Microflex LT Research Articles

Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory

This study compared the diagnostic performance of Bruker's Microflex LT and bioMérieux's Vitek MS matrix-assisted laser desorption ionization–time of flight mass spectrometry systems. A total of 477 isolates were tested on both instruments. Discrepant results were resolved by sequencing. Overall, there was no statistically significant difference between the proportion of isolates correctly identified, miscalled or not called by each instrument. Although both systems were good at identifying yeast (66/69 to species level), the confidence level was high only to genus level for 30% of the isolates on the Bruker. Both systems performed with high accuracy when evaluated solely on Food and Drug Administration–approved organisms for each database. A user-based assessment of the 2 instruments revealed an overall preference for the Vitek MS instrument.

Answer to October 2014 Photo Quiz

Answer: Imported cutaneous diphtheria. The suspected colonies were rapidly identified as Corynebacterium diphtheriae by use of a Microflex LT matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) and subsequently characterized as nontoxigenic Corynebacterium diphtheriae biotype mitis by the National Reference Center for Corynebacterium of the diphtheriae Complex (Institut Pasteur, Paris, France). Treatment with antibiotics was carried on for a total duration of 14 days and allowed for a complete recovery. Nasopharyngeal swabs were taken for all members of the household (i.e., the patient's sister, father, and mother) in order to assess the presence of asymptomatic carriers. All samples were negative for C. diphtheriae growth. Multilocus sequence typing of the isolate generated a new allelic profile, assigned as sequence type 251 (1). Diphtheria is usually considered a life-threatening pharyngitis or laryngitis, and C. diphtheriae is generally not regarded as a skin pathogen. In vaccinated countries, cutaneous diphtheria is discovered as imported cases in travelers coming from areas where the disease is endemic or as autochthonous cases in socioeconomically disadvantaged populations (2,–4). Clinical presentation of skin diphtheria is nonspecific, and most commonly, the organs involved are the lower and upper limbs. Clinical symptoms typically begin as a vesicle, which rapidly progresses to form a punch-out ulcer. At first, these skin lesions may be painful, but they rapidly become insensitive to pain. Diphtheria ulcers generally evolve chronically, and spontaneous recovery can take several weeks. Interestingly, in the present case, the lesions were pruriginous, and scratching them could therefore enhance the diffusion of the bacterium. Definitive diagnosis is generally provided by culture of lesion swabs, but the specific Tinsdale medium is generally not available, as it is expensive and has a shelf life of 1 week. C. diphtheriae colonies are morphologically indistinguishable from those of other Corynebacterium species and thus are likely to be confused with skin flora. Furthermore, most ulcers yield growth of associated pathogens such as Staphylococcus aureus and Streptococcus pyogenes, therefore implying the existence of a causal relationship between the latter pathogens and the skin lesions (5, 6). In fact, these pathogens are usually involved in acute skin infections (e.g., erysipelas or impetigo), while cutaneous diphtheria is chronic and painless. C. diphtheriae identification is challenging and time-consuming with the use of conventional methods, but as it proved to be of valuable assistance in this investigation, MALDI-TOF mass spectrometry would probably simplify and expedite the identification of this pathogen (7, 8). Recommended treatment of skin diphtheria includes thorough cleaning of the lesion, penicillin or macrolide therapy, and, in cases of toxigenic symptoms, antitoxin (2, 9, 10). In conclusion, chronic cutaneous ulcers in specific epidemiological situations should be tested for diphtheria, independent of vaccination status. Their singular clinical and microbiological manifestations probably explain why cutaneous lesions evolve chronically and are more contagious than respiratory infections (3). (See page 3523 in this issue [doi:10.1128/JCM.00779-13] for photo quiz case presentation.)

Stafilokokların Pozitif Kan Kültür Şişelerinden MALDI-TOF MS Sistemi ile Direkt Olarak Tanımlanması

Bloodstream infections are substantial causes of morbidity and mortality worldwide. Staphylococcus species are the most commonly isolated microorganisms from blood cultures in clinical microbiology laboratories. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time- of- Flight Mass Spectrometry) system allows the identification of microorganisms directly from positive blood culture bottles. The aim of this study was to evaluate the MALDI-TOF MS system for the identification of staphylococci directly from the positive blood culture bottles which revealed the presence of gram-positive cocci by staining. A total of 96 positive blood culture bottles that yielded gram-positive cocci by Gram stain were evaluated. These blood cultures were obtained from 69 patients between December 2013-February 2014. Conventional methods and BD Phoenix™ automated bacterial identification system (Becton Dickinson, USA) were used for routine identification. The strains were also identified by real-time Taqman PCR (qPCR) which was considered as the reference method. In MALDI-TOF MS method, MALDI Sepsityper™ Kit was used for the bacterial identification and all measurements were carried out by using Microflex LT instrument and FlexControl 3.0 software (Bruker Daltonics, USA). Of 96 culture bottles positive for gram-positive cocci, 90 were correctly identified as staphylococci at genus level with all the three study methods (qPCR, BD Phoenix, Bruker MALDI-TOF MS). The other six samples were identified as Enterococcus faecium (n= 4) and Streptococcus pyogenes (n= 2) by both Phoenix and the MALDI-TOF systems. Of the 90 samples, 87 were identified at the species level (15 S.aureus, 33 S.epidermidis, 29 S.hominis, 10 S.haemolyticus) and three at the genus level by the reference qPCR method. When comparing the results obtained by qPCR and Bruker MALDI-TOF MS, incompatibility was detected for three isolates. Those isolates were identified as S.hominis by qPCR, however two of them were identified as S.haemolyticus and one as S.epidermidis by MALDI-TOF MS. Compared with real-time Taqman PCR it was detected that Bruker MALDI TOF MS was identified 100% of S.aureus to the genus and species level and 100% and 96.6% of coagulase-negative staphylococci (CNS) to the genus and species level, respectively. In conclusion, it was thought that Bruker MALDI TOF MS system may allow rapid and reliable identification of S.aureus and CNS directly from positive blood culture bottles compared with the routine methods used in the clinical microbiology laboratory.

Novel physico-chemical diagnostic tools for high throughput identification of bovine mastitis associated gram-positive, catalase-negative cocci.

BackgroundThe routine diagnosis of Streptococcus spp. and other mastitis associated gram-positive, catalase-negative cocci is still based upon biochemical tests and serological methods, which frequently provide ambiguous identification results. We therefore aimed to establish an accurate identification system for differential diagnosis of mastitis associated Streptococcus spp. and related species using biophysical techniques such as Fourier-transform infrared (FTIR) spectroscopy and MALDI – TOF/MS.ResultsBased on a panel of 210 isolates from cases of bovine mastitis, an unsupervised FTIR spectral reference library was established and an artificial neural network (ANN) - assisted identification system was developed. All bacterial isolates were previously identified by species-specific PCR and/or 16S rRNA gene sequence analysis. An overall identification rate of 100% at species level for 173 strains unknown to the ANN and the library was achieved by combining ANN and the spectral database, thus demonstrating the suitability of our FTIR identification system for routine diagnosis. In addition, we investigated the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of mastitis associated Streptococcus spp. and related bacteria. Using the Microflex LT System, MALDI Biotyper software™ (V3.3) we achieved an accuracy rate of 95.2%. A blind study, including 21 clinical samples from dairy cows, revealed a 100% correct species identification rate for FTIR and 90.5% for MALDI-TOF MS, indicating that these techniques are valuable tools for diagnosis.ConclusionsThis study clearly demonstrates that FTIR spectroscopy as well as MALDI-TOF MS can significantly improve and facilitate the identification and differentiation of mastitis associated Streptococcus spp. and related species. Although the FTIR identification system turned out being slightly superior to MALDI-TOF MS in terms of identification on species level, both methods offer interesting alternatives to conventional methods currently used in mastitis diagnosis as both of them provide high accuracy at low operating costs once the instrument is acquired.

MALDI-ToF mass spectrometry for the rapid diagnosis of cancerous lung nodules.

Recently, tissue-based methods for proteomic analysis have been used in clinical research and appear reliable for digestive, brain, lymphomatous, and lung cancers classification. However simple, tissue-based methods that couple signal analysis to tissue imaging are time consuming. To assess the reliability of a method involving rapid tissue preparation and analysis to discriminate cancerous from non-cancerous tissues, we tested 141 lung cancer/non-tumor pairs and 8 unique lung cancer samples among the stored frozen samples of 138 patients operated on during 2012.Samples were crushed in water, and 1.5 µl was spotted onto a steel target for analysis with the Microflex LT analyzer (Bruker Daltonics). Spectra were analyzed using ClinProTools software. A set of samples was used to generate a random classification model on the basis of a list of discriminant peaks sorted with the k-nearest neighbor genetic algorithm. The rest of the samples (n = 43 cancerous and n = 41 non-tumoral) was used to verify the classification capability and calculate the diagnostic performance indices relative to the histological diagnosis. The analysis found 53 m/z valid peaks, 40 of which were significantly different between cancerous and non-tumoral samples. The selected genetic algorithm model identified 20 potential peaks from the training set and had 98.81% recognition capability and 89.17% positive predictive value. In the blinded set, this method accurately discriminated the two classes with a sensitivity of 86.7% and a specificity of 95.1% for the cancer tissues and a sensitivity of 87.8% and a specificity of 95.3% for the non-tumor tissues. The second model generated to discriminate primary lung cancer from metastases was of lower quality.The reliability of MALDI-ToF analysis coupled with a very simple lung preparation procedure appears promising and should be tested in the operating room on fresh samples coupled with the pathological examination.

MALDI-TOF mass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131) and D (ST69, ST405, ST393)

Reliable, quick and low-cost methods are needed for the early detection of multidrug-resistant and highly virulent high-risk B2 and D Escherichia coli clones or clonal complexes (HiRCC). Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) seems to have a good discriminatory potential at different subspecies levels, but it was never evaluated for the discrimination of E. coli clones. We assessed the potential of MALDI-TOF MS coupled to multivariate data analysis to discriminate representative E. coli B2 and D HiRCC. Seventy-three E. coli isolates from B2 (including ST131 and B2 non-ST131 clones) and D (ST69, ST393, ST405) with variable pulsed-field gel electrophoresis (PFGE) patterns, origins and dates (1980-2010) were tested. MS spectra were acquired from independent extracts obtained from different plate cultures in two different Microflex LT MALDI-TOF devices (Bruker) after a standard extraction procedure. MALDI-TOF MS fingerprinting analysis revealed a good discriminatory ability between the four HiRCC analysed (ST131, ST69, ST405, ST393) and between B2 ST131 and other B2 non-ST131 isolates. Clusters defined by MALDI-TOF MS were consistent with the clonal complexes assigned by multilocus sequence typing (MLST), although differences were detected regarding the composition of clusters obtained by the comparison of PFGE profiles. We demonstrate, for the first time, that characteristic mass fingerprints of different E. coli HiRCC are sufficiently discriminatory and robust to enable their differentiation by MALDI-TOF MS, which might represent a promising tool for the optimisation of infection control, individual patient management and large-scale epidemiological studies of public health relevance. The good correlation between phenotypic and genotypic features further corroborates phylogenetic relationships delineated by MLST.

Performance of two MALDI-TOF MS systems for the identification of yeasts isolated from bloodstream infections and cerebrospinal fluids using a time-saving direct transfer protocol

The rapid and correct identification of pathogens is of paramount importance for the treatment of patients with invasive infections. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can speed up the identification of bacteria and fungi and has quickly been embraced by medical microbiology laboratories worldwide. Different MALDI-TOF systems have been compared in studies focussing on identification rates of different pathogens. Another aspect that has not been systematically assessed is the performance in daily routine and handling, which is important especially for microbiology routine laboratories. We compared two widespread commercial systems, Microflex LT Biotyper (Bruker) and VitekMS (bioMérieux), for the identification of 210 relevant clinical yeasts under routine conditions, using a time-saving direct transfer protocol. We assessed the need for an additional extraction step, the threshold for species identification and the duration of measurements with the two systems. The tested yeasts included 34 Candida albicans isolates, 144 non-albicans Candida spp. and 32 yeasts of different genera. The results of the two MS systems were compared with that of biochemical identification and, in case of discrepancies, DNA sequencing of the internal transcribed spacer or the large subunit of ribosomal DNA. Both systems correctly identified 96.2% of isolates [202/210, non-significant (n.s.)]. Misidentifications were observed for VitekMS only (n=5, no major errors, n.s.). VitekMS was the slower system (19.8 vs. 8.0min for 10 samples, p=0.002) but had the advantage of a more effective direct transfer protocol with less need for an additional extraction step.

Direct Bacterial Identification in Positive Blood Cultures by Use of Two Commercial Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and fungi was recently introduced in microbiology laboratories. This technology could greatly improve the clinical management of patients and guidance for chemotherapy. In this study, we used a commercial MALDI Sepsityper extraction method to evaluate the performance of two commercial MALDI-TOF MS systems, the Vitek MS IVD (bioMérieux) and the Microflex LT Biotyper (Bruker Daltonics) for direct bacterial identification in positive blood cultures. In 181 monomicrobial cultures, both systems generated genus to species level identifications for >90% of the specimens (Biotyper, 177/181 [97.8%]; Vitek MS IVD, 167/181 [92.3%]). Overall, the Biotyper system generated significantly more accurate identifications than the Vitek MS IVD system (P = 0.016; 177 versus 167 out of 181 specimens). The Biotyper system identified the minority species among polymicrobial blood cultures. We also compared the performance of an in-house extraction method with that of the Sepsityper on both MALDI-TOF MS systems. The in-house method generated more correct identifications at the genus level than the Sepsityper (96.7% versus 93.5%) on the Biotyper system, whereas the two methods exhibited the same performance level (88.0% versus 88.0%) on the Vitek MS IVD system. Our study confirmed the practical advantages of MALDI-TOF MS, and our in-house extraction method reduced the reagent cost to $1 per specimen, with a shorter turnaround time of 3 h, which is highly cost-effective for a diagnostic microbiology service.

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.

Identification of Lactobacilli from Deep Carious Lesions by Means of Species-Specific PCR and MALDI-TOF Mass Spectrometry

The aim of the present study was to compare MALDI-TOF results for the identification of 87 lactobacilli, isolated from soft or hard carious dentin from 70 first molars of 7- to 8-year-old children with those obtained by species-specific PCR. The 87 isolates were analyzed by MALDI-TOF MS (Microflex LT, MALDI Biotyper 3.0, Bruker Daltonik, Bremen, Germany), using a reference data base of 4110 strains including > 90 lactobacillus species. For the identification with species-specific PCR, oligonucleotide primers (16S rRNA) specific for L. casei, L. paracasei, L. rhamnosus, L. gasseri, L. plantarum, and L. acidophilus were used; type strains served as controls. The PCR-products were separated electrophoretically on a 1.5% agarose gel and identified by their position on the gel. For 93% of the strains both methods produced concordant results: 40 strains were identified as L. rhamnosus, 16 as L. paracasei subsp. paracasei, 15 as L. paracasei subsp. tolerans, 4 as L. paracasei, 3 as L. gasseri, 2 as L. plantarum, and 1 as L. casei. In 4.5% of the cases the results were discordant. Of the 3 strains, not identified by species-specific PCR, 1 strain was identified by MALDI-TOF MS as L. spec. and 1 as L. parabuchneri. One strain could not be identified by either method. Both methods are highly sensitive. Limitations can be the precision of the primers (PCR) or the scarcity of strains from a certain habitat in the data base. Additional information is necessary for the strains without or with discordant identification.

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.

Evaluation of a Simple Protein Extraction Method for Species Identification of Clinically Relevant Staphylococci by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

In clinical microbiology, bacterial identification is labor-intensive and time-consuming. A solution for this problem is the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In this study, we evaluated a modified protein extraction method of identification performed on target plates (on-plate extraction method) with MALDI-TOF (Bruker Microflex LT with Biotyper version 3.0) and compared it to 2 previously described methods: the direct colony method and a standard protein extraction method (standard extraction method). We evaluated the species of 273 clinical strains and 14 reference strains of staphylococci. All isolates were characterized using the superoxide dismutase A sequence as a reference. For the species identification, the on-plate, standard extraction, and direct colony methods identified 257 isolates (89.5%), 232 isolates (80.8%), and 173 isolates (60.2%), respectively, with statistically significant differences among the three methods (P < 0.05). In conclusion, the on-plate extraction method is at least as good as standard extraction in identification rate and has the advantage of a shorter processing time.

Carbapenemase-ProducingKlebsiella pneumoniaein Hospital, Singapore, 2011

Carbapenemase-Producing<i>Klebsiella pneumoniae</i>in Hospital, Singapore, 2011

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis of Gram-Positive, Catalase-Negative Cocci Not Belonging to the Streptococcus or Enterococcus Genus and Benefits of Database Extension

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry with a Bruker Daltonics microflex LT system was applied to 90 well-characterized catalase-negative, Gram-positive cocci not belonging to the streptococci or enterococci. Biotyper version 2.0.43.1 software was used singly or in combination with a database extension generated in this study with 51 collection strains from 16 genera. Most strains were identified by using both databases individually, and some were identified only by applying the combined database. Thus, the methodology is very useful and the generated database extension was helpful.

Identification of clinical isolates of anaerobic bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for the rapid identification of anaerobic bacteria that had been isolated from clinical specimens and previously identified by 16s rRNA sequencing. The Bruker Microflex MALDI-TOF instrument with the Biotyper Software was used. We tested 152 isolates of anaerobic bacteria from 24 different genera and 75 different species. A total of 125 isolates (82%) had Biotyper software scores greater than 2.0 and the correct identification to genus and species was made by MALDI-TOF for 120 (79%) of isolates. Of the 12 isolates with a score between 1.8 and 2.0, 2 (17%) organisms were incorrectly identified by MALDI-TOF. Only 15 (10%) isolates had a score less than 1.8 and MALDI-TOF gave the wrong genus and species for four isolates, the correct genus for two isolates, and the correct genus and species for nine isolates. Therefore, we found the Bruker MALDI-TOF MicroFlex LT with an expanded database and the use of bacteria extracts rather than whole organisms correctly identified 130 of 152 (86%) isolates to genus and species when the cut-off for an acceptable identification was a spectrum score ≥1.8.

Comparison of the Microflex LT and Vitek MS Systems for Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

This study compared the performance of three matrix-assisted laser desorption ionization-time of flight mass spectrometry systems: Microflex LT (Bruker Daltonics, Bremen, Germany), Vitek MS RUO (Axima Assurance-Saramis database; bioMérieux, Marcy l'Etoile, France), and Vitek MS IVD (bioMérieux). A total of 1,129 isolates, including 1,003 routine isolates, 73 anaerobes, and 53 bacterial enteropathogens, were tested on the Microflex LT and Axima Assurance devices. The spectra were analyzed using three databases: Biotyper (Bruker Daltonics), Saramis, and Vitek MS (bioMérieux). Among the routine isolates requiring identification to the species level (n = 986), 92.7% and 93.2% were correctly identified by the Biotyper and Vitek MS databases, respectively. The Vitek MS database is more specific for the identification of Streptococcus viridans. For the anaerobes, the Biotyper database often identified Fusobacterium isolates to only the genus level, which is of low clinical significance, whereas 20% of the Bacteroides species were not identified or were misidentified by the Vitek MS database. For the enteropathogens, the poor discrimination between Escherichia coli and Shigella explains the high proportion of unidentified organisms. In contrast to the Biotyper database, the Vitek MS database properly discriminated all of the Salmonella entrica serovar Typhi isolates (n = 5). The performance of the Saramis database was globally poorer. In conclusion, for routine procedures, the Microflex LT and Vitek-MS systems are equally good choices in terms of analytical efficiency. Other factors, including price, work flow, and lab activity, will affect the choice of a system.

Differentiation of division I (cfiA-negative) and division II (cfiA-positive) Bacteroides fragilis strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used in clinical microbiological laboratories to identify bacteria and fungi at a species level and to subtype them. The cfiA gene encoding the unique carbapenemases found in Bacteroides is restricted to division II Bacteroides fragilis strains. The aim of this study was to evaluate whether MALDI-TOF MS is suitable for differentiating B. fragilis strains which harbour the cfiA gene from those that do not. A well-defined collection of 40 B. fragilis isolates with known imipenem MICs (0.062->32 mg l(-1)) were selected for this study. Twelve B. fragilis strains with known cfiA status, including NCTC 9343 (division I) and TAL3636 (division II), were measured by means of microflex LT MALDI-TOF MS and well-defined differences in mass spectra between the cfiA-positive and cfiA-negative strains were found in the interval 4000-5500 Da. A further 28 strains were selected for the blind measurements: 9 cfiA-positive clinical isolates with different imipenem MICs ranging between 0.06 and >32 mg l(-1) (different expressions of the metallo-β-lactamase gene) were clearly separated from the 19 cfiA-negative isolates. The presence or absence of the selected peaks in all tested strains clearly differentiated the strains belonging to B. fragilis division I (cfiA-negative) or division II (cfiA-positive). These results suggest a realistic method for differentiating division II B. fragilis strains (harbouring the cfiA gene) and to determine them at a species level at the same time. Although not all cfiA-positive B. fragilis strains are resistant to carbapenems, they all have the possibility of becoming resistant to this group of antibiotics by acquisition of an appropriate IS element for full expression of the cfiA gene, leading to possible treatment failure.

Pigs as Source for ToxigenicCorynebacterium ulcerans

To the Editor: Toxigenic Corynebacterium ulcerans may cause a zoonotic infection similar to diphtheria caused by C. diphtheriae. Previously, dairy cattle were considered to be the main reservoir for C. ulcerans (1), but recent publications suggest pet dogs and pet cats as carriers (cats often show bilateral nasal discharge) (2). We report a case of severe C. ulcerans diphtheria-like disease in a person who had had contact with pigs. In December 2007, a previously healthy 56-year-old female farmer was admitted to the Ear, Nose and Throat Department of the University Hospital Erlangen with a 1-week history of sore throat and progressive dysphagia. She did not report fever and had not received prior treatment with antimicrobial drugs. She had thick, whitish pseudomembranes on her uvula, pharynx, and both tonsils. Endoscopic examination of her larynx and hypopharynx showed that both vocal cords were mobile and the mucosa was erythematous. Enlarged cervical lymph nodes were palpable on both sides of her neck. She had no signs of cranial nerve palsies. Her temperature was 36.5°C. Because of the extensive oropharyngeal pseudomembranes, diphtheria was suspected and diphtheria antitoxin (30,000 IU) was administered intramuscularly. The patient was isolated and received intravenous penicillin (5 million units 4×/day). A pharyngeal swab obtained from below the whitish pseudomembranes grew toxigenic C. ulcerans. Species identification was achieved by biochemical differentiation (API Coryne code 0111326), rpoB sequencing (3), and MALDI-TOF analysis (Microflex LT and Biotyper 2.0 Software; Bruker Daltonics, Bremen, Germany). Toxigenicity of the strain, named KL126, was verified by using a C. diphtheriae tox–PCR (4–6), a C. ulcerans tox–specific PCR (4), and the Elek test as described previously (4,5). The tox sequence (GenBank accession no. {type:entrez-nucleotide,attrs:{text:FJ858272,term_id:238817560,term_text:FJ858272}}FJ858272) differs from 2 other published C. ulcerans tox sequences ({type:entrez-nucleotide,attrs:{text:AB304279.1,term_id:167216113,term_text:AB304279.1}}AB304279.1 and {type:entrez-nucleotide,attrs:{text:AY703827.1,term_id:56694206,term_text:AY703827.1}}AY703827.1) at only 3 bp. The patient recovered quickly, and the pseudomembranes vanished within 2 days. However, because an allergic rash had developed after her third day of treatment with penicillin, antimicrobial drug treatment was switched to intravenous erythromycin (500 mg 4×/d). When 1 day later the standardized antibiogram showed resistance to erythromycin, the patient received intravenous ceftriaxone (2 g 1×/d) for 12 days. Seven days after initiation of antimicrobial drug therapy, pharyngeal swabs were taken on 3 consecutive days. Because C. ulcerans no longer grew on culture, the patient was discharged from the hospital. However, 2 days later she was readmitted to hospital for severe polyneuropathy with neuralgia and weakness of both arms, acute difficulty swallowing, and hoarseness. Signs of cardiomyopathy, including sinus bradycardia and grade I atrioventricular block, were present. The patient recovered after symptomatic treatment and returned home after 2 weeks. According to her records, the patient had received a basic vaccination against diphtheria in 1960 and a booster in 1998. The literature describes the classic animal sources for toxigenic C. ulcerans as dairy cattle with mastitis (1). Since 2005, toxigenic C. ulcerans carriage in companion animals, e.g., pet cats and dogs, has been reported (2). Two cases of transmission of a toxigenic C. ulcerans strain from pet dogs to their immunocompromised female owners have been documented in France (7,8). In 2008, toxigenic C. ulcerans in 2 dead killer whales from a Japanese zoo was reported (9). To determine the source of our patient’s illness, an outbreak investigation involving her family and their farm animals was conducted. Their medium-sized pig-breeding farm was located in a remote rural village surrounded by woods; they raised ≈500 pigs in a nonindustrialized manner, and no piglets were purchased from outside the farm. Pharyngeal swabs of 3 family members, 19 pigs, and the farm dog were analyzed for C. ulcerans. Although all family members and the dog were negative for C. ulcerans, 1 of the 19 asymptomatic pigs harbored a toxigenic strain of C. ulcerans. Sequencing of rpoB and tox showed 100% homology between the human and the pig strains. Ribotyping (10) confirmed this result, suggesting the identity of both strains; the obtained ribotype is similar to the reported U1 ribotype profile found in humans and cats (2). We report proven transmission of a toxigenic C. ulcerans strain between a livestock animal and a human, as well as harboring of toxigenic C. ulcerans in pigs. Introduction of C. ulcerans from wild animals seems unlikely because the barn doors were reportedly closed at all times. Because handling of C. ulcerans–infected pigs may lead to diphtheria-like illnesses, studies of toxigenic C. ulcerans carriage among pigs are needed. Similar to our case, diphtheria-like disease caused by an erythromycin- and clindamycin-resistant toxigenic C. ulcerans strain in a US patient has been recently (in 2008) reported (1). Because current recommendations based on C. diphtheriae–caused disease consider erythromycin as the second-line option for treatment or postexposure prophylaxis, these findings highlight the importance of antimicrobial-drug susceptibility testing of toxigenic C. ulcerans strains.

Direct Bacterial Profiling by Matrix-Assisted Laser Desorption−Ionization Time-of-Flight Mass Spectrometry for Identification of Pathogenic Neisseria

The present study investigates the suitability of direct bacterial profiling as a tool for the identification and subtyping of pathogenic Neisseria. The genus Neisseria includes two human pathogens, Neisseria meningitidis and Neisseria gonorrhoeae, as well as several nonpathogenic Neisseria species. Here, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling protocol was optimized using a laboratory strain of E. coli DH5alpha to guarantee high quality and reproducible results. Subsequently, mass spectra for both laboratory and clinical strains of N. gonorrhoeae, N. meningitidis, and several nonpathogenic Neisseria species were collected. Significant interspecies differences but little intraspecies diversity were revealed by means of a visual inspection and bioinformatics examination using the MALDI BioTyper software. Cluster analysis successfully separated mass spectra collected from three groups that corresponded to N. gonorrhoeae, N. meningitidis, and nonpathogenic Neisseria isolates. Requiring only one bacterial colony for testing and using a fast and easy measuring protocol, this approach represents a powerful tool for the rapid identification of pathogenic Neisseria and can be adopted for other microorganisms.