance of the spores and high tolerance of the vegetative cells to catechins and chemicals, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and repetitive-PCR (rep-PCR) were evaluated. For this purpose, 28 strains of B. coagulans obtained from various culture collections were tested. DNA sequence analyses of the genes encoding 16S rRNA and DNA gyrase classified the test strains into two and three groups, respectively, regardless of their phenotypes. Both MALDI-TOF MS and rep-PCR methods classified the test strains in great detail. Strains classified in each group showed similar phenotypes, such as carbohydrate utilization determined using API 50CH. In particular, the respective two pairs of strains which showed the same metabolic characteristic were classified into the same group by both MALDI-TOF MS and rep-PCR methods separating from the other strains. On the other hand, the other strains which have the different profiles of carbohydrate utilization were separated into different groups by these methods. These results suggested that the combination of MALDI-TOF MS and rep-PCR analyses was advantageous for the rapid and detailed typing of bacterial strains in respect to both phenotype and genotype.

Application of the matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry method to identify Moraxella spp isolated from cattle eyes

Infectious bovine keratoconjunctivitis (IBK) is an economically significant disease caused by the Gram-negative bacterium Moraxella bovis. Moraxella bovoculi also secretes virulence factors and is the most frequently isolated species from bovine eyes during IBK outbreaks. Whole genome sequencing has presented extensive genetic differences between M. bovoculi isolated from the eye and nasopharynx (NP) of cattle with and without IBK. At present, distinguishing between Moraxella species in clinical laboratory specimens is time consuming and relies on expensive and complex biochemical testing, of which reactions are not always consistent. Nucleic acid assays (PCR RFLP) can distinguish species, but can be costly and do not work on all strains. Identification of the specific etiologic agent during an IBK outbreak is critical to assist veterinarians implement the proper prevention and treatment strategies. Matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry is an emerging tool in bacterial identification that is rapid, inexpensive, and provides advantages over current identification methods. The overall goal of this study was to determine the accuracy of the MALDITOF MS method as applied to Moraxella spp isolated from bovine eyes and compare it to existing identification methods.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of human oral Capnocytophaga species

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for rapid identification of cfxA PCR positive and negative Capnocytophaga strains. Colonies were grown on blood agar, incubated anaerobically at 37 °C for 48 h, and were then evaluated by MALDI-TOF MS and 16S rRNA gene sequencing. Both methods identified all colonies to the genus level. The MALDI-TOF MS method gave the same result, at the species level, as 16S rRNA gene sequencing for 41/53 Capnocytophaga sp. strains (77.4%), but the limit of this technique was the absence of some species (C. leadbetteri, C. AHN) in the Biotyper-Bruker® database used in this study. Distinction between the cefotaxime resistant and susceptible strains was unsuccessful using the MALDI-TOF MS method. This technique had low discriminatory power to rapidly detect beta-lactamase-producing Capnocytophaga strains in clinical samples. However, the results from a score-oriented dendrogram confirmed MALDI-TOF MS is a rapid, inexpensive, and reliable method for Capnocytophaga species identification. Enrichment of the reference database used (Biotyper®) will improve future results.

Comparison of biomarker based Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and conventional methods in the identification of clinically relevant bacteria and yeast

BackgroundMALDI-TOF MS is an analytical method that has recently become integral in the identification of microorganisms in clinical laboratories. It relies on databases that majorly employ pattern recognition or fingerprinting. Biomarker based databases have also been developed and there is optimism that these may be superior to pattern recognition based databases. This study compared the performance of ribosomal biomarker based MALDI-TOF MS and conventional methods in the identification of selected bacteria and yeast.MethodsThe study was a cross sectional study identifying clinically relevant bacteria and yeast isolated from varied clinical specimens submitted to a clinical laboratory. The identification of bacteria using conventional Vitek 2™ automated system, serotyping and MALDI-TOF MS was performed as per standard operating procedures. Comparison of sensitivities were then carried out using Pearson Chi-Square test and p-value of <0.05 was considered statistically significant. Secondary outcomes analyzed included the major and minor error rates.ResultsOf the 383 isolates MALDI-TOF MS and conventional methods identified 97.6 and 95.7% (p = 0.231) to the genus level and 97.4 and 88.0% (p = 0.000) to the species level respectively. Biomarker based MALDI-TOF MS was significantly superior to Vitek 2™ in the identification of Gram negative bacteria and Gram positive bacteria to the species level. For the Gram positive bacteria, significant difference was observed in the identification of Coagulase negative Staphylococci (p = 0.000) and Enterococcus (p = 0.008). Significant difference was also observed between serotyping and MALDI-TOF MS (p = 0.005) and this was attributed to the lack of identification of Shigella species by MALDI-TOF MS. There was no significant difference observed in the identification of yeast however some species of Candida were unidentified by MALDI-TOF MS.ConclusionBiomarker based MALDI-TOF MS had good performance in a clinical laboratory setting with high sensitivities in the identification of clinically relevant microorganisms.

Rapid detection of carbapenemase-producing Klebsiella pneumoniae strains derived from blood cultures by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)

BackgroundCarbapenemase-producing Enterobacteriaceae (CPE), particularly carbapenemase-producing Klebsiella pneumoniae isolates, are important causative agents of nosocomial infections associated with significant mortality rates mostly in critical wards. The rapid detection and typing of these strains is critical either for surveillance purposes and to prevent outbreaks and optimize antibiotic therapy. In this study, the MALDI-TOF MS method was used to detect rapidly these isolates from blood cultures (BCs) and to obtain proteomic profiles enable to discriminate between carbapenemase-producing and non-carbapenemase-producing strains.ResultsFifty-five K. pneumoniae strains were tested. Identification and carbapenemase-production detection assay using Ertapenem were performed both from bacterial pellets extracted directly from BCs flasks and from subcultures of these strains. For all isolates, a complete antimicrobial susceptibility testing and a genotypic characterization were performed.We found 100% agreement between the carbapenemase-producing profile generated by MALDI TOF MS and that obtained using conventional methods. The assay detected and discriminated different carbapenemase-producing K. pneumoniae isolates within 30 min to 3 h after incubation with Ertapenem.ConclusionsMALDI-TOF MS is a promising, rapid and economical method for the detection of carbapenemase-producing K. pneumoniae strains that could be successfully introduced into the routine diagnostic workflow of clinical microbiology laboratories.

Matrix Assisted Laser Desorption Ionization Mass Fingerprinting for Identification of Acacia Gum in Microsamples from Works of Art.

This paper reports an improved method for the identification of Acacia gum in cultural heritage samples using matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) after enzymatic digestion of the polysaccharide component. The analytical strategy was optimized using a reference Acacia gum (gum arabic, sp. A. senegal) and provided an unambiguous MS profile of the gum, characterized by specific and recognized oligosaccharides, from as little as 0.1 μg of material. The enhanced experimental approach with reduced detection limit was successfully applied to the analysis of naturally aged (∼80 year) gum arabic samples, pure and mixed with lead white pigment, and allowed the detection of gum arabic in samples from a late painting (1949/1954) by Georges Braque in the collection of the Art Institute of Chicago. This first application of the technique to characterize microsamples from a painting, in conjunction with analyses by gas chromatography/mass spectrometry (GC/MS), provided important insights into Braque's unusual mixed paint media that are also helpful to inform appropriate conservation treatments for his works. The robustness of the analytical strategy due to the reproducibility of the gum MS profile, even in the presence of other organic and inorganic components, together with the minimal sample size required, demonstrate the value of this new MALDI-TOF MS method as an analytical tool for the identification of gum arabic in microsamples from museum artifacts.

Going off the “Gold-Standard”: Replacing Electrophoretic Methods With Mass Spectrometry for Plasma Cell Disorders

At our institution, protein electrophoretic (PEL) methods have been the mainstay for detecting and monitoring plasma cells disorders (PCD) since 1967. Although widely accepted by hematologist, these electrophoretic methods remain labor intensive and recent developments in treatments for PCDs are pushing electrophoresis to its analytical limits. Our group has recently published a series of articles describing clinically viable methods for M-protein detection and characterization based on mass spectrometry. Two variations of this technology have emerged: a MALDI-TOF MS method (MASS-FIX) and a LC-ESI-Triple TOF MS method (miRAMM) and are currently starting validation in our laboratory. The two methods share the same pre-analytical immunoglobulin purification but differ in their turnaround time, instrument cost, sensitivity and resolution.

Commentary on clinical utility of whole genome sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

3The present study is commentary and aims to evaluate the practical application of Whole Genome Sequencing (WGS) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in clinical microbiology. The MALDI-TOF MS method has been replaced cultural and biochemical tests for species identification in most laboratories worldwide. Moreover, WGS has elevated the discriminatory power by detection of single nucleotide polymorphisms (SNPs), and helped with the identification of microorganisms that were misidentified by previous culture and biochemical tests. Moreover, WGS can be used in epidemiological studies (e.g. clinical outbreaks) by tracking of phylogenetic maps and analyzing SNP distance; antimicrobial resistance studies such as the study of antimicrobial resistance genes, e.g. CTX-M, NDM, KPC, or OXA; and the study of recombination of plasmid or insertion sequence (IS) elements to elucidate the mechanism of antimicrobial resistance. MALDI-TOF technology is used for bacterial or fungal species identification in most laboratories. VITEK MS (bioMerieux) and Biotyper (Bruker Daltonics) are examples of commercially available MALDI-TOF MS systems. The technique has been used for direct identification of organisms from blood culture bottles, markedly shortening the identification time. Beta-lactamases or carbapenemases have been detected by analyzing specific protein peaks using MALDI-TOF MS. Shiga toxin-producing Escherichia coli, Salmonella serotype, and Vibrio phenotypes can also be detected.

A proteomic approach for rapid identification of Weissella species isolated from Korean fermented foods on MALDI-TOF MS supplemented with an in-house database

Weissella are obligate heterofermentative lactic acid bacteria belonging to the Leuconostocaceae family. Some Weissella can be found in salted and fermented foods, such as kimchi and jeotgal, and plays an important role in the fermentation process. In the present study, for the first time, a rapid and accurate identification method for Weissella species from kimchi and jeotgal was developed based on MALDI-TOF MS, supplemented with an in-house database. Of the 135 Weissella spectra aligned with the MALDI bioTyper database, 56 isolates (41.5%) yielded no reliable identification results with low log scores (<1.7). After registering the spectra of six Weissella reference strains, all of the isolates were correctly identified, of which 113 (83.7%) and 22 (16.3%) were identified at the species and genus level, respectively. Moreover, a dendrogram generated by protein profiles of the different Weissella species clearly presented distinctive clusters, and PCA analysis separated the spectra of Weissella species into four clusters. In comparing food origins, different Weissella species were identified from two fermented foods. W. soli and W. cibaria were isolated from kimchi, while W. thailandensis and W. halotolerans were isolated from jeotgal. The results of our proteomic approach confirm that the MALDI bioTyper database, with our in-house Weissella database, is sufficient for Weissella identification. The MALDI-TOF MS method provides fast and reliable discrimination between different species in the genus Weissella and, therefore, will be useful for safety control in fish farms or in the production of fermented foods. This method can also be applied to the control of opportunistic pathogenic Weissella in human clinical infections.

Zein Characterisation of South African Maize Hybrids and Their Respective Parental Lines Using MALDI-TOF MS

Zeins are important storage proteins and play a role in grain texture and impact on processing. Having a technique to accurately quantify the individual zeins and the size of these proteins would allow for more precise understanding of the impact these individual protein have on grain texture and/or processing. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used to characterise zein protein profiles of five South African maize hybrids, grown at three locations, and their respective parental lines. A new, simplified and shortened zein extraction method was used to characterise the zein profiles and to determine any possible relationship between the hybrids and their parents. A matrix solution comprising two matrices, α-cyano-4-hydroxy-cinammic acid (CHCA) and 2-(4-hydroxyphenylazo) benzoic acid (HABA), was required to detect all major zein (α, β, γ and δ) classes. Within the set of hybrids and parents, additional peaks with molecular weights not previously reported were observed. These were identified as belonging to the δ-zein, β-zein and γ-zein. Relationships between the hybrids and their respective parental lines were observed indicating genetic variation for these zein classes exists. The MALDI-TOF MS method identified differences in individual zein proteins and these differences were observed between hybrids. The method shows a potential for accurately quantifying the presence and molecular size of zein proteins which may be important in milling and food processing. Storage proteins play an important role not only in grain composition but also in some processes such as milling, and variation in these individual proteins may impact on efficiency of processing.

Direct identification of trypanosomatids by matrix-assisted laser desorption ionization-time of flight mass spectrometry (DIT MALDI-TOF MS).

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI-TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI-TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI-TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software-assisted identification at the strain level. Overall, this study shows the importance of MALDI-TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry-based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.

Clinical mass spectrometry proteomics (cMSP) for medical laboratory: What does the future hold?

BackgroundMass spectrometry (MS) methods are being widely used these days in medical laboratories for quantifying many small molecular analytes as well as for microbiological purposes. MethodsLittle use has been made so far, however, of MS for analyzing peptides and proteins in clinical laboratory (an approach known as clinical MS proteomics (cMSP)). The explanation for this situation may be that cMSP assays are more complex to implement than conventional assays, require large investments in terms of equipment and training, and have not yet been sufficiently validated for clinical applications. In addition, the protein analysis assays currently used in medical laboratories mostly meet both laboratory and clinical requirements in terms of analytical performances, ease of use, and turn-around-time. ResultsWith the spread of MS methods in laboratories, increasing interest seems to be focusing on the development of MS for quantifying new analytes. MALDI-TOF MS methods have already been replacing classical methods of bacterial classification in clinical laboratories, for example, and this can be said to be an important step in this direction. ConclusionsIn this paper, the literature available on the topic of clinical MS proteomics is reviewed and the pre-analytical, analytical, and post-analytical challenges which will have to be met in connection with this approach are discussed.

Shortcomings of the Commercial MALDI-TOF MS Database and Use of MLSA as an Arbiter in the Identification of Nocardia Species.

Nocardia species are difficult to identify, a consequence of the ever increasing number of species known and their homogeneous genetic characteristics. 16S rRNA analysis has been the gold standard for identifying these organisms, but proteomic techniques such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) and housekeeping gene analysis, have also been explored. One hundred high (n = 25), intermediate (n = 20), and low (n = 55) prevalence (for Spain) Nocardia strains belonging to 30 species were identified via 16S rRNA and MALDI-TOF MS analysis. The manufacturer-provided database MALDI Biotyper library v4.0 (5.627 entries, Bruker Daltonik) was employed. In the high prevalence group (Nocardia farcinica, N. abscessus, N. cyriacigeorgica and N. nova), the 16S rRNA and MALDI-TOF MS methods provided the same identification for 76% of the strains examined. For the intermediate prevalence group (N. brasiliensis, N. carnea, N. otitidiscaviarum and N. transvalensis complex), this figure fell to 45%. In the low-prevalence group (22 species), these two methods were concordant only in six strains at the species level. Tetra-gene multi-locus sequencing analysis (MLSA) involving the concatemer gyrB-16S rRNA-hsp65-secA1 was used to arbitrate between discrepant identifications (n = 67). Overall, the MLSA confirmed the results provided at species level by 16S rRNA analysis in 34.3% of discrepancies, and those provided by MALDI-TOF MS in 13.4%. MALDI-TOF MS could be a strong candidate for the identification of Nocardia species, but only if its reference spectrum database improves, especially with respect to unusual, recently described species and species included in the described Nocardia complexes.

Emerging tools for identification of arthropod vectors.

The rapid and reliable identification of arthropod vector species is an essential component of the fight against vector-borne diseases. However, owing to the lack of entomological expertise required for the morphological identification method, development of alternative and complementary tools is needed. This review describes the main methods used for arthropod identification, focusing on the emergence of protein profiling using MALDI-TOF MS technology. Sample preparation, analysis of reproducibility, database creation and blind tests for controlling accuracy of this tool for arthropod identification are described. The advantages and limitations of the MALDI-TOF MS method are illustrated by emphasizing different hematophagous arthropods, including mosquitoes and ticks, the top two main vectors of infectious diseases.

Utility of the MALDI-TOF MS method to identify nontuberculous mycobacteria.

In comparison to the conventional real-time polymerase chain reaction method (PCR method) or the DNA-DNA hybridization method (DDH method), the utility of NTM identification by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method has seldom been reported. In this study, 75 clinical NTM isolates from our hospital between April 2013 and July 2014 were identified and analyzed using PCR, DDH, and MALDI-TOF MS methods, and the results for the MALDI-TOF MS method were compared with the others. Identification at the species level was in agreement for 71 (94.5%) of the 75 isolates. For further details, identification was possible for 23 (95.8%) of 24 Mycobacterium avium, 11 (100%) of 11 Mycobacterium intracellulare, and 1 (50%) of 2 isolates mixed with M.avium and M. intracellulare. Mycobacterium ksansasii, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium gordonae, and Mycobacterium chelonae identified by DDH method were same result by MALDI-TOF MS. Additionally, Mycobacterium mucogenicum, which could not be identified by the DDH method, was identified by the MALDI-TOF MS method. However, two isolates identified as Mycobacterium terrae by DDH method could not be identified by the MALDI-TOF MS method and were determined to be Mycobacterium arupense by 16S ribosomal RNA (rRNA) sequence analysis. The present findings show that, for rare bacterial species, identification is sometimes not possible, but, in most cases, the results of identification by the MALDI-TOF MS method have a high concordance rate with the results of the PCR and DDH methods.

Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria.

The minimal antibiotic options for carbapenemase-producing Gram-negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI-TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem-resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemase-producing isolates. MALDI-TOF MS and/or molecular methods like microarray, loop-mediated isothermal amplification and real-time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies.

P07.11 Whole genome sequencing characterisation of a unique neisseria strain generating a positive result with the cobas® ct/ng test

<h3>Introduction</h3> <i>Neisseria gonorrhoeae</i> (NG) cause sexually transmitted bacterial infections. The Roche cobas^® CT/NG Test is a qualitative Nucleic Acid Amplification test (NAAT) utilising a dual-target approach to detect sequences within the direct repeat (DR-9) region of the NG genome. To date, clinical performance data show excellent sensitivity and specificity for urogenital specimens. The test is validated for endocervical swabs, vaginal swabs and urine specimens. <h3>Methods</h3> One clinical case report¹ describes a positive test result for an oropharyngeal swab, an off-label specimen type for the cobas^® CT/NG Test. This interesting Neisseria sample was subject to analysis as part of Roche’s Global Surveillance Program. The isolate was tested by culture, phenotyping, whole cell MALDI-TOF MS and molecular methods such as 16S rDNA based typing and whole genome sequencing. <h3>Results</h3> Whole genome sequencing resulted in a complete genome assembly supporting phylogenetic analysis. Sequencing data assembled into a 2,882,113bp contig and genome closure, which identified the sample as a Neisseria commensal species clustering in the same clade with both N. sicca and N. macacae. One partial assay specific DR-9 target region was identified within the genome of this commensal strain in the context of a phage like motif, which can facilitate genomic exchange of DNA fragments between <i>N. gonorrhoeae</i> and commensal strains. BLAST searches for the prophage/DR9 genetic signature of this newly characterised Neisseria strain revealed no matches to sequences in the public domain, suggesting that this strain is rare. Based on the sequence information of the prophage/DR9 genetic signature oropharyngeal swab specimens determined to be positive by the cobas^® CT/NG Test are currently being evaluated for the presence of this sequence motif. <h3>Conclusion</h3> The cobas^® CT/NG Test on the cobas 4800 system is a reliable molecular method for detection of Chlamydia and Gonorrhoeae from genital specimens. <h3>Disclosure of interest</h3> Authors of the abstract are employees of Roche Molecular Systems. <h3>Reference</h3> Upton A, Bromhead C, Whiley DM. <i>Neisseria gonorrhoeae</i> false-positive result obtained from a pharyngeal swab by using the Roche cobas 4800 CT/NG assay in New Zealand in 2012. <i>J Clin Microbiol</i>. 2013;<b>51</b>:1609–10

Identification of strains with phenotypes similar to those of Staphylococcus aureus isolated from table chicken eggs using MALDI-TOF MS and genotyping methods

Abstract The aim of the study was to identify the affinity of 10 Staphylococcus strains isolated from table chicken eggs to specific species. Preliminary analysis performed by API ID32 Staph test identified these strains as S. aureus, but they exhibited a negative reaction in the tube coagulase test. Thus, the analysed strains were initially characterised as Staphylococcus aureus-like (SAL). Further characterisation was performed by genotypic methods, using restriction fragment length polymorphism (RFLP) of the coagulase gene (coa) and sequencing of the gene rpoB. An attempt was also made to identify the isolated Staphylococcus strains by MALDI-TOF mass spectrometry. The results indicated that none of the strains tested belonged to the species S. aureus. The rpoB sequences of five isolates showed the highest sequence similarity to S. haemolyticus, three isolates to S. chromogenes, and one isolate to S. epidermidis. One strain (SAL4) remained unidentified in this analysis. The results obtained using mass spectrometry were comparable to those based on gene sequence analysis. Strain SAL4, which could not be identified by sequencing, was identified by MALDI-TOF as Staphylococcus chromogenes.