Evaluation of VITEK® 2 and MALDI-TOF/MS automated methodologies in the identification of atypical Listeria spp. isolated from food in different regions of Brazil.

Listeria monocytogenes is a pathogen responsible for listeriosis, a foodborne disease with high mortality rates (20-30%). It mainly affects the elderly, pregnant women, and immunocompromised people. Although not pathogenic, the isolation and identification of Listeria innocua are critical since they can indicate L. monocytogenes' presence as they are closely related and widely distributed in the environment and food processing plants. The objective of this study was to evaluate the effectiveness of the automated methods VITEK® 2 and MALDI-TOF/MS in identifying 94 strains of the genus Listeria with atypical identification profile. The resulting identification by Polymerase Chain Reaction (PCR), using specific primers for the most common species of Listeria, was considered the correct identification and presented a total of 31 strains identified as Listeria innocua (LI), 54 as L. monocytogenes (LM), 8 as Listeria welshimeri (LW) and 1 as Listeria grayi (LG). The VITEK® 2 automated system correctly identified, on average, 79% of the LI strains, 16% of the LM strains, and 88.0% of the LW strains. In the analysis by MALDI-TOF/MS, on average, 73% of LM strains were correctly identified, few LW strains were correctly identified, and all LI strains were incorrectly identified. Both VITEK® 2 and MALDI-TOF/MS correctly identified the LG strain in both analyzes. The results demonstrate that automated methodologies could not discriminate atypical strains of the Listeria genus and point to the need for the use of complementary tests, such as PCR and chromogenic media, for the correct identification of these strains.

Endophytic Bacteria Isolated from Common Bean (Phaseolus vulgaris) Exhibiting High Variability Showed Antimicrobial Activity and Quorum Sensing Inhibition.

Endophytic bacteria play a key role in the biocontrol of phytopathogenic microorganisms. In this study, genotypic diversity was analyzed via repetitive element PCR (rep-PCR) of endophytic isolates of the phylum Actinobacteria that were previously collected from leaves of cultivars of common bean (Phaseolus vulgaris). Considerable variability was observed, which has not been reported previously for this phylum of endophytic bacteria of the common bean. Furthermore, the ethanol extracts from cultures of various isolates inhibited the growth of pathogenic bacteria in vitro, especially Gram-positive pathogens. Extracts from cultures of Microbacterium testaceum BAC1065 and BAC1093, which were both isolated from the 'Talismã' cultivar, strongly inhibited most of the pathogenic bacteria tested. Bean endophytic bacteria were also demonstrated to have the potential to inhibit the quorum sensing of Gram-negative bacteria. This mechanism may regulate the production of virulence factors in pathogens. The ability to inhibit quorum sensing has also not been reported previously for endophytic microorganisms of P. vulgaris. Furthermore, M. testaceum with capacity to inhibit quorum sensing appears to be widespread in common bean. The genomic profiles of M. testaceum were also analyzed via pulsed-field gel electrophoresis, and greater differentiation was observed using this method than rep-PCR; in general, no groups were formed based on the cultivar of origin. This study showed for the first time that endophytic bacteria from common bean plants exhibit high variability and may be useful for the development of strategies for the biological control of diseases in this important legume plant.

Avaliação do crescimento de Salmonella enterica em fórmulas lácteas infantis sob diferentes condições de preparo e armazenamento / Evaluation of the growth of Salmonella enterica in infant milk formulas under different preparation and storage conditions

Salmonella enterica é um dos mais importantes patógenos associados a fórmulas lácteas infantis (FLI). Neste trabalho, foi avaliada a sobrevivência e crescimento de uma estirpe padrão de Salmonella enterica sorotipo Typhi em duas FLI reconstituídas (F1 e F2) sob diferentes condições de preparo e armazenamento similares às utilizadas em lactários hospitalares. Para ambas as FLI inoculadas e incubadas à temperatura ambiente, observou-se um aumento de 3,3 log na população de S. enterica logo nas primeiras 24 horas de incubação e um aumento superior a 4,5 log ao término de 72 horas. A 4ºC, nas primeiras 48 h de incubação, houve leve aumento populacional (< 1,0 log) e, após 72 horas, o crescimento foi similar ao observado sob temperatura ambiente. Em relação ao aquecimento em banho-maria, verificou-se que a 60ºC/ 5 min houve uma redução de um pouco mais de 1 log UFC/ml, entretanto, a 60ºC/ 10 min, a queda observada foi de 2,8 log em F1 e de 2,3 log em F2. Já a 70ºC/ 5 min, ocorreu redução de cerca de 3 log UFC/ml para F1 e F2, enquanto que por 10 min, essa redução foi cerca de 1 log maior. O aquecimento em forno de micro-ondas mostrou ser a forma mais rápida e eficiente de redução da população de Salmonella nas FLI, uma vez que não foi detectada contagem celular após nenhum dos tratamentos utilizados. Os resultados sugerem que FLI contaminadas durante a etapa de preparo podem apresentar um crescimento bacteriano mesmo sob temperatura de refrigeração se mantidos por tempo prolongado e que determinados métodos de tratamento térmico não são suficientes para inibição completa de S. enterica.

Salmonella enterica is one of the most important pathogens associated with infant milk formulas (IMFs). In this study, the survival and growth of an S. enterica serovar Typhi strain in two reconstituted IMFs (F1 and F2) under different conditions of preparation and storage, similar to those used in hospital lactaries, was evaluated. For both IMFs inoculated at room temperature, there was 3.3 log increase in the population of S. enterica by the first 24 h of incubation and > 4.5 log increase at the end of 72 h. At 4ºC, in the first 48 h of incubation, there was a slight increase in population (< 1.0 log) and after 72 h, growth was similar to that observed at room temperature. A heated water bath was used to test the effect of thermal treatment methods on bacterial viability. At 60ºC for 5 min, there was a reduction of slightly more than 1 log CFU/mL; however, at 60ºC for 10 min, the observed decrease was 2.8 log for F1 and 2.3 log for F2. At 70ºC for 5 min, there was a reduction of approximately 3 log CFU/ml for both IMFs and for 10 min, this reduction was approximately 1 log higher. Heating in a microwave oven was the most efficient way of reducing populations of Salmonella in the IMFs, as bacterial cell counts were not detected after any of the treatments used. Our results suggest that IMF contamination during the preparation step may support bacterial growth even under refrigeration if kept for a prolonged time and that some thermal treatment methods are not sufficient for the complete inhibition of S. enterica.

Antibiotic resistance versus antimicrobial substances production by gram-negative foodborne pathogens isolated from minas frescal cheese: heads or tails?

In this study, 15 Gram-negative isolates from Minas Frescal cheese sold in commercial establishments in Rio de Janeiro, Brazil, were able to produce antimicrobial substances (AMSs). Seven, four, two, one, and one isolates identified as Yersinia, Acinetobacter, Enterobacter, Escherichia, and Hafnia genera, respectively, were considered potentially pathogenic. All 15 AMS(+) isolates were resistant to at least 1 antibiotic; however, 7 strains presented resistance to at least 3 antibiotics from different classes, exhibiting multiresistance profiles. The strains were also subjected to plasmid profile analysis. All isolates presented different plasmid forms with most ranging in size from 1 to 10 kb. Activity against various pathogens associated with food was tested and all 15 AMS(+) showed the same activity spectrum, inhibiting all Escherichia coli and Salmonella strains that were tested. Although restricted, the action spectrum of AMS-producing strains is extremely relevant to the food industry because Gram-negative bacteria such as E. coli and Salmonella spp. are most often associated with foodborne illnesses. The findings of this study reveal that even AMS produced by pathogens can have potential applications against other foodborne pathogens.

Isolation and characterization of endophytic bacteria isolated from the leaves of the common bean (Phaseolus vulgaris)

The common bean is one of the most important legumes in the human diet, but little is known about the endophytic bacteria associated with the leaves of this plant. The objective of this study was to characterize the culturable endophytic bacteria of common bean (Phaseolus vulgaris. leaves from three different cultivars (Vermelhinho, Talismã, and Ouro Negro) grown under the same field conditions. The density of endophytic populations varied from 4.5 x 10² to 2.8 x 10³ CFU g-1 of fresh weight. Of the 158 total isolates, 36.7% belonged to the Proteobacteria, 32.9% to Firmicutes, 29.7% to Actinobacteria, and 0.6% to Bacteroidetes. The three P. vulgaris cultivars showed class distribution differences among Actinobacteria, Alphaproteobacteria and Bacilli. Based on 16S rDNA sequences, 23 different genera were isolated comprising bacteria commonly associated with soil and plants. The genera Bacillus, Delftia, Methylobacterium, Microbacterium, Paenibacillus, Staphylococcus and Stenotrophomonas were isolated from all three cultivars. To access and compare the community structure, diversity indices were calculated. The isolates from the Talismã cultivar were less diverse than the isolates derived from the other two cultivars. The results of this work indicate that the cultivar of the plant may contribute to the structure of the endophytic community associated with the common bean. This is the first report of endophytic bacteria from the leaves of P. vulgaris cultivars. Future studies will determine the potential application of these isolates in biological control, growth promotion and enzyme production for biotechnology.