Method For Routine Identification Research Articles

Multiple skin abscesses due to Nocardia neocaledoniensis: a case report and literature review

N. neocaledoniensis is a very rare infectious pathogen that causes human disease, particularly in immunocompromised individuals. In this case report, we describe the successful diagnosis of N. neocaledoniensis in a patient confirmed by mNGS and the treatment of multiple skin abscesses due to N. neocaledoniensis infection. mNGS is an important diagnostic method complementary to routine bacterial culture and identification methods, especially for rare, novel, co-infected pathogens, and pathogens that are difficult to culture. This report may provide a reference for the clinical treatment and diagnosis of N. neocaledoniensis infection in humans.

Evaluation of Geh gene expression in Staphylococcus aureus isolated from acne patients

Acne vulgaris is a chronic skin disease that affects the hair follicle and sebaceous glands. Staphylococcus aureus colonizes human skin and causes infections ranging from local skin infections to systemic infections that may threaten human life due to its ability to produce many virulence factors and antibiotic resistance. The objective of this study is to measure the genetic expression of Geh gene responsible for producing the lipase enzyme from S. aureus isolated from acne patients. This study was conducted by collecting 130 swabs from acne patients at Tikrit Teaching Hospital. Using routine bacterial isolation and identification methods, 50 samples (38.4%) of the total samples were identified as S. aureus through its ability to grow in mannitol salt agar medium, the fermentation of mannitol sugar, and the change of the medium color to yellow. The Geh gene expression of ten S. aureus isolates from acne patients was examined using RT-PCR. The results showed that Geh gene was expressed in all isolates, with an expression ranging from 1.5 to 7.2, and that the mean folding of the Geh gene for ten samples was 4.2. This indicates the presence of high gene expression of the Geh gene in S. aureus isolates. Therefore, it is concluded that Lipase enzyme plays an important role in the pathogenesis of S. aureus in acne patients.

Evaluation of Newly Modified Potassium Nitrate Containing Media in Detection and Antibiogram of Mycobacterium tuberculosis

Background: Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis) and the classic Hansen's strain of leprosy (Mycobacterium leprae) [1]. Tuberculosis is the most common life-threatening opportunistic infection (OI) and AIDS defining illness in developing countries [2]. The routine identification methods of tuberculosis includes, Tuberculin skin test, Chest X-ray, Growth and physical characteristics of the bacteria, automated based system such as BACTEC 460 TB method, Mycobacterium growth indicator tube (MGIT) method, MB/BacT system, as well as molecular based techniques as in Amplification of nucleic acid for detection of Mycobacterium tuberculosis [3-6]. Objectives: To evaluate the validity of newly modified potassium nitrate containing medium (NMK) for rapid detection and antibiogram of M tuberculosis. Material and method: This was a health facility based observational diagnostics retrospective study. 125 sputum specimens were investigated to evaluate the validity of newly modified potassium nitrate containing medium for rapid detection and antibiogram of M tuberculosis. Results: 83/125 (66.4%) showed growth after 5-9 days with a mean of 6.58± 0.977 days NMK - containing medium. Conclusions: The NMK - containing medium showed promising results, it showed a positive growth sings and susceptibility results within 5-9 days of inoculation showing much superior than the golden standard LJ medium.

Naïve Bayes Classifiers and accompanying dataset for Pseudomonas syringae isolate characterization

The Pseudomonas syringae species complex (PSSC) is a diverse group of plant pathogens with a collective host range encompassing almost every food crop grown today. As a threat to global food security, rapid detection and characterization of epidemic and emerging pathogenic lineages is essential. However, phylogenetic identification is often complicated by an unclarified and ever-changing taxonomy, making practical use of available databases and the proper training of classifiers difficult. As such, while amplicon sequencing is a common method for routine identification of PSSC isolates, there is no efficient method for accurate classification based on this data. Here we present a suite of five Naïve bayes classifiers for PCR primer sets widely used for PSSC identification, trained on in-silico amplicon data from 2,161 published PSSC genomes using the life identification number (LIN) hierarchical clustering algorithm in place of traditional Linnaean taxonomy. Additionally, we include a dataset for translating classification results back into traditional taxonomic nomenclature (i.e. species, phylogroup, pathovar), and for predicting virulence factor repertoires.

A-279 Comparison of Direct Identification and Short Incubation Time Methods of MALDI-TOF MS from Positive Blood Vials

Abstract Background MALDI-TOF analysis after a short incubation time of positive blood culture is currently one of the most widely used methods for rapidly identifying microorganisms. This method can identify most pathogenic bacteria within 4 to 6 h after blood culture. This experiment aims to use direct identification for MALDI-TOF analysis, which can shorten the identification time of bacteria and analyze the advantages and disadvantages of the two methods. Method We collected from January 2022 to December 2022, a total of 295 bottles of a single infection. After pretreatment methods such as removing cells and high-speed centrifugation to collect bacteria, carry out direct identification by MALDI-TOF then culture for 5 h for short incubation time method. The culture medium was continued incubated for 18–20 h, to confirm the final identification results of the strains and compare the identification accuracy of direct identification and short incubation time methods. Definition: confidence score cut-off values > = 1.7 of direct identification method. Result The identification rate of score > = 1.7 were 67%(199/295). Gram-negative bacilli were identified in 89%, followed by anaerobes in 60% and Gram-positive species in 56%. The yeast could not be identified either by the short incubation time method or the direct identification method. The short incubation time method identified 90% of Gram-negative bacilli, followed by 79% of Gram-positive bacteria and 7% of anaerobic bacteria. The rapid identification of anaerobic bacteria was significantly better than the identification results of the short incubation time method (60%:7%) (P value = 0.01). In addition, the results can be obtained within one hour by direct identification, which can save at least 5-fold times required for the short incubation time method of about 5 h, although the effect is slightly worse than short incubation time method (67 %:76%). Especially for Gram-negative bacilli, there is no statistically significant difference between direct identification and short incubation time methods; it proves that direct identification method can indeed replace short incubation time method. Conclusion Patients with septic shock who were treated with antibiotics within one hour had an 80% chance of survival; however, for every hour of delay within six hours of onset, the survival rate decreased by 7.6%. Compared with the short incubation time method, the direct identification method can report the correct strain to clinician 5-fold times earlier. This rapid, simplified extraction direct identification method is cost-effective, time-saving, and easy to use. The SSC sepsis guidelines in 2016 pointed out that extending the course of antibiotic treatment not only has no additional benefits but also is prone to drug resistance, resulting in side effects of antibiotics. Therefore, the direct identification method can significantly improve the optimal utilization of antibiotics for effective targeted treatment and reduce mortality, especially for the identification of anaerobic bacteria, which can provide faster identification results. Therefore, it is recommended that clinical laboratories include a direct identification method for routine identification of blood culture, and if they cannot be identified, they can be supplemented by a short incubation time method, to shorten the time of blood culture identification and effectively improve the treatment of bloodstream infections.

Clinical Application of Small Extracellular Vesicles in Gynecologic Malignancy Treatments.

Small extracellular vesicles (sEVs) are the key mediators of intercellular communication. They have the potential for clinical use as diagnostic or therapeutic biomarkers and have been explored as vectors for drug delivery. Identification of reliable and noninvasive biomarkers, such as sEVs, is important for early diagnosis and precise treatment of gynecologic diseases to improve patient prognosis. Previous reviews have summarized routine sEVs isolation and identification methods; however, novel and unconventional methods have not been comprehensively described. This review summarizes a convenient method of isolating sEVs from body fluids and liquid biopsy-related sEV markers for early, minimally invasive diagnosis of gynecologic diseases. In addition, the characteristics of sEVs as drug carriers and in precision treatment and drug resistance are introduced, providing a strong foundation for identifying novel and potential therapeutic targets for sEV therapy. We propose potential directions for further research on the applications of sEVs in the diagnosis and treatment of gynecologic diseases.

Stomatal Analysis of <i>Allium wallichii</i> Kunth Leaves: An Experimental Finding through Quantitative Microscopy

The use of microscopic techniques for the identification of medicinal plants is one of the best practices and an important quality control tool for standardization. Stomata are important organs for the study of phylogenetic relationships including plant origin, evolution, and classification. Allium wallichii (A. wallichii) Kunth, is one of the threatened medicinal herbs having important nutraceutical, medicinal and therapeutic value. The plant is one of the neglected species of Allium and has not been thoroughly investigated pharmacognostically. Therefore, current research work was designed to investigate the stomata types, stomata number and stomatal index through quantitative microscopy. The result presented in the current research will provide a routine identification method for A. wallichii with the allied species of Allium.

Evaluation of the MALDI-TOF MS method for routine identification of Candida species in a tertiary care hospital in Thailand

Evaluation of the MALDI-TOF MS method for routine identification of Candida species in a tertiary care hospital in Thailand

Classification of 27 Corynebacterium kroppenstedtii-Like Isolates Associated with Mastitis in China and Descriptions of C. parakroppenstedtii sp. nov. and C. pseudokroppenstedtii sp. nov.

ABSTRACTCorynebacterium, particularly Corynebacterium kroppenstedtii, has been increasingly recognized as an important pathogen causing mastitis. However, no clear taxonomic, microbiological, or clinical identification for C. kroppenstedtii-related Corynebacterium species is recognized. During the investigation of isolates cultured from female patients with mastitis, 27 lipophilic C. kroppenstedtii-like isolates were obtained from clinical breast specimens from 2017 to 2019 in Guangzhou, China. These isolates were identified by phenotypic characterization, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), partial sequencing of the 16S rRNA, rpoB, and fusA genes, and whole-genome sequencing methods. By phylogenetic analyses, two major clusters were identified that were closely related to C. kroppenstedtii DSM 44385T. Comparative genome analyses suggested that these isolates formed two distinct genospecies within the genus Corynebacterium. The digital DNA–DNA hybridization (dDDH) values for the two genospecies were 45.5 to 47.8% between them and 47.4 to 47.7% and 49.9% to C. kroppenstedtii DSM 44385T, respectively. Based on these results, it can be concluded that these isolates need to be recognized as two new species of the genus Corynebacterium, for which we proposed the names Corynebacterium parakroppenstedtii sp. nov. and Corynebacterium pseudokroppenstedtii sp. nov. The type strain for the novel species Corynebacterium parakroppenstedtii is MC-26T (NBRC 115146T; CCTCC AB 2020210T), and that for Corynebacterium pseudokroppenstedtii is MC-17XT (NBRC 115143T; CCTCC AB 2020199T).IMPORTANCE In this study, we characterized two novel species that were closely related to but hard to distinguish from C. kroppenstedtii by routine identification methods used in clinical laboratories. Since all 27 C. kroppenstedtii-like isolates were obtained from breast specimens of female patients with mastitis, they may be potential pathogens causing mastitis. We hope to perform further epidemiological investigation of these strains and explore their role in mastitis.

Candida auris Identification and Profiling by MALDI-ToF Mass Spectrometry.

MALDI-ToF MS has become the standard method for routine identification of most medically important yeasts in clinical and public health laboratories and has largely replaced phenotypic identification methods as a first-line identification tool. Fungal identification is based on extensive and well-curated mass spectra libraries usually provided by the manufacturer of the MALDI-ToF MS platform; however, many centers do create specialized or in-house database collections to aid analysis. Most MALDI-ToF MS systems offer simple and standardized workflows for the identification of clinically relevant yeasts to species level with a high throughput, high accuracy, and a low overall cost per test. Thismakes MALDI-ToF MS an ideal platformfor use in routine clinical, diagnostic, and research microbiology laboratories which may lack experience or expertise in the identification of pathogenic fungi.In this chapter we review three standard protocols for the proteomic-based identification of Candida auris isolated from cultures of clinical or environmental surveillance samples in diagnostic and research laboratories.

Application of individual response factors for accurate quantitation of alkylated PAH homologs in complex environmental samples using gas chromatography/triple quadrupole mass spectrometry (GC–MS/MS)

Alkylated polycyclic aromatic hydrocarbons (alkyl PAHs) are abundant in the environment. They can be more toxic and persistent than their non-alkylated parent PAH compounds, and thus alkyl PAHs can pose an increased threat to environmental quality. However, the US EPA has not listed alkyl PAHs as priority pollutants, which is primarily due to a lack of reliable and accurate analytical method for routine identification and quantification of alkyl PAHs in complex environmental samples. A multiple reaction monitoring (MRM) method in GC–MS/MS has been proposed for more accurate quantification of target alkyl PAHs in complex sample matrices. However, the lack of certified analytical standards for alkyl PAH isomers and the historic use of relative response factors (RFs) of parent PAH to quantify its alkylated PAH isomers can compromise the quantitative results obtained from MRM analyses. In this study, we used commercially available parent and alkyl PAH standards to develop and optimize both SIM and MRM analytical methods. The optimized methods were used to determine and compare RFs of alkyl PAHs to their respective parent PAHs in both SIM and MRM modes. The results showed that the conventional approach of quantifying alkyl PAHs by using the RFs of corresponding parent PAHs underestimates alkyl PAHs by 1.2–6.4 (2.2 ± 1.2) and 1.2–6.7 (3.4 ± 2.0) times, respectively, for SIM and MRM analyses. The results further demonstrated that the use of RFs of parent PAHs to quantify their alkylated homologs affects MRM-based analysis more than SIM-based analysis, underestimating the alkyl PAHs in MRM mode to as low as only 15% of the actual concentrations obtained by using individual RFs for each alkylated PAH homologs. The results showed that a GC–MS/MS in MRM mode, with baseline separation and higher selectivity, is a powerful analytical tool for the analysis of alkyl PAHs in complex matrices, however, the use of at least one of the individual RFs for target PAH analyte clusters is critical for accurate quantitative MRM analysis. An accurate quantification of alkyl PAHs is important for determining their transport, fate, and environmental health impacts for next generation of oil spill studies as well as for updating existing EPA priority pollutant list by including alkyl PAHs in the list.

First Record of Dactylogyrus Vastator (Nybelin 1924) (Monogenea: Dactylogyridae) in Common Carp (Cyprinus Carpio Linnaeus, 1758) from Aquaculture Facilities In Macedonia

The representatives of the class Monogenea are of great importance in fish pathology. Dactylogyrus vastator (Nybelin 1924) (Monogenea: Dactylogyridae) is a common pathogenic monogenean parasite in common carp (Cyprinus carpio Linnaeus, 1758) from freshwater lakes and cyprinid ponds, worldwide. In our study, a total of 133 specimens of common carp from the cage culture system on Mladost Reservoir (N. Macedonia) were subjected to a parasitological investigation, by seasons. Only fresh fish were subjected to routine identification, dissection, and observation methods. Parasite identification was performed morphologically, based on the character of significant organs, using referent keys for determination. Dactylogyrus vastator was found in winter, on gills of 27 specimens of common carp, with a prevalence of 2.818%, and mean intensity of 6.850. During our research, the presence of Dactylogyrus vastator was found in an aquaculture facility with a very high fish stock density. This parasite species would lead to fish mortality and it depends primarily on the mean intensity, the fish condition, and size, as well as the water temperature and oxygen content. The records of Dactylogyrus vastator in common carp in the present study are considered as the first records in N. Macedonia. The common carp is regarded as a new host for these parasite species in Macedonian aquaculture.

First preliminary study on identification of bacterial fish pathogens with Raman spectroscopy

Accurate and rapid determination of bacterial disease agents of fish is an important step for sustainable and efficient aquaculture production. In general, biochemical and molecular methods are used for pathogen detection but they are usually time-consuming and required qualified personnel. Recently spectroscopic methods are preferred in clinical and food microbiology and declared as a promising alternative method for pathogens diagnosis with many advantages. In this study, the significant spectra of three important bacterial fish pathogens (Lactococcus garvieae, Vibrio anguillarum and Yersinia ruckeri) were determined by Raman spectroscopy. The first data of the pathogens were obtained and the distinctive differences in polysaccharides, nucleic acids, fatty acids and amino acids were identified. This preliminary study aimed to be pioneer for further studies in aquaculture and veterinary microbiology toward developing an alternative method for routine identification.

Direct Detection of Streptococcus suis from Cerebrospinal Fluid, Positive Hemoculture, and Simultaneous Differentiation of Serotypes 1, 1/2, 2, and 14 within Single Reaction.

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future.

An evaluation of the species and subspecies of the genus Salmonella with whole genome sequence data: Proposal of type strains and epithets for novel S. enterica subspecies VII, VIII, IX, X and XI

Species and subspecies within the Salmonella genus have been defined for public health purposes by biochemical properties; however, reference laboratories have increasingly adopted sequence-based, and especially whole genome sequence (WGS), methods for surveillance and routine identification. This leads to potential disparities in subspecies definitions, routine typing, and the ability to detect novel subspecies. A large-scale analysis of WGS data from the routine sequencing of clinical isolates was employed to define and characterise Salmonella subspecies population structure, demonstrating that the Salmonella species and subspecies were genetically distinct, including those previously identified through phylogenetic approaches, namely: S. enterica subspecies londinensis (VII), subspecies brasiliensis (VIII), subspecies hibernicus (IX) and subspecies essexiensis (X). The analysis also identified an additional novel subspecies, reptilium (XI). Further, these analyses indicated that S. enterica subspecies arizonae (IIIa) isolates were divergent from the other S. enterica subspecies, which clustered together and, on the basis of ANI analysis, subspecies IIIa was sufficiently distinct to be classified as a separate species, S. arizonae. Multiple phylogenetic and statistical approaches generated congruent results, suggesting that the proposed species and subspecies structure was sufficiently biologically robust for routine application. Biochemical analyses demonstrated that not all subspecies were distinguishable by these means and that biochemical approaches did not capture the genomic diversity of the genus. We recommend the adoption of standardised genomic definitions of species and subspecies and a genome sequence-based approach to routine typing for the identification and definition of novel subspecies.

MALDI-TOF MS Overcomes Misidentification of the Uncommon Human Pathogen Candida famata by Routine Phenotypic Identification Methods.

Candida famata has been associated with the identifiable Candida infections that takes place in human and the identification error of this species possibly will result in misinterpretation of antifungal susceptibility and improper diagnosis; which will have a major effect on the prognosis and therapy of patients. Our objective is to correctly identify Candida spp. collected from patients at the intensive care units, New Cairo University teaching hospital in Cairo-Egypt using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Hundred clinically isolated yeast strains were identified using API 20C AUX obtained from patients receiving care at intensive care units. ATB FUNGUS 3strips were used to detect the minimum inhibitory concentration. Thirty-three non duplicate strains identified as C. famata were subjected to re-identification by MALDI-TOF MS. Our results revealed that isolates were initially identified as C. famata 33%, C. tropicalis 15%, C. albicans12% and C. parapsillosis 10% using the phenotypic techniques. MALDI-TOF MS analyses results showed that the 33 C. famata isolates are C. tropicalis (n = 29), Trichosporon asahii (n = 2), C. parapsilosis (n = 1), and Aeromonas sobria (n = 1). Antifungal resistance was low in the Candida species, except for reduced susceptibility to itraconazole among C. krusei strains. This report shows that misidentification of C. famata is frequent when using conventional phenotypic methods of identification which result in challenges in treating fungal infections. MALDI-TOF MS is an accurate convenient substitute to classical approaches for fungal identification. In general, antifungal multidrug resistance is uncommon in our studied Candida species and yeast isolates.

First Case of Brucellosis Caused by an Amphibian-type Brucella.

We report the first case of brucellosis caused by an isolate whose genome is identical that of a frog isolate from Texas, demonstrating the zoonotic potential of amphibian-type Brucella. Importantly, with such atypical Brucella, correct diagnosis cannot be performed using routine serological tests or identification methods.

Characteristics of phenotype and molecular type of Vibrio cholerae O1 in Beijing, 2018

ObjectiveTo understand the characteristics of the phenotype and molecular type of Vibrio cholerae O1 in Beijing in 2018. MethodsThe strains of V. cholerae were isolated from cholera cases and external environments. Serotyping was performed for the strains with routine identification methods, and V. cholerae toxin gene were detected by using real-time PCR. The sensitivities of the strains to 21 kinds of antibiotics was conducted with micro-broth dilution method. Pulsed field gel electrophoresis (PFGE) with Xba Ⅰ and Not Ⅰ restriction enzymes was performed for the molecular typing of V. cholerae O1 strains. ResultsIn 29 V. cholerae O1 strains isolated, the proportions of non-toxin producing Ogawa strains, non-toxin producing Inaba strains and toxin producing Ogawa strains were 62.07% (18/29), 24.14% (7/29) and 13.79% (4/29) respectively. The resistance rate of the strains to cefazolin was highest (82.76%), all the strains were sensitivity to meropenem (100.00%). Twenty eight strains were drug resistant with 9 drug resistance types, the highest was resistant to 13 antibiotics; there were 13 resistance categories to 11 classes of antibiotics, the highest was resistant to 7 classes of antibiotics. Results from PFGE indicated that the 29 V. cholerae strains were classified into 16 molecular types. ConclusionV. cholerae O1 was mainly non-toxic in Beijing, and multi drug resistance of V. cholerae O1was severe. The results of PFGE molecular typing suggested that the attention should be paid to food safety, especially to strengthen the monitoring of aquatic products.

Performance of MALDI-TOF Mass Spectrometry for the Identification of the HACEK Group and Other Fastidious Gram-Negative Rods

Objective: The aim of this study was to determine the capacity of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to identify 155 HACEK clinical isolates and other fastidious or infrequently isolated Gram-negative rods (e.g., Actinobacillus, Capnocytophaga, Pasteurella, Neisseria, Moraxella, Dysgonomonas, among others). Methods: All the isolates were identified by standard biochemical tests and MALDI-TOF MS. Two different extraction methods (direct transfer formic acid method on spot and ethanol formic acid extraction method) and different cut-offs for genus/specie level identification were used. MALDI-TOF MS identification was considered correct when the result obtained from the MS database agreed with the phenotypic identification result. When both the methods gave discordant results, the 16S rDNA gene sequencing was considered as the gold standard identification method. Results: Employing the score cut-offs suggested by the manufacturer, 93.55% and 69.03% isolates were correctly identified at the genus and species level, respectively. On the contrary , employing lower cut-off scores for identification, 98.06% and 92.09% isolates were properly identified at the genus and species level respectively and no significant differences between the results obtained with two extraction methods were observed . Conclusion: The accurate identification of 14 genera showed the reliability of MALDI-TOF MS as an optional methodology to the routine identification methods currently used in laboratories.

Comparison of VITEK 2, MALDI-TOF MS, 16S rRNA gene sequencing, and whole-genome sequencing for identification of Roseomonas mucosa

The bacterial species Roseomonas mucosa is pathogenic in humans, and although it is rarely detected during routine diagnostics, it is becoming increasingly important clinically. For a long time, R. mucosa was regarded as a classic environmental bacterium. Recent studies, however, revealed that it is part of the physiological human skin flora and mainly affects immunocompromised patients. Furthermore, the use of catheter systems may increase the risk of contracting R. mucosa infections. The bacterium has been linked to severe infections, such as bacteraemia, osteomyelitis and cellulitis. Therefore, it is important to discern the best method of identifying R. mucosa in routine laboratory testing. To facilitate this testing, we compared three suitable methods for routine bacterial identification in the laboratory: VITEK 2, MALDI-TOF MS and 16S rRNA gene sequencing. Additionally, we conducted whole-genome sequencing (WGS) and calculated the average nucleotide identity (ANI). ANI is seen as the gold standard of strain identification; therefore, we decided to use it as a reference method. Both MALDI-TOF MS and 16S rRNA gene sequencing confidently identified the species. However, when using the VITEK 2 technique, isolates were misidentified as Roseomonas gilardii, Rhizobium radiobacter, or Sphingomonas paucimobilis. When conducting WGS and determining the ANI, it became obvious that one isolate belonged to the species R. gilardii rather than R. mucosa. Therefore (although not yet applicable in routine diagnostics), we suggest that WGS is presently the most appropriate technique to reliably identify Roseomonas mucosa. However, after expanding the Biotyper database, MALDI-TOF MS could also be an applicable method.