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 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.

Diversity and epidemiological profile of Pseudomonas aeruginosa from drinking water in Brazil genotyped using multi-locus sequence typing.

Pseudomonas aeruginosa is a Gram-negative bacillus associated with waterborne diseases. The objective of this study was to determine whether particular P. aeruginosa sequence types (STs) were associated with drinking water contamination in Brazil. This was achieved by searching the Pseudomonas PubMLST database, which contains the records for 8358 strains collected between 1938 and 2023. The majority (97.2%) had the complete 7-loci multilocus sequence typing profile and were assigned to 3486 STs. After eBURST (an algorithm used to infer patterns of evolutionary descent among clusters), 1219 groups with single-locus variant and 575 groups with double-locus variant were formed. Brazil was the South American country with the most isolates (n = 219, 58.24%), and the Simpson's index was 0.9392. Of the 219 Brazilian isolates, eight were isolated in water and identified as STs 252, 1417, 1605, 2502, 2620, 3078, and 3312. ST252, 1417, and 3078 have already been isolated from clinical cases worldwide. Furthermore, ST1605 and 2620, after the eBURST, they were grouped in the same clonal complex as STs involved in human infections. In conclusion, P. aeruginosa STs involved in human infections were found in bottled drinking water commercialized in Brazil, revealing that these types of drinking waters can be a vehicle of contamination.

Environmental assessment of sewage contamination in the surroundings of a marine outfall combining human mastadenovirus and fecal indicator bacteria.

This study assessed the microbiological contamination of the marine area of a metropolitan region, where a marine outfall is used as a sanitary solution for domestic sewage. For human mastadenovirus (HAdV) quantification 134 water samples were concentrated by skimmed milk flocculation method and analyzed with qPCR and PMAxx-qPCR, being the latter to assess the capsid integrity viral. HAdV with intact capsids were detected in 10 % (16/102) of samples classified as suitable for bathing using at least one fecal bacterial indicator. Spatial analysis of the results showed that the drainage channels of the basin that flow into the sea are the main sources of microbiological contamination in the foreshore zone, where intact HAdV reached a concentration of up to 3 log genomic copies/L. HAdV serotypes A12, D, F40 and F41 were characterized. Our results suggest the use of intact HAdV as a complementary parameter to assess the quality of recreational waters.

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.

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 certified reference material for the potency assay in yellow fever vaccine quality control, in accordance with International Standards Organization guidance.

The attenuated yellow fever vaccine (YFV) is offered free of charge to the Brazilian population through the National Immunization Program (NIP). One of the specifications for quality control analyses of the vaccine is the potency determination. This test determines the number of plaque forming units (PFU) in Vero cells. In order to validate the results, the reference material (RM) is analysed in parallel with an established reference vaccine. The aim of this study was to establish certified RM for use as an internal control in the potency assay for the production chain of YFV. The candidate RM homogeneity and stability were determined, and characterized by a collaborative study for further certification. The RM was considered sufficiently homogeneous with average 4.68 log10 IU/HD and stable at (-20 ± 10) ºC and (22.5 ± 2.5) ºC for 715 and 183 days, respectively. When reconstituted and stored in aliquots of 0.6 mL, it was stable at (-20 ± 10) ºC for eight days. But it was not stable at (5 ± 3) ºC for three days. In a collaborative study, two independents' laboratories gave an averaged value of 4.56 ± 0.030 log10 IU/HD. After determining the expanded uncertainty of homogeneity, stability, and characterization, the certified RM lot: 195VFA020Z presented a property value of 4.56 ± 0.22 log10 IU/HD. It was concluded that the new certified RM can be used in routine analysis of a YFV producer, since it has its property value established and it is stable. The possibility of using it in aliquots after reconstitution will also allow the RM to have a much longer shelf life.

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.

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.

Evaluation of hydrogen peroxide virucidal efficacy against yellow fever virus 17DD vaccine strain for application in a vaccine manufacturing industry.

The aim of this study was to evaluate the inactivation performance of hydrogen peroxide to the yellow fever virus 17DD vaccine strain, used for the production of attenuated yellow fever vaccine, in two matrixes: formulated yellow fever vaccine (FYV) and yellow fever viral suspension - active pharmaceutical ingredient (API). The samples were dried on stainless steel and exposed to hydrogen peroxide liquid (HPL) at concentrations of 30, 10, 3 and 1% for 20 and 60 min; and to hydrogen peroxide vapour (HPV) in an isolator. The exposure to HPL 30 and 10 %, within 20 min, reduced the virus titre at least 3.85 log10 PFU/mL (74.8 %). During 60 min of exposure, the HPL 30, 10 and 3% reduced the virus titre by at least 3.18 log10 PFU/mL (62.6 %). HPV exposure resulted in complete virus inactivation in FYV (≥ 4.42 log10 PFU/mL reduction) and for API samples 3.17 log10 PFU/mL (64.3 %) reduction. Hydrogen peroxide showed to be a promising disinfectant for elimination of yellow fever virus. However, the optimum concentration and contact time will vary depending on the type of application, and as such may complement individual risk assessments of biological production processes.

Diversity of Cronobacter genus isolated between 1970 and 2019 on the American continent and genotyped using multi-locus sequence typing.

This study aimed to evaluate the Cronobacter spp. strains isolated on the American continent and characterized using multi-locus sequence typing (MLST) available in the PubMLST database and current literature. From 465 Cronobacter spp. strains, the majority (n = 267, 57.4%) was from North America, mainly from USA (n = 234) and 198 (42.6%) were from South America, mainly from Brazil (n = 196). A total of 232 (49.9%) were isolated from foods, 102 (21.9%) from environmental, 87 (18.7%) from clinical, 27 (5.8%) from PIF, one from water (0.2%) and 16 (3.5%) from unknown sources. A total of five species were represented: Cronobacter sakazakii (374, 80.4%), Cronobacter malonaticus (41, 8.8%), Cronobacter dublinensis (29, 6.2%), Cronobacter turicensis (16, 3.5%) and Cronobacter muytjensii (5, 1.1%). The strains with complete MLST profile (n = 345) were assigned to 98 STs, a ratio of 3.5 strain by ST found and the calculated Simpson`s index was 0.93. The strains showed a high diversity and after eBURST analysis, 30 STs (n = 189) formed 12 single and/or double-locus variant clonal complexes (CC). A total of 38 STs (38.7%) were associated with clinical cases of infection, including well established C. sakazakii CC 1, 4, 8 and 83; C. malonaticus ST60, 307, 394 and 440; and C. sakazakii ST 12 and 494.

Assessment of the microbiological quality of natural mineral waters according to the manufacturing time of 20 L returnable packs in Brazil.

This study aimed to assess the microbiological quality of natural mineral waters commercialized in 20 L returnable packs in Brazil by investigating the presence of bacteria and viruses in packs with different manufacturing times (Tm). With this purpose, 99 water samples from 33 lots (n = 3/batch) of 15 brands, obtained from packs with three intervals of Tm, were analyzed. Total coliforms (16.2%), Pseudomonas aeruginosa (9.9%), sulphite-reducing Clostridium (5.0%) and Escherichia coli (2.0%) were detected but enterococci and norovirus GII not. Regarding brands, 11 (73.3%) presented unsatisfactory results for at least one of the lots analyzed. Pseudomonas aeruginosa analysis revealed six sequence types and strains were susceptible to all antibiotics tested and were able to produce biofilms. Human adenovirus (4) and norovirus GI (9) were also identified in nine samples randomly selected. Natural mineral waters commercialized in 20 L packs with Tm ≥ 2 years presented more microbiological contamination (P ≤ 0.012) than ones with a Tm of 0-1 year or a Tm of 1-2 years. These results suggest that the validity period of reusable 20 L packs should be reduced or that they can no longer be reused.

Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil.

The aim of this study was to evaluate the microbiological quality of 45 samples of corn-based farinaceous foods commercialized in Brazil. The bacteriological analysis performed were: detection of Salmonella and Cronobacter, and enumeration of faecal coliforms and Bacillus cereus. The Cronobacter isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene and MLST. No sample presented contamination by Salmonella or B. cereus (<102 UFC/g). Faecal coliforms were detected in two (4.4%) samples but in low concentration (≤23.0 MPN/g), and 20 samples (44.4%) contained Cronobacter. Twenty-nine unique Cronobacter isolates were identified as C. sakazakii (n = 18), C. malonaticus (n = 2); that presented 11 different fusA alleles, including new fusA 183. MLST analysis revealed 17 sequence types (STs), six of which were newly identified (ST687-690, 693, and 694). Resistance or intermediary resistance were found to ceftazidime (15.0%), aztreonam (15.0%), nalidixic acid (15.0%), nitrofurantoin (15.0%), cefepime (10.0%), gentamicin (5.0%), and tetracycline (5.0%). The presence of Cronobacter in corn-based farinaceous foods could be a significant risk to infants as these products are used as alternatives to commercially available infant formula. Strategies to manage the risk of Cronobacter infections due to the consumption of these alternative feeds need to be developed by the regulatory agencies.

Efficacy of complex decongestive therapy for lymphedema of the lower limbs: a systematic review.

Lymphedema is a chronic condition that negatively affects function and quality of life. There is currently no definitive treatment. However, some options have been proposed to mitigate its consequences. Complex Decongestive Therapy (CDT) stands out as one of the main treatment methods of choice. This systematic review aimed to evaluate the effectiveness of this technique for treating lower extremity lymphedema. The results revealed that CDT was effective in reducing the volume of affected limbs. However, some questions have not yet been answered, such as: How long do patients benefit from using CDT? and How to maintain the gains obtained? It was not possible to perform a meta-analysis because of heterogeneity, unsatisfactory methodological quality of the available studies, and the lack of a gold-standard protocol for administration of the technique. Further studies are needed to advance lymphedema research and therapy.

Eficácia da terapia complexa descongestiva para linfedema nos membros inferiores: revisão sistemática / Efficacy of complex decongestive therapy for lymphedema of the lower limbs: a systematic review

Resumo O linfedema é uma condição crônica que afeta negativamente a função e a qualidade de vida. Atualmente, não existe tratamento definitivo. Todavia, algumas opções foram propostas para amenizar suas consequências. Entre elas, destaca-se a terapia complexa descongestiva (TCD), que se configura como um dos principais métodos de escolha de tratamento. Esta revisão sistemática teve por objetivo avaliar a eficácia dessa técnica no tratamento de linfedemas nos membros inferiores. Os resultados revelaram que a TCD foi eficaz na redução do volume das extremidades afetadas. No entanto, algumas questões ainda não foram elucidadas, tais como: por quanto tempo os pacientes se beneficiam com o uso da TCD? Como manter os ganhos obtidos? Diante da heterogeneidade, da insatisfatória qualidade metodológica dos trabalhos disponíveis e da inexistência de protocolo padrão para aplicação da técnica, não foi possível realizar a metanálise, o que demonstra que há muito a se avançar na investigação e na terapêutica do linfedema.

Abstract Lymphedema is a chronic condition that negatively affects function and quality of life. There is currently no definitive treatment. However, some options have been proposed to mitigate its consequences. Complex Decongestive Therapy (CDT) stands out as one of the main treatment methods of choice. This systematic review aimed to evaluate the effectiveness of this technique for treating lower extremity lymphedema. The results revealed that CDT was effective in reducing the volume of affected limbs. However, some questions have not yet been answered, such as: How long do patients benefit from using CDT? and How to maintain the gains obtained? It was not possible to perform a meta-analysis because of heterogeneity, unsatisfactory methodological quality of the available studies, and the lack of a gold-standard protocol for administration of the technique. Further studies are needed to advance lymphedema research and therapy.

Glicose como causa e tratamento de necrose cutânea / Glucose as a cause of and treatment for cutaneous necrosis

A escleroterapia continua sendo um dos procedimentos mais executados pelos cirurgiões vasculares brasileiros. O conhecimento das suas complicações é imprescindível para que possamos evitá-las. Os efeitos colaterais graves desse método de tratamento para as telangiectasias dos membros inferiores são raros e frequentemente associados a um erro técnico ou à dosagem injetada. São predominantemente locais, apresentando-se, algumas vezes, como uma situação de difícil resolução. Relatamos um caso de formação de necrose cutânea após escleroterapia química com glicose hipertônica (75%) e sua cicatrização utilizando preparação tópica contendo vaselina e glicose 60%, cujo resultado estético foi satisfatório

Sclerotherapy remains one of the procedures most frequently performed by Brazilian vascular surgeons. Knowledge of its complications is indispensable to enable us to avoid them. The severe side effects of this method of treatment for telangiectasias of the lower limbs are rare and are often associated with technical errors or the dose injected. Complications are predominantly local, but are sometimes difficult to resolve. We report a case of formation of cutaneous necrosis after chemical sclerotherapy using hypertonic glucose (75%), which healed when treated with a topical preparation containing vaseline and 60% glucose, with satisfactory esthetic results

Fatal Cronobacter sakazakii Sequence Type 494 Meningitis in a Newborn, Brazil.

We describe a case of infection with Cronobacter sakazakii sequence type 494 causing bacteremia and meningitis in a hospitalized late premature infant in Brazil. We conducted microbiological analyses on samples of powdered infant formula from the same batch as formula ingested by the infant but could not identify the source of contamination.

Evaluation of skimmed milk flocculation method for virus recovery from tomatoes.

This study aimed to evaluate the elution-concentration methodology based on skimmed milk flocculation from three varieties of tomatoes (Solanum lycopersicum L. [globe], Solanum lycopersicum var. cerasiforme [cherry] and hybrid cocktail [grape tomato]) for further monitoring of field samples. Spiking experiments were performed to determine the success rate and efficiency recovery of human norovirus (NoV) genogroup II, norovirus murine-1 (MNV-1) used as sample process control virus and human adenovirus (HAdV). Mean values of 18.8%, 2.8% and 44.0% were observed for NoV GII, MNV-1 and HAdV, respectively with differences according to the types of tomatoes, with lower efficiency for cherry tomatoes. Analysis of 90 samples, obtained at commercial establishments in the metropolitan region of Rio de Janeiro State, revealed 4.5% positivity for HAdV. Bacterial analysis was also performed with no detection of Salmonella spp., L. monocytogenes and fecal coliforms. Data demonstrated that the skimmed milk flocculation method is suitable for recovering HAdV from tomatoes and highlights the need for considering investigation in order to improve food safety.