Evaluation of phenotypical and genotypical methods for the identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility.

AIMS: Evaluate methods for identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility. METHODS AND RESULTS: From 270 S. maltophilia strains identified by VITEK®2, 40 were selected and submitted to MALDI TOF-MS, 16S and 23S rRNA gene analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and an antimicrobial susceptibility profile. 16S rRNA sequencing was able to identify 39 (97.5%) strains as Stenotrophomonas spp. and one (2.5%) as Luteimonas huabeiensis. MALDI TOF-MS identified 37 (92.5%) strains as S. maltophilia, and three (7.5%) were not identified. PCR targeting 23S rRNA yielded a positive result for 39 (97.5%) strains. However, after sequencing, two strains were identified as Stenotrophomonas rhizophila, showing false-positive results. The confirmed S. maltophilia strains (n = 37) showed 35 distinct ERIC-PCR profiles and exhibited sensitivity to minocycline and levofloxacin, and six (16.3%) showed intermediate resistance to sulfamethoxazole-trimethoprim. CONCLUSION: Matrix-assisted laser desorption lonization-time of flight mass spectrometry (MALDI-TOF MS) was a satisfactory methodology for the identification of S. maltophilia, but expansion of the database is necessary for the identification of other species. 16S rDNA sequencing showed low resolution for Stenotrophomonas species differentiation. PCR targeting 23S rRNA could not differentiate S. maltophilia from S. rhizophila. ERIC-PCR was shown to be a useful tool for the microbial source tracking of S. maltophilia.

Phenotypical and molecular characterization of Acinetobacter spp. isolated from a pharmaceutical facility.

Characterizing microorganisms according to different criteria is useful when investigating sources of microbiological contamination in the pharmaceutical industry. The aim of this study was to characterize 38 Acinetobacter baumannii complex strains isolated from a biopharmaceutical industry by 16S rRNA sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), multilocus sequence typing (MLST), antimicrobial susceptibility profile, biofilm formation, and sensibility to disinfectants. Thirty-three (86.9%) strains were identified by 16S rRNA gene sequencing as A. seifertii/pitti/nosocomialis/lactucae, four (10.5%) as A. baumannii, and one (2.6%) as A. vivianii/courvalini. MALDI-TOF/MS did not identify one strain, and incorrectly identified 30/37 (81.1%) strains as A. baumannii. Strains were assigned to 12 different STs, of which nine were newly defined in this study (STs 2091-2099). Twenty-six (68.4%) strains showed resistance to amikacin and gentamicin. Thirty-three (86.8%) strains were classified as moderately or strongly adherent on polystyrene. Alcohol 70%/15 min and quaternary ammonium 0.08%/20 min were not able to eliminate the biofilm formed, but sodium hypochlorite 0.1%/15 min was efficient. In conclusion, improved methods are needed to improve the identification of Acinetobacter strains in pharmaceutical industries. This organism is of particular concern as it forms recalcitrant biofilms, leading to persistence in the manufacturing environment and increased risk of product contamination.

Identification of Sutcliffiella horikoshii strains in an immunobiological pharmaceutical industry facility.

The pharmaceutical industry must comply with the requirements for good manufacturing practices to reduce inherent contamination risks in the production process. Bacillus and related genera are among the main bacterial isolated from clean areas, raw material, and products in the pharmaceutical industries, but the correct identification of these species is still a challenge. The aim of this study was to characterize by phenotyping, protein profiling, and 16S rRNA gene sequencing Sutcliffiellahorikoshii strains (n = 6) isolated from an immunobiological pharmaceutical facility, and to propose the reclassification of Bacillus tianshenii to the genus Sutcliffiella, and Sutcliffiella tianshenii sp. nov. The strains were characterized by VITEK®2, matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) using VITEK®MS, and 16S rRNA gene sequencing analysis. MALDI-TOF/MS did not identify any strains that were identified by 16S rRNA as S. horikoshii. VITEK®2 showed false-positive results, with misidentification as B. sporothermodurans (reclassified as Heyndrickxia sporothermodurans) and Geobacillus thermoleovorans. After MALDI-TOF/MS database expansion, with the creation of SuperSpectrum, the strains were correctly identified as S. horikoshii. This study is the first report of isolation of S. horikoshii strains from a pharmaceutical industry. More studies are necessary to better understand the ability of S. horikoshii to contaminate the environment and products.

Establishment of a Reference Material in Quality Control for Use in Infectivity and Identity Assays of Recombinant COVID-19 Vaccine, in Accordance with International Standards Organization Guidance.

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), began in 2019. One of the strategies for pandemic control was mass vaccination. In Brazil, the recombinant COVID-19 vaccine (RCV) was produced on a large scale and offered at no charge to the population. The specifications for quality control analyses of RCV included identity and infectivity determination. To validate the results, a reference material (RM) must be analyzed in parallel with the sample vaccine. This research aimed to establish the RM for use in the identity and infectivity assay for RCV. The candidate RM was analyzed using homogeneity and stability studies. The RM was considered homogeneous for identity (cycle threshold (Ct) ≤ 25.19) and infectivity (average x- was 9.25 log10 infectious units/mL). The RM was considered adequately stable for identity during the total period in all studies, being stable at -70, 5, and 22.5 °C for 380, 313, and 14 days, respectively (Ct ≤ 21.81). For infectivity, the RM was stable at -70, 5, and 22.5 °C for 380, 97, and three days, respectively. Since the property identity and infectivity values of the RM were established, the new RM could be used in quality control analysis.

Characterization by MALDI-TOF MS and 16S rRNA Gene Sequencing of Aerobic Endospore-Forming Bacteria Isolated from Pharmaceutical Facility in Rio de Janeiro, Brazil.

Bacillus and related genera are among the most important contaminants in the pharmaceutical production environment, and the identification of these microorganisms at the species level assists in the investigation of sources of contamination and in preventive and corrective decision making. The aim of this study was to evaluate three methodologies for the characterization of endospore-forming aerobic bacterial strains isolated from a pharmaceutical unit in Rio de Janeiro, Brazil. MALDI-TOF MS was performed using MALDI Biotyper® and VITEK® MS RUO systems, and complete 16S rRNA gene sequencing was performed using the Sanger methodology. The results showed the prevalence of the genera Bacillus (n = 9; 36.0%), Priestia (n = 5; 20.0%), and Paenibacillus (n = 4; 16.0%). Three (20.0%) strains showed <98.7% of DNA sequencing similarity on the EzBioCloud Database, indicating possible new species. In addition, the reclassification of Bacillus pseudoflexus to the genus Priestia as Priestia pseudoflexus sp. nov. is proposed. In conclusion, 16S rRNA and MALDI TOF/MS were not sufficient to identify all strains at the species level, and complementary analyses were necessary.

Evaluation of Antimicrobial Resistance Patterns of Pseudomonas aeruginosa Strains Isolated among COVID-19 Patients in Brazil Typed by Fourier-Transform Infrared Spectroscopy.

This study aimed to characterize Pseudomonas aeruginosa strains isolated from hospitalized patients during the COVID-19 pandemic. This was achieved using phenotypic and molecular techniques, including their antimicrobial resistance profile and biofilm formation. Eighteen strains were isolated from a hospital in Rio de Janeiro, Brazil, and identified by VITEK®2, MALDI-TOF/MS (VITEK MS® and MALDI Biotyper®), and 16S rRNA sequencing. Fourier-transform infrared (FTIR) spectroscopy, antimicrobial susceptibility testing, and biofilm formation and disinfectant tolerance tests were applied to evaluate the virulence characteristics of the strains. VITEK®2 (≥99%), VITEK MS® (≥82.7%), and MALDI Biotyper® (score ≥ 2.01) accurately identified the P. aeruginosa strains, but 16S rRNA sequencing did not differentiate the species P. aeruginosa from P. paraeruginosa. FTIR typing identified three different clusters, but no correlation between the phenotypical or antimicrobial susceptibility testing patterns was found. Most strains exhibited resistance to various antimicrobials. The exceptions were sensitivity to amikacin and norfloxacin, and consequently, these could be considered potential treatment options. Most strains (n = 15, 83.3%) produced biofilms on polystyrene. Sodium hypochlorite treatment (0.5%/15 min) was shown to be the most effective disinfectant for biofilm elimination. P. aeruginosa biofilm formation and tolerance to disinfectants demonstrate the need for effective cleaning protocols to eliminate contamination by this organism in the hospital environment and medical equipment.

Evaluation of MALDI-TOF MS, sequencing of D2 LSU rRNA and internal transcribed spacer regions (ITS) for the identification of filamentous fungi isolated from a pharmaceutical facility.

The identification of filamentous fungi through culture characterization may be hampered by phenotypic variability. Information obtained from the identification of microorganisms are important for investigation of sources of contamination of a product or process. The aim of this study was to identify filamentous fungal strains (n = 50) isolated from a pharmaceutical facility by using Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), as well as D2 domain of the large-subunit (LSU) ribosomal RNA gene and internal transcribed spacer regions (ITS) sequencing. MALDI-TOF MS system only identified five strains at the species level, while 45 were not identified. The analysis through GenBank allowed the identification of up to 19 strains at the species level, while MycoBank allowed the identification of up to nine strains at the species level. The databases identified up to 11 genera: Penicillium, Aspergillus, Cladosporium, Chaetomium, Coniochaeta, Curvularia, Diaporthe, Fusarium, Trichoderma, Rhizopus and Microdochium. MALDI-TOF MS showed an insufficient database to identify the species of fungi. DNA sequencing was the best methodology to identify to the genus level but was unable to differentiate between closely related species. Therefore further methods for the identification of filamentous fungi from pharmaceutical areas at species level need to be developed.