Molecular characterization and whole-genome sequencing of Corynebacterium diphtheriae causing skin lesion.

We present a case of skin lesion caused by nontoxigenic Corynebacterium diphtheriae. Genomic taxonomy analyses corroborated the preliminary identification provided by mass spectrometry. The strain showed a susceptible phenotype with increased exposure to penicillin, the first drug of choice for the treatment. An empty type 1 class integron carrying only the sul1 gene, which encodes sulfonamide resistance, was found flanked by transposases. Virulence factors involved in adherence and iron uptake, as well as the CRISPR-Cas system, were predicted. MLST analysis revealed the ST-681, previously reported in French Guiana, a European territory.

Treat Me Well or Will Resist: Uptake of Mobile Genetic Elements Determine the Resistome of Corynebacterium striatum.

Corynebacterium striatum, a bacterium that is part of the normal skin microbiota, is also an opportunistic pathogen. In recent years, reports of infections and in-hospital and nosocomial outbreaks caused by antimicrobial multidrug-resistant C. striatum strains have been increasing worldwide. However, there are no studies about the genomic determinants related to antimicrobial resistance in C. striatum. This review updates global information related to antimicrobial resistance found in C. striatum and highlights the essential genomic aspects in its persistence and dissemination. The resistome of C. striatum comprises chromosomal and acquired elements. Resistance to fluoroquinolones and daptomycin are due to mutations in chromosomal genes. Conversely, resistance to macrolides, tetracyclines, phenicols, beta-lactams, and aminoglycosides are associated with mobile genomic elements such as plasmids and transposons. The presence and diversity of insertion sequences suggest an essential role in the expression of antimicrobial resistance genes (ARGs) in genomic rearrangements and their potential to transfer these elements to other pathogens. The present study underlines that the resistome of C. striatum is dynamic; it is in evident expansion and could be acting as a reservoir for ARGs.

Novel mutations in the QRDR region gyrA gene in multidrug-resistance Corynebacterium spp. isolates from intravenous sites.

The resistance to fluoroquinolones in corynebacteria is due to mutations occurring in the quinolone-resistance-determining region (QRDR) of the gyrA gene encoding the enzyme gyrase A subunit. In recent years we can observe an increasing number of infections caused by multidrug-resistant Corynebacterium striatum, Corynebacterium jeikeium and Corynebacterium urealyticum, including wide range of disorders, such as invasive infections. In this study 14 Corynebacterium spp. isolated from intravenous sites were sequenced and new combinations of mutations in the QRDR of the gyrA gene were found in C. jeikeium and C. urealyticum. Nowadays, no study comparing mutations in this region and the susceptibility to fluoroquinolones in C. jeikeium and C. urealyticum has been described. All the isolates that showed double mutation (position 87 and 91) in the QRDR gyrA gene had high MIC to the fluoroquinolones tested.

Bloodstream and catheter-related infections due to different clones of multidrug-resistant and biofilm producer Corynebacterium striatum.

BACKGROUND: Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen associated with immunocompromised and chronically ill patients, as well as nosocomial outbreaks. In this study, we characterized 23 MDR C. striatum isolated of bloodstream and catheter-related infections from a hospital of Rio de Janeiro. METHODS: C. striatum isolates were identified by 16S rRNA and rpoB genes sequencing. The dissemination of these isolates was accomplished by pulsed-field gel electrophoresis (PFGE). All isolates were submitted to antimicrobial susceptibility testing by disk diffusion and by minimum inhibitory concentration using E-test strips methods. Antimicrobial resistance genes were detected by polymerase chain reaction. Quantitative tests were performed on four different abiotic surfaces and the ability to produce biofilm on the surface of polyurethane and silicone catheter was also demonstrated by scanning electron microscopy. RESULTS: Eleven PFGE profiles were found. The PFGE profile I was the most frequently observed among isolates. Five different MDR profiles were found and all PFGE profile I isolates presented susceptibility only to tetracycline, vancomycin, linezolid and daptomycin. Only the multidrug-susceptible isolate did not show mutations in the quinolone-resistance determinant region (QRDR) of the gyrA gene and was negative in the search of genes encoding antibiotic resistance. The other 22 isolates were positive to resistance genes to aminoglycoside, macrolides/lincosamides and chloramphenicol and showed mutations in the QRDR of the gyrA gene. Scanning electron microscopy illustrated the ability of MDR blood isolate partaker of the epidemic clone (PFGE profile I) to produce mature biofilm on the surface of polyurethane and silicone catheter. CONCLUSIONS: Genotyping analysis by PFGE revealed the permanence of the MDR PFGE profile I in the nosocomial environment. Other new PFGE profiles emerged as etiologic agents of invasive infections. However, the MDR PFGE profile I was also found predominant among patients with hematogenic infections. The high level of multidrug resistance associated with biofilm formation capacity observed in MDR C. striatum is a case of concern.

Genome sequence of a multidrug-resistant Corynebacterium striatum isolated from bloodstream infection from a nosocomial outbreak in Rio de Janeiro, Brazil.

Multidrug-resistant (MDR) Corynebacterium striatum has been cited with increased frequency as pathogen of nosocomial infections. In this study, we report the draft genome of a C. striatum isolated from a patient with bloodstream infection in a hospital of Rio de Janeiro, Brazil. The isolate presented susceptibility only to tetracycline, vancomycin and linezolid. The detection of various antibiotic resistance genes is fully consistent with previously observed multidrug-resistant pattern in Corynebacterium spp. A large part of the pTP10 plasmid of MDR C. striatum M82B is present in the genome of our isolate. A SpaDEF cluster and seven arrays of CRISPR-Cas were found.

Sphygmomanometers and thermometers as potential fomites of Staphylococcus haemolyticus: biofilm formation in the presence of antibiotics.

BACKGROUND: The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES: In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS: The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS: ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS: Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.

Description of Endozoicomonas arenosclerae sp. nov. using a genomic taxonomy approach.

The taxonomic position of strains Ab112(T) (CBAS 572(T)) and Ab227_MC (CBAS 573) was evaluated by means of genomic taxonomy. These isolates represent the dominant flora cultured from the healthy marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro. Strains CBAS 572(T) and CBAS 573 shared >98 % 16S rRNA sequence identity with Endozoicomonas numazuensis and Endozoicomonas montiporae. In silico DNA-DNA Hybridization, i.e. genome-to-genome distance (GGD), amino acid identity (AAI) and average nucleotide identity (ANI) further showed that these strains had <70 %, at maximum 71.1 and 78 % of identity, respectively, to their closest neighbours E. numazuensis and E. montiporae. The DNA G+C content of CBAS 572(T) and CBAS 573 were 47.6 and 47.7 mol%, respectively. Phenotypic and chemotaxonomic features also allowed a separation from the type strains of their phylogenetic neighbours. Useful phenotypic features for discriminating CBAS 572(T) and CBAS 573 from E. numazuensis and E. montiporae species include C8 esterase, N-acetyl-ß-glucosaminidase, citric acid, uridine and siderophore. The species Endozoicomonas arenosclerae sp. nov. is proposed to harbour the new isolates. The type strain is CBAS 572(T) (=Ab112(T)).

Biofilm formation and fibrinogen and fibronectin binding activities by Corynebacterium pseudodiphtheriticum invasive strains.

Biofilm-related infections are considered a major cause of morbidity and mortality in hospital environments. Biofilms allow microorganisms to exchange genetic material and to become persistent colonizers and/or multiresistant to antibiotics. Corynebacterium pseudodiphtheriticum (CPS), a commensal bacterium that colonizes skin and mucosal sites has become progressively multiresistant and responsible for severe nosocomial infections. However, virulence factors of this emergent pathogen remain unclear. Herein, we report the adhesive properties and biofilm formation on hydrophilic (glass) and hydrophobic (plastic) abiotic surfaces by CPS strains isolated from patients with localized (ATCC10700/Pharyngitis) and systemic (HHC1507/Bacteremia) infections. Adherence to polystyrene attributed to hydrophobic interactions between bacterial cells and this negatively charged surface indicated the involvement of cell surface hydrophobicity in the initial stage of biofilm formation. Attached microorganisms multiplied and formed microcolonies that accumulated as multilayered cell clusters, a step that involved intercellular adhesion and synthesis of extracellular matrix molecules. Further growth led to the formation of dense bacterial aggregates embedded in the exopolymeric matrix surrounded by voids, typical of mature biofilms. Data also showed CPS recognizing human fibrinogen (Fbg) and fibronectin (Fn) and involvement of these sera components in formation of "conditioning films". These findings suggested that biofilm formation may be associated with the expression of different adhesins. CPS may form biofilms in vivo possibly by an adherent biofilm mode of growth in vitro currently demonstrated on hydrophilic and hydrophobic abiotic surfaces. The affinity to Fbg and Fn and the biofilm-forming ability may contribute to the establishment and dissemination of infection caused by CPS.

Biofilm production by multiresistant Corynebacterium striatum associated with nosocomial outbreak.

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum 1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatum bound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

Corynebacterium ulcerans isolates from humans and dogs: fibrinogen, fibronectin and collagen-binding, antimicrobial and PFGE profiles.

Corynebacterium ulcerans has been increasingly isolated as an emerging zoonotic agent of diphtheria and other infections from companion animals. Since pets are able to act as symptomless carriers, it is also essential to identify virulence potential for humans of these isolates. In this work the ability of C. ulcerans to bind to fibrinogen (Fbg), fibronectin (Fn) and Type I collagen as well the genetic relationship among strains isolated from human and asymptomatic dogs in Rio de Janeiro (Brazil) were analyzed. Five pulsed-field gel electrophoresis (PFGE) profiles were demonstrated (I, II, III, IV and V). In addition, the IV and V profiles exhibiting ≥85 % similarity were expressed by the BR-AD41 and BR-AD61 strains from companion dogs living in the same neighborhood. Independent of the PFGE-types, human and dog isolates showed affinity to Fbg, Fn and collagen. Heterogeneity of PFGE profiles indicated endemicity of C. ulcerans in the Rio de Janeiro metropolitan area. Differences in the expression of adhesins to the human extracellular matrix may contribute to variations in the virulence and zoonotic potential of C. ulcerans strains.

Description of Corynebacterium hiratae sp. nov. isolated from a human tissue bone a novel member of Corynebacterium Genus.

BACKGROUND: Corynebacterium spp. are widely disseminated in the environment, and they are part of the skin and mucosal microbiota of animals and humans. Reports of human infections by Corynebacterium spp. have increased considerably in recent years and the appearance of multidrug resistant isolates around the world has drawn attention. OBJECTIVES: To describe a new species of Corynebacterium from human tissue bone is described after being misidentified using available methods. METHODS: For taxonomic analyses, phylogenetic analysis of 16S rRNA and rpoB genes, in silico DNA-DNA hybridization, average nucleotide and amino acid identity, multilocus sequence analysis, and phylogenetic analysis based on the complete genome were used. FINDINGS: Genomic taxonomic analyzes revealed values of in silico DNA-DNA hybridization, average nucleotide and amino acids identity below the values necessary for species characterization between the analyzed isolates and the closest phylogenetic relative Corynebacterium aurimucosum DSM 44532T. MAIN CONCLUSIONS: Genomic taxonomic analyzes indicate that the isolates analyzed comprise a new species of the Corynebacterium genus, which we propose to name Corynebacterium hiratae sp. nov. with isolate 332T (= CBAS 826T = CCBH 35,014T) as the type strain.

Clonal multidrug-resistant Corynebacterium striatum within a nosocomial environment, Rio de Janeiro, Brazil.

Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases.

Novel configurations of type I-E CRISPR-Cas system in Corynebacterium striatum clinical isolates.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a prokaryotic adaptive immune system that, through Cas proteins, promote the degradation of foreign nucleic acids such as phages and plasmids. We analyzed 10 genomes of Corynebacterium striatum clinical isolates from a public hospital in Rio de Janeiro, Brazil, the most emergent multidrug-resistant Corynebacterium species. All isolates were submitted to antimicrobial susceptibility testing. The occurrence and diversity of the CRISPR system were investigated by bioinformatics tools. Our analysis revealed that the isolates exhibited type I-E gene arrangements, and 3 more multidrug-resistant isolates, alternative type I-E gene arrangements, showing a divergent gene arrangement within the cas operon. Phylogenetic analysis of the cas1 gene of this type I-E CRISPR-Cas system alternative arrangement, termed here type I-E', showed a cluster in a distinct clade of the type I-E CRISPR-Cas system. The systems' guanine-cytosine (GC) content is lower than the genomic DNA's GC content, and mobile genetic elements were found in some isolates near the CRISPR-Cas system. Most CRISPR spacers are unknown indicating that there is a reservoir of unexplored corynebacteriophages and plasmids. Some spacers showed perfect homologies with phage and plasmid sequences. Intact phage regions were found in 3 of our isolates, ranging from 9.1 to 43.8 kb, with regions showing similarity to Rhodococcus and Corynebacterium phages. Our results may contribute to research about the CRISPR-Cas system diversity in C. striatum, where there are no published data to date.

Phenotypic and molecular characterization and complete genome sequence of a Corynebacterium diphtheriae strain isolated from cutaneous infection in an immunized individual.

Diphtheria is an infectious disease potentially fatal that constitutes a threat to global health security, with possible local and systemic manifestations that result mainly from the production of diphtheria toxin (DT). In the present work, we report a case of infection by Corynebacterium diphtheriae in a cutaneous lesion of a fully immunized individual and provided an analysis of the complete genome of the isolate. The clinical isolate was first identified by MALDI-TOF Mass Spectrometry. The commercial strip system and mPCR performed phenotypic and genotypic characterization, respectively. The antimicrobial susceptibility profile was determined by the disk diffusion method. Additionally, genomic DNA was sequenced and analyzed for species confirmation and sequence type (ST) determination. Detection of resistance and virulence genes was performed by comparisons against ResFinder and VFDB databases. The isolate was identified as a nontoxigenic C. diphtheriae biovar Gravis strain. Its genome presented a size of 2.46 Mbp and a G + C content of 53.5%. Ribosomal Multilocus Sequence Typing (rMLST) allowed the confirmation of species as C. diphtheriae with 100% identity. DDH in silico corroborated this identification. Moreover, MLST analyses revealed that the isolate belongs to ST-536. No resistance genes were predicted or mutations detected in antimicrobial-related genes. On the other hand, virulence genes, mostly involved in iron uptake and adherence, were found. Presently, we provided sufficient clinical data regarding the C. diphtheriae cutaneous infection in addition to the phenotypic and genomic data of the isolate. Our results indicate a possible circulation of ST-536 in Brazil, causing cutaneous infection. Considering that cases of C. diphtheriae infections, as well as diphtheria outbreaks, have still been reported in several regions of the world, studies focusing on taxonomic analyzes and predictions of resistance genes may help to improve the diagnosis and to monitor the propagation of resistant clones. In addition, they can contribute to understanding the association between variation in genetic factors and resistance to antimicrobials.

Corynebacterium guaraldiae sp. nov.: a new species of Corynebacterium from human infections.

Non-diphtheria Corynebacterium species (NDC) belonging to the human skin and mucosa microbiota are frequently neglected as contaminants. However, reports of human infections by Corynebacterium spp. have increased considerably in recent years. In this study, a group of six NDC isolates of urine (n = 5) and sebaceous cyst (n = 1) from two South American countries were identified at genus level or misidentified based on API® Coryne and genetic/molecular analyses. The 16S rRNA (99.09-99.56%) and rpoB (96.18-97.14%) gene sequence similarities of the isolates were higher when compared with Corynebacterium aurimucosum DSM 44532 T. Multilocus sequence analysis (MLSA) indicated that these six NDC isolates compose a distinctive phylogenetic clade. Genome-based taxonomic analysis with the whole-genome sequences was able to separate these six isolates from other known Corynebacterium type strains. Average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between closely related type strains and the six isolates were considerably lower than the currently recommended threshold values for species circumscription. Phylogenetic and genomic taxonomy analyses indicated these microorganisms as a novel Corynebacterium species, for which we formally propose the name Corynebacterium guaraldiae sp. nov. with isolate 13T (= CBAS 827T = CCBH 35012T) as type strain.

Molecular characterization and phylogenetic analysis of the first Corynebacterium rouxii strains isolated in Brazil: a recent member of Corynebacterium diphtheriae complex.

BACKGROUND: Corynebacterium diphtheriae complex was formed by the species C. diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in the recent past. In addition to C. diphtheriae, C. ulcerans and C. pseudotuberculosis species can carry the tox gene, which encodes diphtheria toxin. Currently, three new species have been included in the complex: Corynebacterium rouxii, Corynebacterium silvaticum, and Corynebacterium belfantii. C. rouxii is derived from the ancient Belfanti biovar of C. diptheriae. We provide the complete genome sequences of two non-toxigenic strains C. rouxii isolated from a cat with a purulent infection in Brazil. The taxonomic status and sequence type, as well as the presence of resistance and virulence genes, and CRISPR-Cas system were additionally defined. RESULTS: The genomes showed an average size of 2.4 Mb and 53.2% GC content, similar to the type strain of the species deposited in Genbank/NCBI. Strains were identified as C. rouxii by the rMLST database, with 95% identity. ANI and DDH in silico were consistent with values above the proposed cut-off points for species limit, corroborating the identification of the strains as C. rouxii. MLST analyses revealed a new ST, which differs from ST-537 only by the fusA allele. No horizontal transfer resistance gene was predicted in both genomes and no mutation was detected in the constitutive genes gyrA and rpoB. Some mutations were found in the seven penicillin-binding proteins (PBPs) detected. The tox gene was not found, but its regulatory gene dtxR was present. Among the predicted virulence genes are those involved in iron uptake and adherence, in addition to the DIP0733 protein involved in epithelial cell adhesion and invasion. The CRISPR-Cas type I-E system was detected in both genomes, with 16 spacer sequences each. Of them, half are unknown according to the databases used, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. CONCLUSIONS: This is the first genomic study of C. rouxii reported in Brazil. Here we performed taxonomic analysis and the prediction of virulence factors. The genomic analyses performed in this study may help to understand the potential pathogenesis of non-toxigenic C. rouxii strains.

Virulence potential of the first Corynebacterium mycetoides strain isolated from human urine: a rare species of Corynebacterium.

For many years, the potential pathogenic of non-diphtheriae corynebacteria were underestimated. Nowadays, a growing number of Corynebacterium species are recognized as opportunistic agents of human infections, mainly in hospital settings. In addition, multidrug-resistant Corynebacterium isolates from clinical specimens, have been reported and the role of Corynebacterium spp. in urinary tract infections (UTIs) has been highlighted. Several studies have reported Corynebacterium species as the agent of UTIs especially in patients with risk factors. Thus, the present work aimed to report the first isolation of Corynebacterium mycetoides from human urine and an initial study on its virulence properties. The isolate, initially characterized by phenotypical tests as a multidrug-resistant Corynebacterium sp., was recovered from the urine of a female transplant patient. Mass spectrometry and 16S rRNA and rpoB genes sequencing identified the isolate as C. mycetoides. The isolate was found able to adhere to and survive into epithelial cells (Vero cells), and its pathogenic potential was confirmed when tested against Caenorhabditis elegans nematode. The results obtained suggest that C. mycetoides is a potential pathogen for the urinary tract in humans and for a better understanding of the multifactorial mechanisms of virulence, studies about this species should be continued.

Assessment of VITEK® 2, MALDI-TOF MS and full gene 16S rRNA sequencing for aerobic endospore-forming bacteria isolated from a pharmaceutical facility.

VITEK®2, MALDI-TOF MS and 16S rRNA sequencing were evaluated for the identification of aerobic endospore-forming bacteria (AEB) from a pharmaceutical facility. MALDI-TOF MS demonstrated higher accuracy compared to VITEK®2, although both databases were insufficient to identify AEB species. Sequencing was the best methodology, but unable to identify closely related species.

MALDI-TOF MS database expansion for identification of Bacillus and related genera isolated from a pharmaceutical facility.

Bacillus and related genera are among the main bacterial groups isolated from pharmaceutical production areas. The identification of Bacillus species and related genera by classical methods is particularly difficult, due to similarities between closely related species. The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is one of the most promising techniques for chemotaxonomic characterization of microorganisms, being an alternative to genotypic methods. This study aimed to identify Bacillus strains and related genera isolated from immunobiological production areas by phylogenetic analysis of housekeeping genes and expand the database associated with MALDI-TOF MS to improve their identification. In a previous study, 97 aerobic endospore-forming bacteria isolated from a pharmaceutical facility were analyzed by MALDI-TOF MS and 16S rRNA gene full-length sequencing. All strains were identified as Bacillus and related genera by the latest methodology. Among the 97 strains, 22 were unidentified and 2 strains were misidentified by MALDI-TOF MS. In the present study, these 24 strains were subjected to 16S rRNA gene phylogenetic analysis. Strains not identified at species level by this methodology were submitted to rpoB gene phylogenetic analysis. After identifying the strains, 19 of the 24 strains were incubated for 24, 48, and 72 h on Tryptic Soy Agar and Sheep Blood Agar and subjected to analysis by MALDI-TOF MS. A SuperSpectrum for each strain was created and entered into the equipment database. Finally, the 24 strains were again submitted to proteomic analysis by MALDI-TOF MS, and, at this time, all were correctly identified. The genotypic identification of in-house isolated strains and the introduction of these spectra in MALDI-TOF MS, in order to obtain a customized database, proved to be an extremely effective tool in the identification of Bacillus and related genera from pharmaceutical industry origin.