Microflex LT Mass Spectrometer Research Articles

Molecular Сharacterization of <i>Neisseria meningitidis</i> Serogroup W in St. Petersburg

The study included 14 isolates of Neisseria meningitidis serogroup W, isolated from patients with generalized forms of meningococcal infection aged 0–17 years at the Pediatric Research and Clinical Center for Infectious Diseases during the period from 2009 to 2020, as well as from carriers. The isolates from carriers (17–18 years old) were isolated in 2016–2019. The isolates were identified by MALDI-TOF mass spectrometry using a Microflex LT mass spectrometer (Bruker Daltonics, Germany). Whole genome sequencing of N. meningitidis was carried out on the basis of the Miseq Illumina platform; it was established that they belong to ST11 (cc11) sequence type. The results of the whole genome sequencing of the studied meningococci showed that there are 2 groups of the subcluster belonging to the Hajj cluster in St. Petersburg. In addition, they show close affinity with meningococci of the Anglo-French and Swedish clusters of the Hajj line.

Antibiotic Sensitivity of <i>Neisseria meningitidis</i> Isolated From Patients with Generalized Forms of Meningococcal Infection and From Healthy Carriers

The study included 50 isolates of Neisseria meningitidis isolated from patients with generalized forms of meningococcal infection and 48 isolates, which were isolated from carriers. The isolates were identified by MALDI-TOF mass spectrometry using a Microflex LT mass spectrometer (Bruker Daltonics, Germany). Minimum inhibitory concentrations (MICs) of benzylpenicillin, ampicillin, ceftriaxone, meropenem, ciprofloxacin, azithromycin, rifampicin, and chloramphenicol were assessed by the method of serial agar dilution with interpretation of the results according to EUCAST 2021 criteria. Clinical resistance to penicillin was detected in 7% of isolates. However, all isolates with MIC >0.064 µg/ml (n=26) had mutations in the penA gene. Decreased sensitivity to rifampicin was found in four isolates isolated from patients and four isolated from carriers. Seven out of eight isolates with reduced sensitivity to rifampicin had mutations in the rpoB gene. Resistance to ceftriaxone, meropenem, ciprofloxacin was not detected.

MALDI-TOF mass spectrometry for direct KPC detection among Enterobacterales.

In this study, we evaluate a method for the KPC enzyme detection, using MALDI-TOF MS, for Enterobacterales. A total of 300 clinical Enterobacterales isolates were selected. The collection included 259 carbapenemase-producing (157 KPC and 102 non-KPC) and 41 carbapenemase non-producing isolates. Bacterial proteins were extracted from Mueller-Hinton agar plates using formic acid, isopropyl alcohol, and water (17:33:50). Samples were prepared with a double layer of synapinic acid. Analyses were performed using a Microflex LT mass spectrometer (Bruker Daltonics) and flexAnalysis 4.0 software (Bruker Daltonics). Statistical analyses were performed using SPSS Software. A distinctive peak at m/z 28,643-28,731 was found in all 157 KPC-producing isolates, and it was consistently absent in the 143 KPC non-producing group. KPC-producing peak intensities ranged from 77 to 3893. Considering an intensity cutoff value ≥ 120 for the presence of KPC, this methodology presented 98.09% and 97.90% of sensitivity and specificity, respectively.

Rare species in the structure of acid-resistent members of the order Actinomycetales isolated from clinical material.

The article presents data on the structure of acid-resistant members of the order Actinomycetales and rare species that have been isolated and identified using various methods. The study included strains of non-tuberculous mycobacteria (NTM) isolated from clinical material during examination for tuberculosis in the period from 2016 to 2019. The total number of samples with signs of NTMs growth that were included in the study was 316 samples. Primary isolation on Levenshtein-Jensen, Finn II, and MGIT media and NTMs identification by DNA-hybridization. All strains that were not identified prior to the species and culture, identified as microorganisms with a high G+C content (High GC GR +) were re-identified using a MALDI-ToF Microflex LT mass spectrometer (Bruker®). By the method of DNA-hybridization, 188 strains isolated by NTM were successfully identified to form 58.5% of all selected cultures. Among the selected species, representatives of slowly growing NTMs (M. avium complex, M. gordonae, M. kansasii) predominated, which amounted to 67.0% of all NTM strains identified to the species. Among the cultures for which DNA hybridization failed to carry out acceptable identification, predominantly NTMs were found, among which M. gordonae, M. avium, M. kansasii dominated. A number of NTMs were represented by rare species: M. iranicum and M. pseudoshottsii. Among this group of microorganisms, other acid-resistant aerobic actinomycetes were isolated, including those of potential clinical significance: Gordonia spp., Tsukamurella spp., Rhodococcus spp., Nocardia spp. When identifying cultures containing high concentrations of G+C, the maximum number of microbial associations was revealed, including those consisting of two types of NTMs (M. monacense + M. flavescens, M. avium + M. kansasii), as well as associations of M. gordonae with staphylococci. The same group included rare NTM species: M. fredericbergense, M. szulgai, M. malmoense, M. bohemicum, M. septicum, as well as representatives of the genera Nocardia, Gordonia, Tsukamurella.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for accurate discrimination of Staphylococcus schleiferi subspecies

S. schleiferi is one of the main species isolated from canine otitis externa, pyoderma and from apparently healthy dogs. The species is divided into two subspecies, S. schleiferi schleiferi and S. schleiferi coagulans. MALDI-TOF MS does not distinguish correctly these two subspecies. This study aimed to identify biomarkers that could possibly discriminate Staphylococcus schleiferi subspecies by MALDI-TOF MS. Twelve strains (eight S. schleiferi schleiferi and four S. schleiferi coagulans) were firstly identified. Each isolate was submitted to a protein extraction protocol and subjected to spectrometry on Bruker Microflex LT mass spectrometer. Spectra were analyzed with the BioNumerics software v7.6. Our results showed that spectra clustered according to subspecies, and a set of five MALDI-TOF MS biomarkers were selected to enable the discrimination of S. schleiferi subspecies. In addition, these biomarkers were predicted to represent highly conserved proteins, which could contribute to the identification of subspecies-specific proteins that could be used for improved subspecies identification in clinical samples.

Combination of Coral UTI ScreenTM system, gram-stain and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for diagnosis of urinary tract infections directly from urine samples

This study proposes an algorithm for microbiological diagnosis of urinary tract infections based on screening by luminometry and Gram-stain, followed by identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Positive urine samples detected with the luminometry screening Coral UTI ScreenTM system underwent Gram staining and identification of the causative organism was performed by MALDI-TOF Microflex LT mass spectrometer (Bruker Daltonics, Germany). Subsequently, the results were compared with those of conventional culture identification using WIDER MIC/id system (Francisco Soria Melguizo SA, Spain). Considering the conventional approach as the gold standard, the proposed algorithm presented both a high specificity (98.1%) and a positive likelihood ratio of 37.42. The implementation of this algorithm would allow diagnosis of urinary tract infection in less than an hour in 92.4% of positive samples. This combination of techniques would be useful particularly for patients with severe UTI, pyelonephritis or urinary sepsis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) proteomic profiling of cerebrospinal fluid in the diagnosis of enteroviral meningitis: a proof-of-principle study.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for diagnosing viral infections by directly testing clinical specimens has not previously been explored. In this proof-of-principle study, we tested the hypothesis that proteomic profiling of cerebrospinal fluid (CSF) by mass spectrometry may be useful in the diagnosis of enteroviral (EV) meningitis. A total of 114 cryopreserved CSF samples were analyzed, of which 47 were positive for EV and 67 were negative. Total CSF proteins were precipitated and subjected to MALDI-TOF-MS analysis in a low (2-20kDa) molecular weight range using a MicroFlex LT mass spectrometer. The whole data set was randomly split into a training set (n = 76 specimens) and a validation set (n = 38 samples). Backward/forward stepwise logistic regression analyses identified 30 peaks that were differentially present in EV-positive and EV-negative specimens. These were used to build a model which displayed an overall classification accuracy of 93%. The discriminative ability of the model was confirmed by using a validation sample set (overall accuracy 83%). In fact, the model was able to correctly classify 61 out of 67 EV-negative samples and 42 out of 47 EV-positive specimens. EV meningitis is associated with a distinctive protein profile that may be directly detectable in CSF specimens by MALDI-TOF-MS.

Potential of MALDI-TOF MS as an alternative approach for capsular typing Streptococcus pneumoniae isolates

Streptococcus pneumoniae can be classified in more than 90 capsular types, as traditionally determined by serological methods and more recently by PCR-based techniques. Such methods, however, can be expensive, laborious or unable to accurately discriminate among certain serotypes. Therefore, determination of capsular types, although extremely important for epidemiological purposes and for estimating the impact of pneumococcal conjugate vaccines, is mainly restricted to research laboratories, being rarely performed in the clinical setting. In the present study, MALDI-TOF MS was evaluated as an alternative tool to characterize 416 pneumococcal isolates belonging to serotypes 6A, 6B, 6C, 9N, 9V or 14. For MALDI-TOF MS analysis, each isolate was submitted to an extraction protocol using formic acid and acetonitrile. Measurements were performed with a Bruker Microflex LT mass spectrometer using default parameters and generating spectra in the range of 2,000–20,000 m/z. Spectra were analyzed with the BioNumerics software v7.6. Isolates were mainly distributed according to the capsular type in a Neighbor Joining tree and serotypes investigated were successfully discriminated by the presence/absence of 14 selected biomarkers. The results suggest that MALDI-TOF MS is a promising alternative for typing pneumococcal strains, highlighting its usefulness for rapid and cost-effective routine application in clinical laboratories.

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.

Rapid identification of bacteria from bioMerieux BacT/ALERT blood culture bottles by MALDI-TOF MS

Several studies have reported poor results when trying to identify microorganisms directly from the bioMerieux BacT/ALERT blood culture system using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry. The aim of this study is to evaluate two new methods, Sepsityper and an enrichment method for direct identification of microorganisms from this system. For both methods the samples were processed using the Bruker Microflex LT mass spectrometer (Biotyper) using the Microflex Control software to obtain spectra. The results from direct analysis were compared with those obtained by subculture and subsequent identification. A total of 350 positive blood cultures were processed simultaneously by the two methods. Fifty-three cultures were polymocrobial or failed to grow any organism on subculture, and these results were not included as there was either no subculture result, or for polymicrobial cultures it was known that the Biotyper would not be able to distinguish the constituent organisms correctly. Overall, the results showed that, contrary to previous reports, it is possible to identify bacteria directly from bioMerieux blood culture bottles, as 219/297 (74%) correct identifications were obtained using the Bruker Sepsityper method and 228/297 (77%) were obtained for the enrichment method when there is only one organism was present. Although the enrichment method was simpler, the reagent costs for the Sepsityper method were approximately £4.00 per sample compared to £0.50. An even simpler and cheaper method, which was less labour-intensive and did not require further reagents, was investigated. Seventy-seven specimens from positive signalled blood cultures were analysed by inoculating prewarmed blood agar plates and analysing any growth after 1-, 2-and 4-h periods of incubation at 37° C, by either direct transfer or alcohol extraction. This method gave the highest number of correct identifications, 66/77 (86%), and was cheaper and less labour-intensive than either of the two above methods.

Streptococcus constellatus Lymphadenitis in Chronic Granulomatous Disease

We read with interest the article titled “Streptococcal infections in Patients with Chronic Granulomatous Disease” by Falcone et al in August issue of the journal. We would like to report a case of lymphadenitis and pneumonia in a 14 year-old boy with chronic granulomatous disease due to Streptococcus constellatus a member of the Streptococcus anginosus group. This 14 year-old patient presented to our clinic with history of an enlarging neck mass for one week and two days of mild nonproductive cough. He did not have any constitutional symptoms. His physical examination demonstrated a 2×4 cm non-fluctuant and non-tender mass in the left anterior cervical area. The ultrasound of neck revealed conglomerated nodes without any suppuration. His chest xray demonstrated a left upper lobe consolidation. He underwent biopsy of the lymph node and bronchial alveolar lavage washings. Tissue cultures revealed alpha hemolytic colonies after 72 h on sheep blood agar, with a distinctive butterscotch smell. The API 20 Strep system (bioMerieux, Inc. Hazelwood, MO) did not yield an acceptable identification score. Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) using a MALDI-TOF MicroFlex LT mass spectrometer (Bruker Daltonics, Billerica, MA) identified Streptococcus constellatus with high score (∼2.0). We did not test our isolate for production of hydrogen peroxide. Histology of the node revealed acute and chronic inflammation and bronchial alveolar lavage washings yielded no growth. The patient is being treated with high dose amoxicillin and his clinical symptoms have improved. Our case not only extends the observations of Falcone et al but also records lymphadenitis due to Streptococcus anginosus group which is uncommon and has not been previously reported to our knowledge in CGD patients. It is however unclear in the few published cases of S.viridans infections in CGD , if further speciation was attempted or not. Our case not only illustrates the rarity of lymphadenitis due to S.anginosus group but also underscores the importance of observing the cultures for longer than 72 h.

The value of MALDI-TOF MS for the identification of clinically relevant anaerobic bacteria in routine laboratories

Between 2010 and 2011, 283 clinically relevant non-duplicate anaerobic isolates were analysed by MALDI-TOF MS and the results were compared with conventional identification. Immediately after isolation, an ethanol precipitation was carried out on isolated colonies and the stabilized samples were anonymized and sent to the laboratory of Bruker Daltonik, Bremen, Germany, where the identification was done using the standard protocol for micro-organism identification on a Microflex LT mass spectrometer equipped with the MALDI Biotyper 3.0 software. Of 283 isolates, 218 (77 %) were identified at species level [log(score) ≥2.0], 31 isolates (10.95 %) were identified at genus level [log(score) 1.7-2.0] and 34 (12 %) gave non-reliable identification [log(score) <1.7]. Out of the 31 isolates with log(score) 1.7-2.0, in the case of 24 isolates the species name given by the MALDI Biotyper was accepted if it was the same as for the classical identification. Of 218 isolates identified at species level, 40 results were discordant with phenotypic identification, and of the 31 isolates identified at genus level according to the manufacturer's score cut-off, four gave results discordant with the phenotypic method. For the 44 discordant results, 16S rRNA gene sequencing confirmed MALDI-TOF MS identification in 41 cases, leaving three isolates (0.7 %) that had been misidentified by MALDI-TOF MS.