MALDI-TOF MS-based Identification Research Articles

Quantification of β-lactamase producing bacteria in German surface waters with subsequent MALDI-TOF MS-based identification and β-lactamase activity assay

Environmental oligotrophic bacteria are suspected to be highly relevant carriers of antimicrobial resistance (AMR). However, there is a lack of validated methods for monitoring in the aquatic environment. Since extended-spectrum β-lactamases (ESBLs) play a particularly important role in the clinical sector, a culturing method based on R2A-medium spiked with different combinations of β-lactams was applied to quantify β-lactamase-producing environmental bacteria from surface waters. In German surface water samples (n = 28), oligotrophic bacteria ranging from 4.0 × 103 to 1.7 × 104 CFU per 100 mL were detected on the nutrient-poor medium spiked with 3rd generation cephalosporins and carbapenems. These numbers were 3 log10 higher compared to ESBL-producing Enterobacteriales of clinical relevance from the same water samples. A MALDI-TOF MS identification of the isolates demonstrated, that the method leads to the isolation of environmentally relevant strains with Pseudomonas, Flavobacterium, and Janthinobacterium being predominant β-lactam resistant genera. Subsequent micro-dilution antibiotic susceptibility tests (Micronaut-S test) confirmed the expression of β-lactamases. The qPCR analysis of surface waters DNA extracts showed the presence of β-lactamase genes (blaTEM, blaCMY-2, blaOXA-48, blaVIM-2, blaSHV, and blaNDM-1) at concentrations of 3.7 (±1.2) to 1.0 (±1.9) log10 gene copies per 100 mL. Overall, the results demonstrate a widespread distribution of cephalosporinase and carbapenemase enzymes in oligotrophic environmental bacteria that have to be considered as a reservoir of ARGs and contribute to the spread of antibiotic resistance.

A large-scale genomically predicted protein mass database enables rapid and broad-spectrum identification of bacterial and archaeal isolates by mass spectrometry

MALDI-TOF MS-based microbial identification relies on reference spectral libraries, which limits the screening of diverse isolates, including uncultured lineages. We present a new strategy for broad-spectrum identification of bacterial and archaeal isolates by MALDI-TOF MS using a large-scale database of protein masses predicted from nearly 200,000 publicly available genomes. We verify the ability of the database to identify microorganisms at the species level and below, achieving correct identification for > 90% of measured spectra. We further demonstrate its utility by identifying uncultured strains from mouse feces with metagenomics, allowing the identification of new strains by customizing the database with metagenome-assembled genomes.

MALDI-TOF MS-based identification and antibiotics profiling of Salmonella species isolated from retail chilled chicken in Saudi Arabia

ObjectivesSalmonella is a well-known foodborne pathogen that is spread around the world. It causes diseases both in animals and humans. The development of antibiotic-resistant Salmonella strains results in the failure of formerly effective drugs in humans and animals and poses a serious threat to world health. In the Kingdom of Saudi Arabia, the rise in Salmonella prevalence in poultry is seen as a serious problem. Saudi Arabia has endured several epidemics of Salmonella infections with varied patterns of drug resistance in the last few decades. MethodsA total of 112 chilled chicken carcass of five different local poultry companies were procured from retail outlets in Jeddah. The ISO 6579:2002 standard was followed to isolate and identify Salmonella. The isolates were identified using cultural and biochemical features and were further confirmed using (MALDI-TOF MS). Antibiotic susceptibility for each Salmonella isolate was determined using the automated MicroScan WalkAway plus System. ResultsOut of the 112 samples, 35 (31.25%) samples harboured Salmonella spp. According to MALDI-TOF MS identification, 34 isolates were recognized as S. Typhimurium or S. Enteritidis with high confidence levels (log (score) values between 2.00 and 3.00), while one isolate was characterized as a Salmonella sp. with a low confidence level (log (score) < 2.00). The antibiotic sensitivity pattern demonstrated resistance to fluoroquinolones, cephalosporin, and penicillin, however, carbapenem was effective against all the isolated Salmonella spp. Out of the 35 isolates, 23 (65.71%) isolates resisted three or more than three different antibiotics and thus were regarded as multi-drug resistant (MDR) strains. ConclusionsThe findings of this study indicated the presence of MDR Salmonella species in chilled chicken marketed in Jeddah, Saudi Arabia which highlights potential public health risks. Accordingly, a thorough investigation of the veterinary service, safety and hygienic system of poultry industry, as well as vendors is needed to safeguard the consumer health.

Sepsis in Critically Ill Patients: Epidemiology, Risk Factors, and Role of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry for Identification of Sepsis-Causing Organisms.

BackgroundMatrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid mass spectrometry technology for species identification. It is a useful, fast, and accurate tool for routine laboratory analysis. This study aimed to investigate the epidemiology of sepsis-causing organisms in patients admitted to tertiary-level intensive care units (ICU), the role of MALDI-TOF MS in species identification, and patients’ clinical outcomes.MethodologyA prospective observational study was performed in a tertiary-level ICU for one year. The first blood, urine, and endotracheal (ET) aspirate samples were sent before starting antibiotics. We received the antibiotic sensitivity report within 48 hours or earlier using MALDI-TOF MS. Treatment was modified based on MALDI-TOF MS reports. Response to treatment was monitored, and clinical outcomes were noted based on ICU stay. Patients were followed up until discharge, shifting to referring parent unit, or death.ResultsThis study included 200 patients admitted to ICUs who at the time of admission did not have a fever. The most common organisms were Acinetobacter baumannii, Klebsiella pneumonia, and Escherichia coli in ET aspirates; Candida albicans and Enterococcus faecium in urine; and Pseudomonas aeruginosa, K. pneumonia, and A. baumaniiin blood. Of the 200 patients, 130 (65%) shifted to the parent unit ward, and 70 (35%) patients died, with an ICU stay of 12.89 ± 6.51 days. There was no significant difference in mortality when organisms grew from either ET or urine compared with sterile samples. If organisms resistant to all primary antibiotics grew from ET, mortality was 60.6%. Mortality was 56.8% if isolates were in the blood.ConclusionsEarly MALDI-TOF MS-based species identification and appropriate antibiotics initiation play a key role in the treatment and care for critically ill patients with sepsis. MALDI-TOF MS has the potential to significantly aid sepsis management.

Phylogenetic diversity and plant growth-promoting activities of rhizobia nodulating fenugreek (Trigonella foenum-graecum Linn.) cultivated in different agroclimatic regions of India.

Fenugreek (Trigonella foenum-graecum Linn.), is an extensively cultivated legume crop used as a herb, spice, and traditional medicine in India. The symbiotic efficiency and plant growth-promoting potential of fenugreek rhizobia depend on the symbiont strain and environmental factors. We isolated 176 root-nodulating bacteria from fenugreek cultivated in different agroclimatic regions of India. MALDI-TOF MS-based identification and phylogenetic analyses based on 16S rRNA and five housekeeping genes classified the fenugreek-rhizobia as Ensifer (Sinorhizobium) meliloti. However, the strains represent separate sub-lineages of E. meliloti, distinct from all reported sub-lineages across the globe. We also observed the spatial distribution of fenugreek rhizobia, as the three sub-lineages of E. meliloti recorded during this study were specific to their respective agroclimatic regions. According to the symbiotic gene (nodC and nifH) phylogenies, all three sub-lineages of E. meliloti harboured symbiotic genes similar to symbiovar meliloti; as with the housekeeping genes, these also revealed a spatial distribution for different clades of sv. meliloti. The strains could nodulate fenugreek plants and they showed plant growth-promoting potential. Significant differences were found in the plant growth parameters in response to inoculation with the various strains, suggesting strain-level differences. This study demonstrates that fenugreek rhizobia in India are diverse and spatially distributed in different agro-climatic regions.

Current Scenario and Challenges in the Direct Identification of Microorganisms Using MALDI TOF MS.

MALDI TOF MS-based microbial identification significantly lowers the operational costs because of minimal requirements of substrates and reagents for extraction. Therefore, it has been widely used in varied applications such as clinical, food, military, and ecological research. However, the MALDI TOF MS method is laced with many challenges including its limitation of the reference spectrum. This review briefly introduces the background of MALDI TOF MS technology, including sample preparation and workflow. We have primarily discussed the application of MALDI TOF MS in the identification of microorganisms. Furthermore, we have discussed the current trends for bioaerosol detection using MALDI TOF MS and the limitations and challenges involved, and finally the approaches to overcome these challenges.

MALDI-TOF MS-Based identification of bacteria and a survey of fresh vegetables with pathogenic bacteria in Beijing, China

The Codex Alimentarius Commission has established quantitative risk assessment standards and monitoring protocols for foodborne bacterial pathogens. Here, a simple protocol and a time and cost-effective platform were investigated based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify 4 pathogens, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes, which were obtained from 7 types of vegetables. This protocol and platform will be helpful for routine monitoring of foodborne bacterial pathogens. Furthermore, the samples obtained from the Beijing region of China were monitored and reviewed, and the survey results showed 26.0% of samples tested positive for microorganisms. E. coli was present most often (14.0%) and showed similar positive proportions among all of the vegetable products. In addition, exogenous contamination from the transport, storage or primary processing of vegetables requires special attention.

Insufficient repeatability and reproducibility of MALDI-TOF MS-based identification of MRSA.

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is essential for proper initial antibiotic therapy and timely set up of hygienic measures. Recently, detection of MRSA using MALDI-TOF mass spectrometer mediated by the peptide-phenol-soluble modulin (PSM-mec)-linked to the class A mec gene complex present in SCCmec cassettes types II, III, and VIII of MRSA strains, has been commercially available. We present here a multicentre study on MALDI-TOF MS detection of MRSA evincing a poor repeatability and reproducibility of the assay. The sensitivity of the assay varies between 50 and 90% in strains carrying psmMEC and psmδ genes encoding for PSM-mec and δ-toxin (a member of the PSM peptide family), respectively. No false positive results were found. The very major error calculation was 30% and the major error achieved 0%. Interlaboratory repeatability varies between 0 and 100%. No significant difference was observed with the use of different cultivation media. Our data showed a poor sensitivity of the method excluding it from the use in routine laboratory testing.

Early adjustment of empirical antibiotic therapy of bloodstream infections on the basis of direct identification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Gram staining results

IntroductionTo assess the potential added value of rapid MALDI-TOF MS-based identification of bacteria in positive blood cultures to the information provided by Gram staining for adequate empirical antibiotic treatment adjustments in patients with bloodstream infections (BSI). MethodsWe conducted a retrospective, single-center, pre-post quasi-experimental study. In the pre-MALDI-TOF MS phase of the study antibiotic adjustments were made on the basis of Gram stain results, whereas in the MALDI-TOF MS phase they were based on information provided by Gram staining and MALDI-TOF MS results. No antimicrobial stewardship program for BSI was in place within the study period. Antibiotic regimens were categorized as correct, improvable or incorrect. ResultsCohorts were matched for demographics, clinical characteristics of patients and bacterial species involved. Enterobacteriales were the most represented in both study periods (67%), followed by Non-fermenting Gram-negative bacilli and Gram-positive cocci. The number of patients receiving correct, improvable and incorrect empirical antibiotic treatments was comparable for both study periods (P = 0.45, P = 0.57, P = 0.87, respectively). The percentage of patients who ended up receiving correct treatment following modified empirical antibiotic regimens was significantly higher (P = 0.008) in the MALDI-TOF MS phase (27 patients/38.6%) than in the pre-MALDI-TOF MS phase of the study (11 patients/15.7%), although overall adequate coverage of the bacteria causing the infection was comparable across the study periods (90%). ConclusionGram stain results offer valuable information for early adjustment of empirical antibiotic therapies for BSI. Nevertheless, rapid identification of bacteria involved in BSI by MALDI-TOF MS provides added value to achieve this aim.

Direct detection of intact Klebsiella pneumoniae carbapenemases produced by Enterobacterales using MALDI-TOF MS.

A MALDI-TOF MS-based identification method for KPC-producing Enterobacterales was developed. The molecular mass of the intact KPC-2 polypeptide was estimated for blaKPC-2 transformants using MALDI Microflex and the exact mass was confirmed by LC and a high-resolution MS/MS system. A total of 1181 clinical Enterobacterales strains, including 369 KPC producers and 812 KPC non-producers, were used to set up the methodology and the results were compared with those from PCR analyses. For external validation, a total of 458 Enterobacterales clinical isolates from a general hospital between December 2018 and April 2019 were used. The exact molecular mass of the intact KPC-2 protein was 28 718.13 Da and KPC peaks were observed at m/z 28 708.87-28 728.34 using MALDI Microflex. Most of the KPC-2 (99.1%, 335/338) and KPC-3 (100%, 6/6) producers presented a clear peak via this method, while 12.0% (3/25) of the KPC-4 producers had a peak of weak intensity associated with low levels of gene expression. It took less than 20 min for the entire assay to be performed with colonies on an agar plate. External validation showed that the analytical sensitivity and specificity of the method compared with PCR were 100% (59/59) and 99.50% (397/399), respectively. The MALDI-TOF MS-based method for directly detecting the intact KPC protein is applicable to routine tests in clinical microbiology laboratories, supported by its speed, low cost and excellent sensitivity and specificity.

Time to Positivity as a Prognostic Tool in the Performance of Short-Term Subculture for MALDI-TOF MS-Based Identification of Microorganisms from Positive Blood Cultures in Pediatric Patients.

The present study aimed to investigate the relationship between the time to positivity (TTP) of blood cultures and the performance of short-term subculture for MALDI-TOF MS-based identification of microorganisms from positive blood cultures in pediatric patients. The study was conducted between April 2018 and July 2019 at a tertiary children's hospital in Eastern China and the TTP of all the blood cultures included was retrospectively collected at the same time. In total, 332 monomicrobial blood cultures were included in the study. Blood cultures that were identified at the genus level (score ≥ 1.700) by the short-term subculture protocol had shorter TTP (median TTP: 17.5h) than of those not identified (median TTP: 24.6h; P < 10-3). Those that were identified at the species level (score ≥ 2.000) by the short-term subculture protocol also had shorter TTP (median TTP: 16.7h) than of those not identified (median TTP: 21.7h; P < 10-3). ROC curve analysis indicated that the TTP cutoff value to the genus level of the short-term subculture protocol was 18.2h (area under the curve (AUC): 0.801; 95% confidence interval (CI) 0.741-0.861; P < 10-3). At the same time, the TTP cutoff value to the species level of the short-term subculture protocol was 18.1h (AUC: 0.747; 95% CI 0.694-0.800; P < 10-3). TTP is a convenient and valuable prognostic tool for the determination of the performance of short-term subculture for MALDI-TOF MS-based identification of microorganisms from positive blood cultures in pediatric patients.

MALDI-TOF MS-Based Identification of Melanized Fungi is Faster and Reliable After the Expansion of In-House Database.

Invasive fungal infections caused by melanized fungi are a growing concern. Rapid and reliable identification plays an important role in optimizing therapy. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based identification has emerged as a faster and more accurate diagnostic technique. However, lack of a protein extraction protocol and limited database restricts the identification of melanized fungi by MALDI-TOF MS. The study is designed to standardize protein extraction protocol, to enrich the existing, and to create an in-house database for the rapid identification of melanized fungi. In this study, 59 sequence-confirmed, melanized fungi were used to expand and to create an in-house database using a modified protein extraction protocol. A total of 117 clinical isolates are further used to validate the created database. Using existing Bruker database, only 29(24.8%) out of 117 moulds could be identified. However, all the isolates are identified accurately by supplementing the Bruker database with the created in-house database. MALDI-TOF MS takes significantly lesser time for identification compared to DNA sequencing. An expanded database with modified protein extraction protocol can reduce significant time to identify melanized fungi by MALDI-TOF MS.

Application of the biocopolymer preparation system, rapid BACpro® II kit, for mass-spectrometry-based bacterial identification from positive blood culture bottles by the MALDI Biotyper system

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet.Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit.Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with >1.7 score and >2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P < 0.05 for >1.7 and P < 0.05 for >2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with >1.7 score and 69.4% with >2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P < 0.05 for >1.7).These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification.

An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-μm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min.Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test.

Comparison of Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry with VITEK2 Gram Positive System for Identification of Beta Haemolytic Streptococci

Background: Matrix–assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is known as a successful tool for bacterial identification and suitable for clinical diagnostics. The aim of our study was to evaluate diagnostic accuracy of MALDI-TOF MS system and VITEK2 Gram Positive Identification (GP ID) system for the identification of Beta Hemolytic Streptococci (BHS). Materials and Methods: A total 148 BHS which were isolated from various clinical specimens were included in the study. Isolates were identified by Bruker Biotyper 3.4 and VITEK2 GP ID coupled to the agglutination test. Identification results for Bruker Biotyper 3.4 and VITEK2 GP ID systems were classified in three different categories as follows: correct identification, low level of discrimination, no identification/misidentification. Identification results of BHS were evaluated according to these categories. Results: BHS isolates were defined as Streptococcus pyogenes (n: 69), Streptococcus agalactiae (n: 59), Streptococcus dysgalactiae serogroup C (n: 5), and Streptococcus dysgalactiae serogroup G (n: 15) according to latex agglutination assay. MALDI-TOF MS analysis of 148 BHS isolates yielded correct identification for 89.9% of S. pyogenes , 64.4% of S. agalactiae , and 95.0% S. dysagalactiae serogroup C and G; while VITEK2 GP ID gave correct identification for 62.3% of S. pyogenes , 67.8% of S. agalactiae , and 65.0% of S. dysagalactiae serogroup C and G. Conclusions: MALDI-TOF MS-based identification provides faster and more accurate identification of beta-hemolytic streptococcal species than VITEK2 GP ID system.

Validation of a novel automatic deposition of bacteria and yeasts on MALDI target for MALDI-TOF MS-based identification using MALDI Colonyst robot.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) -based identification of bacteria and fungi significantly changed the diagnostic process in clinical microbiology. We describe here a novel technique for bacterial and yeast deposition on MALDI target using an automated workflow resulting in an increase of the microbes’ score of MALDI identification. We also provide a comparison of four different sample preparation methods. In the first step of the study, 100 Gram-negative bacteria, 100 Gram-positive bacteria, 20 anaerobic bacteria and 20 yeasts were spotted on the MALDI target using manual deposition, semi-extraction, wet deposition onto 70% formic acid and by automatic deposition using MALDI Colonyst. The lowest scores were obtained by manual toothpick spotting which significantly differ from other methods. Identification score of semi-extraction, wet deposition and automatic wet deposition did not significantly differ using calculated relative standard deviation (RSD). Nevertheless, the best results with low error rate have been observed using MALDI Colonyst robot. The second step of validation included processing of 542 clinical isolates in routine microbiological laboratory by a toothpick direct spotting, on-plate formic acid extraction (for yeasts) and automatic deposition using MALDI Colonyst. Validation in routine laboratory process showed significantly higher identification scores obtained using automated process compared with standard manual deposition in all tested microbial groups (Gram-positive, Gram-negative, anaerobes, and yeasts). As shown by our data, automatic colony deposition on MALDI target results in an increase of MALDI-TOF MS identification scores and reproducibility.

Clinical Strains of Chryseobacterium and Elizabethkingia spp. Isolated from Pediatric Patients in a University Hospital: Performance of MALDI-TOF MS-Based Identification, Antimicrobial Susceptibilities, and Baseline Patient Characteristics.

Our objective was to evaluate the performance of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of the Chryseobacterium and Elizabethkingia spp. isolated from pediatric patients at Hacettepe University Hospital using 16S rRNA gene sequencing as the gold standard and to determine the antimicrobial susceptibility patterns of the isolates and baseline characteristics of patients. All stored Chryseobacterium and Elizabethkingia spp. isolated from various clinical specimens (sputum, blood, and urine) of pediatric patients at Hacettepe University Hospital between 2012 and 2016 were included in this study. Minimum inhibitory concentrations of 10 antimicrobial agents were determined by Etest for all isolates. To determine the baseline characteristics of patients, medical records of all patients were retrospectively reviewed. In total, 18 isolates of Chryseobacterium spp. (16 C. indologenes, 2 C. gleum) and 5 isolates of Elizabethkingia spp. (3 E. meningoseptica, 2 E. anophelis) were identified by 16S rRNA sequencing. MALDI-TOF MS correctly identified 19 (82.6%) isolates to the species level. The quinolones (ciprofloxacin and levofloxacin), trimethoprim/sulfamethoxazole and piperacillin/tazobactam showed the highest spectrum of activity against the overall collection of isolates. Cystic fibrosis (CF) was the underlying disease in 81.8% of patients. To our knowledge, this study includes the largest number of Chryseobacterium spp. isolated from clinical specimens of pediatric patients in Turkey. In this study, we also report the first clinical isolate of E. anophelis in Turkey. Since, the majority of strains were isolated from patients with CF; larger, prospective clinical studies are needed to establish whether chryseobacteria could be considered as an emerging opportunistic pathogen in patients with CF.

MALDI-TOF mass spectrometry: An emerging tool for unequivocal identification of non-fermenting Gram-negative bacilli.

Background & objectives:Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been instrumental in revolutionizing microbiological identification, especially in high-throughput laboratories. It has enabled the identification of organisms like non-fermenting Gram-negative bacilli (NFGNB), which has been a challenging task using conventional methods alone. In this study an attempt was made to validate MALDI-TOF MS for the identification of clinical isolates of each of the three most common NFGNB, other than Pseudomonas spp., taking molecular methods as the gold standard.Methods:One hundred and fifty clinical isolates of NFGNB, confirmed by molecular methods such as Acinetobacter baumannii[oxa-51 polymerase chain reaction (PCR)], Burkholderia cepacia complex (expanded multilocus sequence typing) and Stenotrophomonas maltophilia (species-specific PCR), were taken. Isolated colonies from fresh cultures of all 150 isolates were smeared onto ground steel plate, with and without formic acid extraction step. The identification was carried out using MALDI-TOF MS Biotyper database.Results:A concordance of 100 and 73.33 per cent was found between the molecular techniques and MALDI-TOF MS system in the identification of these isolates up to genus and species levels, respectively. Using a cut-off of 1.9 for reliable identification, rate of species identification rose to 82.66 per cent. Principal component analysis dendrogram and cluster analysis further increased discrimination of isolates.Interpretation & conclusions:Our findings showed MALDI-TOF MS-based identification of NFGNB as a good, robust method for high-throughput laboratories.

Applications of copolymer for rapid identification of bacteria in blood culture broths using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify pathogens in blood culture samples. However, sample pretreatment is needed for direct identification of microbes in blood culture bottles. Conventional protocols are complex and time-consuming. Therefore, in this study, we developed a method for collecting bacteria using polyallylamine–polystyrene copolymer for application in wastewater treatment technology. Using representative bacterial species Escherichia coli and Staphylococcus capitis, we found that polyallylamine–polystyrene can form visible aggregates with bacteria, which can be identified using MALDI-TOF MS. The processing time of our protocol was as short as 15min. Hemoglobin interference in MALDI spectra analysis was significantly decreased in our method compared with the conventional method. In a preliminary experiment, we evaluated the use of our protocol to identify clinical isolates from blood culture bottles. MALDI-TOF MS-based identification of 17 strains from five bacterial species (E. coli, Klebsiella pneumoniae, Enterococcus faecalis, S. aureus, and S. capitis) collected by our protocol was satisfactory. Prospective large-scale studies are needed to further evaluate the clinical application of this novel and simple method of collecting bacteria in blood culture bottles.

Decision criteria for MALDI-TOF MS-based identification of filamentous fungi using commercial and in-house reference databases

BackgroundSeveral Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry protocols, which differ in identification criteria, have been developed for mold and dermatophyte identification. Currently, the most widely used approach is Bruker technology, although no consensus concerning the log(score) threshold has been established. Furthermore, it remains unknown how far increasing the number of spots to compare results might improve identification performance.In this study, we used in-house and Bruker reference databases as well as a panel of 422 isolates belonging to 126 species to test various thresholds. Ten distinct identification algorithms requiring one to four spots were tested.ResultsOur findings indicate that optimal results were obtained by applying a decisional algorithm in which only the highest score of four spots was taken into account with a 1.7 log(score) threshold. Testing the entire panel enabled identification of 87.41% (in-house database) and 35.15% (Bruker database) of isolates, with a positive predictive value (PPV) of 1 at the genus level for both databases as well as 0.89 PPV (in-house database) and 0.72 PPV (Bruker database) at the species level. Applying the same rules to the isolates for which the species were represented by at least three strains in the database enabled identification of 92.1% (in-house database) and 46.6% (Bruker database) of isolates, with 1 PPV at the genus level for both databases as well as 0.95 PPV (in-house database) and 0.93 PPV (Bruker database) at the species level.ConclusionsDepositing four spots per extract and lowering the threshold to 1.7, a threshold which is notably lower than that recommended for bacterial identification, decreased the number of unidentified specimens without altering the reliability of the accepted results. Nevertheless, regardless of the criteria used for mold and dermatophyte identification, commercial databases require optimization.