The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.

Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacEΔ1, a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacEΔ1. The qacEΔ1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacEΔ1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacEΔ1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacEΔ1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa. As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacEΔ1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.

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This study aimed to assess the microbiological quality of natural mineral waters commercialized in 20 L returnable packs in Brazil by investigating bacteria and virus's presence in packs with different time of manufacturing (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 II. Regarding brands, 11 (73.3%) presented unsatisfactory results for at least one of the lots analyzed. P. aeruginosa analysis revealed six sequence types (ST), 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 Tm 0–1 year and Tm 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.

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping)

Pseudomonas aeruginosa é um patógeno oportunista associado a doenças transmitidas por águas e, possui a capacidade de formar biofilme em superfícies. Neste estudo, seis cepas de P. aeruginosa isoladas de águas minerais naturais em embalagens reutilizáveis de 20L foram submetidas à caracterização molecular por ERIC-PCR e avaliadas quanto à formação de biofilme. O ERIC-PCR revelou que as seis cepas apresentaram perfis genotípicos distintos. Nenhuma cepa apresentou formação de biofilme na matriz aço inox. Na matriz poliestireno houve formação de biofilme e a exposição ao hipoclorito de sódio a nas concentrações de 1% e 2% eliminou o biofilme formado. Contudo, na concentração 0,02% o biofilme formado não foi eliminado mesmo após 60 min de contato. O consumo de águas minerais contaminadas por P. aeruginosa e a possibilidade de formação de biofilmes em bebedouros de plástico podem representar um risco para indivíduos pertencentes ao grupo de risco, como grávidas, imunossuprimidos e idosos.

No abstract