Marcelo Luiz Lima Brandão

Biofilm formation by Staphylococcus hominis in pharmaceutical environments can promote microbial persistence on surfaces and hinder cleaning and disinfection, posing a risk to product quality and safety. This study aimed to standardize an in vitro adaptive methodology to evaluate biofilm for-mation on surfaces of interest to the pharmaceutical industry and analyze the impact of different interpretation criteria. Fifty-four S. hominis strains isolated between 2014 and 2022 from a pharma-ceutical production chain were evaluated. Biofilm formation was analyzed on stainless steel, acrylic, and vinyl coating using the Cell Counting Kit-8 (CCK-8) assay to quantify bacterial cell viability, as well as on polystyrene using the crystal violet method. Qualitative and quantitative interpretations applied different equations to define the negative control cutoff. Vinyl coating showed the highest formation, with 31 (57.41%) strains classified as moderate or strong producers, followed by stainless steel (n=20; 37.01%), acrylic (n=16; 29.63%), and polystyrene (n=2; 3.70%). Quantitative analysis identified surface-dependent hyper-producing strains, reaching maximum values from 49.75 to 61.60%. Applying three standard deviations to the negative control generated reclassifications without statistically significant differences between criteria. The proposed standardized method supports the CCK-8 assay as an adaptive methodology to evaluate biofilm formation by different bacterial strains and surfaces.

Background/Objectives: The global crisis of antimicrobial resistance and limited discov-ery of new antimicrobial classes have intensified the search for alternative sources of bio-active natural products. Environmental bacteria harbor largely unexplored biosynthetic diversity, much of which is encoded in biosynthetic gene clusters (BGCs). This review examines current strategies for genome-guided BGC bioprospecting, emphasizing antimi-crobial and environmental applications, and the transition from computational prediction to experimental validation. Methods: We review advances in bacterial genome mining, protein structure prediction, molecular docking, and experimental approaches for evalu-ating antimicrobial and bioremediation potential. Emphasis is placed on representative environmental bacterial genera and the integration of genomic, structural, metabolomic, microbiological, and environmental approaches. Results: Genome mining has revealed substantial biosynthetic potential across diverse environmental bacteria. Comparative ge-nomics and BGC similarity analyses support dereplication and prioritization, while AI-based approaches can expand the search toward atypical and poorly characterized bi-osynthetic systems. Protein structure prediction and molecular docking provide mecha-nistic hypotheses but cannot independently demonstrate metabolite production or biolog-ical activity. Experimental approaches, including One Strain-Many Compounds, heterol-ogous expression, metabolomics, functional genetics, antimicrobial assays, and environ-mentally relevant microcosms, are therefore essential for linking genomic potential to me-tabolite production and biological function. Conclusions: BGC-driven bioprospecting pro-vides a powerful framework for expanding microbial natural product discovery beyond conventional screening. Its greatest potential lies in multi-omics approaches and experi-mental validation. Future progress will depend on improving the prioritization and acti-vation of cryptic BGCs and establishing stronger gene-to-metabolite-to-function relation-ships, ultimately supporting the development of novel antimicrobials and environmental-ly relevant biotechnological applications.

As doenças de transmissão hídrica e alimentar (DTHA) são relevantes para a saúde pública devido à ampla circulação de microrganismos patogênicos em alimentos, exigindo que laboratórios de apoio à vigilância sanitária executem métodos analíticos confiáveis. O objetivo deste estudo foi realizar uma verificação das metodologias de Pesquisa de Salmonella spp., Contagem de estafilococos coagulase positiva (ECP) e Bacillus cereus presuntivo utilizadas no Laboratório de Microbiologia de Alimentos do Instituto Municipal de Vigilância Sanitária, Vigilância de Zoonoses e Inspeção Agropecuária do Rio de Janeiro (IVISA RIO), conforme a ABNT NBR ISO/IEC 17025:2017. Amostras de alimentos prontos para consumo foram contaminadas artificialmente com cepas de material de referência certificado dos microrganismos alvo (Salmonella Typhimurium, ATCC MRC-14028, Staphylococcus aureus ATCC MRC-6538 e Bacillus cereus ATCC MRC-11778), além de um interferente (Escherichia coli ATCC MRC-8739), avaliando seletividade, limite de detecção (LD) e quantificação (LQ). Os resultados mostraram seletividade satisfatória para as três metodologias, com ausência de resultados falso-positivos. O LD para Salmonella spp. foi de 1,0 unidade formadora de colônia (UFC)/25 g; e os LQ de ECP e B. cereus presuntivo foram de 6,0 e 10² UFC/g, respectivamente. Estes resultados obtidos foram compatíveis com os parâmetros estabelecidos para alimentos prontos para consumo, evidenciando a capacidade do laboratório de fornecer resultados confiáveis e tecnicamente robustos, de forma a atender à legislação brasileira vigente. Dessa forma, os valores obtidos contribuem para fortalecer o papel dos laboratórios de saúde pública como ferramentas essenciais na geração de informações primárias, fundamentais para a tomada de decisão em políticas de prevenção e controle de DTHA. A consolidação de metodologias confiáveis não apenas assegura a qualidade dos laudos emitidos, mas também promove maior proteção à saúde da população, especialmente dos grupos mais vulneráveis, como crianças, idosos e imunocomprometidos.

O presente artigo analisa normas sanitárias brasileiras instituídas em um Estado Democrático de Direito marcado por profundas desigualdades socioeconômicas. Com o objetivo de contribuir para o debate do modelo regulatório na área de alimentos no Brasil, utiliza o exemplo das cozinhas solidárias, cujo Programa é instituído pela Lei n.º 14.628/2023, e discute as diretrizes das RDC Anvisa n° 216/2004 e da RDC Anvisa nº 49/2013. A investigação classifica-se como qualitativa, exploratória e descritiva. As normas escolhidas para análise versam sobre os temas inclusão econômica e social, regularização para o exercício de atividade de interesse sanitário do microempreendedor individual, do empreendimento familiar rural e do empreendimento econômico solidário e Boas Práticas para Serviços de Alimentação. Observou-se que essas normas contribuem com diferentes olhares para o atual modelo regulatório de alimentos no Brasil. Sob a perspectiva das cozinhas solidárias, o cenário é desafiador e aponta para a necessidade de articulação entre os entes federados no âmbito do SNVS, com necessidade de atuar por meio de ações estruturantes, tais como a formulação e publicação de regulamentos técnicos complementares aos federais em âmbito estadual e municipal, investimento na educação permanente e na capacitação, padronização dos instrumentos de trabalho que priorizem a gestão do risco epidemiológico real e o controle de pontos críticos, garantindo ao mesmo tempo o direito à alimentação adequada, como preconizado no SUS.

Inadequate surface sanitization represents a significant risk to sterility assurance and regulatory compliance. Therefore, an effective cleaning and disinfection program is a critical component of contamination control strategies in pharmaceutical facilities manufacturing sterile medicinal products. This study aimed to standardize a carrier-based methodology for evaluating the efficacy of disinfectants against in-house environmental isolates recovered from a pharmaceutical industry facility. Nine representative strains were selected from five different groups—Gram-positive non-spore-forming bacteria (Micrococcus luteus and Kocuria spp.), Gram-positive spore-forming bacteria (two Bacillus spp. strains), Gram-negative bacteria (Pseudomonas aeruginosa and Acinetobacter haemolyticus), yeasts (Candida parapsilosis and Rhodotorula mucilaginosa), and filamentous fungus (Penicillium spp.)—based on historical environmental monitoring data (2012–2022), and were characterized using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF MS) and molecular sequencing (16S rRNA or D2 LSU rDNA). Disinfectant efficacy was assessed on stainless-steel and low-density polyethylene surfaces using NF T 72-281:2014 with adaptations, testing alcohol 70%, sodium hypochlorite 0.5%, quaternary ammonium 0.05%, peracetic acid 0.5%, and accelerated hydrogen peroxide wipes. All agents demonstrated ≥5 log₁₀ reductions against vegetative bacteria and fungi on both surfaces. However, variable sporicidal performance was observed, particularly for one Bacillus cereus group strain (B1342/15), which showed limited viability reduction on stainless steel. These findings highlight inter-strain variability and the greater tolerance of surface-associated spores. The study reinforces the importance of carrier-based testing using in-house isolates to ensure realistic validation of disinfectants and to strengthen microbiological risk management within pharmaceutical contamination control strategies.

Inadequate surface sanitization represents a significant risk to sterility assurance and regulatory compliance. Therefore an effective cleaning and disinfection programme is a critical component of contamination control strategies in pharmaceutical facilities manufacturing sterile medicinal products. This study aimed to standardize a carrier-based methodology for evaluating the efficacy of disinfectants against in-house environmental isolates recovered from a pharmaceutical industry facility. Nine representative strains (six bacteria and three fungi), selected based on historical environmental monitoring data (2012–2022), were characterized using matrix-assisted laser desorption/ionization - time-of-flight / mass spectrometry (MALDI-TOF MS) and molecular sequencing (16S rRNA or D2 LSU rDNA). Disinfectant efficacy was assessed on stainless-steel and low-density polyethylene surfaces using NF T 72-281:2014 with adaptations, testing alcohol 70%, sodium hypochlorite 0.5%, quaternary ammonium 0.05%, peracetic acid 0.5%, and accelerated hydrogen peroxide wipes. All agents demonstrated ≥5 log₁₀ reductions against vegetative bacteria and fungi on both surfaces. However, variable sporicidal performance was observed, particularly for one Bacillus cereus group strain (B1342/15), which showed limited reduction on stainless-steel. These findings highlight inter-strain variability and the greater tolerance of surface-associated spores. The study reinforces the importance of carrier-based testing using in-house isolates to ensure realistic validation of disinfectants and to strengthen microbiological risk management within pharmaceutical contamination control strategies.

The genus Burkholderia , particularly the Burkholderia cepacia complex (Bcc), encompasses Gram-negative bacteria of recognized ecological, biotechnological, and clinical relevance. Accurate identification of species within this complex is challenging due to high phenotypic and genetic similarity, impacting clinical diagnostics and epidemiological surveillance. This review addresses phenotypic, proteomic, and molecular methods for species- and clone-level identification, highlighting their advantages and limitations. Classical methods, including selective media, Burkholderia cepacia selective agar (BCSA), Oxidation-Fermentation Polymyxin Bacitracin Lactose agar (OFPBLA), and Pseudomonas cepacia agar (PCA), and biochemical tests are useful for initial screening but have limited resolution for differentiating closely related Bcc species. Automated systems, such as VITEK® 2, provide rapid genus-level identification, whereas Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Fourier Transform Infrared Spectroscopy (FTIR) improve accuracy for species differentiation and clone typing, depending on the quality and currency of reference databases. Molecular methods, including Multilocus Sequence Typing (MLST), which sequences housekeeping genes such as gyrB and recA , and Whole-Genome Sequencing (WGS), offer high-resolution species- and clone-level identification, enabling detailed epidemiological monitoring. WGS represents the current gold standard, providing comprehensive information on identification, antimicrobial resistance, virulence factors, and clone typing. A tiered strategy is recommended, combining accessible methods for initial screening with advanced techniques for research centers and genomic surveillance. This study emphasizes the critical need for methodological standardization to enhance clinical diagnosis, epidemiological surveillance, and management of infections caused by species of the genus Burkholderia .

Accurate and reproducible descriptions of novel prokaryotic taxa are essential for understanding their evolutionary relationships and ecological roles. This editorial review provides detailed guidelines for standardizing the description of novel taxa, in alignment with the International Code of Nomenclature of Prokaryotes (ICNP). The recommendations focus on essential requirements for documentation, designation, and nomenclature of prokaryotic taxa. This structured approach supports researchers in producing consistent, well-defined descriptions that meet current standards for valid publication and effective classification of novel prokaryotic taxa.

The characterization of environmental Pseudomonas spp. from pharmaceutical industries is a relatively underexplored area of research. This study used 40 isolates of Pseudomonas from a pharmaceutical company that had been presumptively identified as Pseudomonas aeruginosa by VITEK^®2. The isolates were characterized using 16S rRNA sequencing, Multilocus Sequence Typing (MLST), Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass (MALDI–TOF MS), Fourier Transform Infrared (FT-IR) spectroscopy, ERIC-PCR, antimicrobial susceptibility profiling, and biofilm formation assessment on stainless steel surfaces. Twenty isolates were confirmed as P. aeruginosa. Sixteen isolates were only identified to the genus level of Pseudomonas, and the remaining four isolates were identified as Enterococcus faecalis (n = 2), Proteus spp. (n = 1), and Staphylococcus saprophyticus subsp. bovis/S. edaphicus/S. saprophyticus subsp. saprophyticus/S. pseudoxylosus/S. xylosus/S. caeli/S. ureilyticus. Typing of the 20 P. aeruginosa strains yielded 18 distinct FT-IR profiles and 19 ERIC-PCR profiles. The MLST analysis identified eight new sequence types (ST4292–ST4299). All strains were classified as multidrug-resistant (MDR), exhibiting resistance to multiple antimicrobial classes. Biofilm formation was observed in 28 (77.8%) Pseudomonas spp. strains on polystyrene surfaces, classified as moderately or strongly adherent, while all P. aeruginosa strains formed biofilms on stainless steel. Of the three disinfectants tested, sodium hypochlorite at 0.01% showed the best performance, reducing or eliminating biofilm formation in 24 (85.7%) strains. The regular evaluation of disinfection effectiveness in pharmaceutical industries is essential, as the presence of biofilm-forming strains can compromise production and contaminate final products.