Encapsulation of Cinnamaldehyde and Vanillin as a Strategy to Increase Their Antimicrobial Activity.

Many studies have suggested that the encapsulation of natural antimicrobials increases their antimicrobial activity. In this sense, the objective was to study the inactivation of microorganisms with encapsulated cinnamaldehyde and vanillin (E-CIN and E-VN), in comparison with the unencapsulated antimicrobials (CIN and VN) in protein beverages. Additionally, the microbial response was quantified through mathematical modeling. Cinnamaldehyde and vanillin were encapsulated using whey protein concentrate (WPC) as the encapsulating agent. The effectiveness at inactivating Escherichia coli, Listeria innocua, and Saccharomyces cerevisiae was evaluated in a protein-apple juice beverage during storage (4 °C). Encapsulation increased the effectiveness of cinnamaldehyde, reaching reductions of 1.8, 3.3, and 5.3 log CFU/mL in E. coli, L. innocua, and S. cerevisiae, respectively, while vanillin encapsulation had little effect on antimicrobial activity, reducing by 0.5, 1.4, and 1.1 log cycles, respectively. The combined treatments (E-CIN + E-VN) had an additive effect in reducing E. coli and a synergistic effect against S. cerevisiae. The Gompertz model was more versatile and better described the biphasic curves, whereas the Weibull model complemented the information regarding the spectrum of resistances within the microbial population. In conclusion, the encapsulation of cinnamaldehyde with WPC enhanced its activity. However, further studies are necessary to improve the antimicrobial activity of vanillin.

Listeria innocua and serotypes of Listeria monocytogenes isolated from clinical cases in small ruminants in the northwest of Uruguay / Listeria innocua e serotipos do Listeria monocytogenes isoladas dos casos clínicos em pequenos ruminantes no noroeste do Uruguai

Listeriosis is an infectious disease caused by bacteria of the genus Listeria, the neurological form being more common in ruminants. There are many reports of listeriosis in small ruminants in the region that includes Brazil, Argentina and Uruguay. However, these diagnoses were mainly based on histological lesions in the central nervous system (CNS) without the isolation and characterization of the involved Listeria strains. The aim of this study was to report sheep and goats listeriosis cases from 2016 to 2021 in northwestern Uruguay. The diagnosis was made according to lesions observed at histopathology, plus Listeria isolation in CNS, identifying it at specie and serotype level. Nine animals (n=9) of three outbreaks and five sporadic cases of listeriosis were studied. Sheep was the species with more cases in relation to goats, and adults were the category most affected. Cases occurred in spring and less frequently in winter. All presented neurological clinical signs and the lesions in the CNS were consistent with suppurative meningoencephalitis and micro-abscesses in the brainstem. In eight of nine CNS samples, Listeria strains were isolated (seven L. monocytogenes and one L. innocua). All the L. monocytogenes isolates carried the inlA gene; serotyping showed that four strains belonged to serotype 1/2b, two isolates belonged to serotype 4b, and one to serotype 1/2a. Considering that listeriosis is a common disease in this region and the fact that isolates are scarcely recovered from small ruminants, it would be important to emphasize the need for Listeria isolation to better characterize the strains that affect animals. Not only to improve knowledge about the epidemiology of disease but also with the objective of developing serotype specific vaccines for animal use.

Listeriose uma doença bacteriana causada pelo gênero Listeria, a forma nervosa é a mais comum em ruminantes. No Brasil, Argentina e Uruguai há vários relatos de listeriose em pequenos ruminantes com diagnóstico baseado na histopatologia do sistema nervoso central (SNC), sem o isolamento e a caracterização do agente. O objetivo deste trabalho foi relatar uma série de casos diagnosticados em ovinos e caprinos no período 2016-2021 no noroeste do Uruguai. O diagnóstico foi feito basado nas lesões observadas na histopatologia, e caracterização das cepas de Listeria recuperadas do SNC quanto à espécie e sorotipo. Nove animais (n=9) do três surtos e cinco casos isolados de listeriose foram estudados. Os ovinos foram a espécie com o maior número de casos em relação aos caprinos, sendo os animais adultos a categoria mais afetada em ambas espécies. A doença ocorreu principalmente na primavera com alguns casos observados no inverno. Todos os casos apresentavam sinais clínicos nervosos e as lesões no SNC caracterizavam-se por meningoencefalite supurativa com presença de microabscessos no tronco encefálico. Em oito de nove amostras do SNC foram isoladas cepas de Listeria (sete L. monocytogenes e uma L. innocua). Todos os isolados de L. monocytogenes continham o gene inlA; a sorotipagem apresentou quatro cepas do serotipo 1/2b, duas cepas serotipo 4b e uma cepa 1/2a. Levando em consideração que nesta região a listeriose é uma doença frequente e que existem poucos isolados recuperados de casos clínicos em pequeño ruminantes, torna-se relevante o isolamento deste agente para caracterização das cepas que afetam os animais. Não só para melhorar o conhecimento sobre a epidemiologia da doença, mas também com o objetivo de desenvolver vacinas sorotipo-especificas para uso animal.

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.

Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry.

Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar), in a range from 1 to 4 µg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics.

Effect of citral and carvacrol on the susceptibility of Listeria monocytogenes and Listeria innocua to antibiotics.

UNLABELLED: The aim of this study was to evaluate the antibiotic susceptibility of Listeria innocua (L. innocua) and Listeria monocytogenes (L. monocytogenes) cells in the presence of citral and carvacrol at sublethal concentrations in an agar medium. The presence of terpenes in the L. monocytogenes and L. innocua culture medium provided a reduction in the minimal inhibitory concentration (MIC) of all the antibiotics tested. These effects were dependent on the concentration of terpenes present in the culture medium. The combination of citral and carvacrol potentiated antibiotic activity by reducing the MIC values of bacitracin and colistin from 32.0 and 128.0 µg ml⁻¹ to 1.0 and 2.0 µg ml⁻¹, respectively. Thus, both Listeria species became more susceptible to these drugs. In this way, the colistin and bacitracin resistance of L. monocytogenes and L. innocua was reversed in the presence of terpenes. Results obtained in this study show that the phytochemicals citral and carvacrol potentiate antibiotic activity, reducing the MIC values of cultured L. monocytogenes and L. innocua. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytochemicals citral and carvacrol potentiate antibiotic activity of erythromycin, bacitracin and colistin by reducing the MIC values of cultured Listeria monocytogenes and Listeria innocua. This effect in reducing the MIC values of the antibiotics tested in both micro-organisms was increased when natural antimicrobials were combined. This finding indicated that the combination among terpenes and antibiotic may contribute in reducing the required dosage of antibiotics due to the possible effect of terpenes on permeation barrier of the micro-organism cell membrane.

Biological activities of a mixture of biosurfactant from Bacillus subtilis and alkaline lipase from Fusarium oxysporum

In this study, we investigate the antimicrobial effects of a mixture of a biosurfactant from Bacillus subtilis and an alkaline lipase from Fusarium oxysporum (AL/BS mix) on several types of microorganisms, as well as their abilities to remove Listeria innocua ATCC 33093 biofilm from stainless steel coupons. The AL/BS mix had a surface tension of around 30 mN.m-1, indicating that the presence of alkaline lipase did not interfere in the surface activity properties of the tensoactive component. The antimicrobial activity of the AL/BS mix was determined by minimum inhibitory concentration (MIC) micro-assays. Among all the tested organisms, the presence of the mixture only affected the growth of B. subtilis CCT 2576, B. cereus ATCC 10876 and L. innocua. The most sensitive microorganism was B. cereus (MIC 0.013 mg.mL-1). In addition, the effect of the sanitizer against L. innocua attached to stainless steel coupons was determined by plate count after vortexing. The results showed that the presence of the AL/BS mix improved the removal of adhered cells relative to treatment done without the sanitizer, reducing the count of viable cells by 1.72 log CFU.cm-2. However, there was no significant difference between the sanitizers tested and an SDS detergent standard (p<0.05).

Biological Activities of a Mixture of Biosurfactant from Bacillus subtilis and Alkaline Lipase from Fusarium oxysporum.

In this study, we investigate the antimicrobial effects of a mixture of a biosurfactant from Bacillus subtilis and an alkaline lipase from Fusarium oxysporum (AL/BS mix) on several types of microorganisms, as well as their abilities to remove Listeria innocua ATCC 33093 biofilm from stainless steel coupons. The AL/BS mix had a surface tension of around 30 mN.m(-1), indicating that the presence of alkaline lipase did not interfere in the surface activity properties of the tensoactive component. The antimicrobial activity of the AL/BS mix was determined by minimum inhibitory concentration (MIC) micro-assays. Among all the tested organisms, the presence of the mixture only affected the growth of B. subtilis CCT 2576, B. cereus ATCC 10876 and L. innocua. The most sensitive microorganism was B. cereus (MIC 0.013 mg.mL(-1)). In addition, the effect of the sanitizer against L. innocua attached to stainless steel coupons was determined by plate count after vortexing. The results showed that the presence of the AL/BS mix improved the removal of adhered cells relative to treatment done without the sanitizer, reducing the count of viable cells by 1.72 log CFU.cm(-2). However, there was no significant difference between the sanitizers tested and an SDS detergent standard (p<0.05).

Biological activities of a mixture of biosurfactant from Bacillus subtilis and alkaline lipase from Fusarium oxysporum

In this study, we investigate the antimicrobial effects of a mixture of a biosurfactant from Bacillus subtilis and an alkaline lipase from Fusarium oxysporum (AL/BS mix) on several types of microorganisms, as well as their abilities to remove Listeria innocua ATCC 33093 biofilm from stainless steel coupons. The AL/BS mix had a surface tension of around 30 mN.m-1, indicating that the presence of alkaline lipase did not interfere in the surface activity properties of the tensoactive component. The antimicrobial activity of the AL/BS mix was determined by minimum inhibitory concentration (MIC) micro-assays. Among all the tested organisms, the presence of the mixture only affected the growth of B. subtilis CCT 2576, B. cereus ATCC 10876 and L. innocua. The most sensitive microorganism was B. cereus (MIC 0.013 mg.mL-1). In addition, the effect of the sanitizer against L. innocua attached to stainless steel coupons was determined by plate count after vortexing. The results showed that the presence of the AL/BS mix improved the removal of adhered cells relative to treatment done without the sanitizer, reducing the count of viable cells by 1.72 log CFU.cm-2. However, there was no significant difference between the sanitizers tested and an SDS detergent standard (p 0.05).

Listeria sp. presence in Brazilian colonial cheese: potential risk to public health / Presença de Listeria sp. em queijos tipo colonial no Brasil: risco potencial para a saúde pública

This work was carried out aiming to isolate microorganisms belonging to the Listeria genus. For that purpose, 161 samples of commercial colonial cheese obtained in several geographic regions of Rio Grande do Sul, from 2003 to 2006, were bacteriologicaly examined according to the official protocol of Brazilian Ministry of Agriculture. Isolated of Listeria sp. from 12 samples were examined regarding to phenotypic characteristics based on colonial morphology, elective culture media and blood agar, type of hemolysis, sugar fermentation and CAMP test. Listeria innocua was the most frequent isolate from 10 cheese samples, followed by L. monocytogenes which was isolated from 2 samples.

Este trabalho foi conduzido com o objetivo de isolar microrganismos do gênero Listeria. Para tanto foram analisadas 161 amostras de queijos comercializados em diversos pontos de venda do Estado do Rio Grande do Sul, no período compreendido entre os anos 2003 e 2006. As análises microbiológicas para o isolamento do agente seguiram a metodologia oficial do Ministério da Agricultura Pecuária e Abastecimento do Brasil. Em 12 amostras bacterianas inicialmente classificadas como pertencentes ao gênero Listeria foram observadas as características relativas à morfologia colonial em meios seletivos e Ágar-sangue, morfologia e coloração dos microrganismos, fermentação de açúcares e reação no CAMP teste. Os resultados obtidos mostraram que a espécie Listeria innocua foi a mais prevalente, tendo sido isolada de 10 amostras, seguida de L. monocytogenes, isolada em duas amostras.

Listeria sp. presence in Brazilian colonial cheese: potential risk to public health / Presença de Listeria sp. em queijos tipo colonial no Brasil: risco potencial para a saúde pública

This work was carried out aiming to isolate microorganisms belonging to the Listeria genus. For that purpose, 161 samples of commercial colonial cheese obtained in several geographic regions of Rio Grande do Sul, from 2003 to 2006, were bacteriologicaly examined according to the official protocol of Brazilian Ministry of Agriculture. Isolated of Listeria sp. from 12 samples were examined regarding to phenotypic characteristics based on colonial morphology, elective culture media and blood agar, type of hemolysis, sugar fermentation and CAMP test. Listeria innocua was the most frequent isolate from 10 cheese samples, followed by L. monocytogenes which was isolated from 2 samples.

Este trabalho foi conduzido com o objetivo de isolar microrganismos do gênero Listeria. Para tanto foram analisadas 161 amostras de queijos comercializados em diversos pontos de venda do Estado do Rio Grande do Sul, no período compreendido entre os anos 2003 e 2006. As análises microbiológicas para o isolamento do agente seguiram a metodologia oficial do Ministério da Agricultura Pecuária e Abastecimento do Brasil. Em 12 amostras bacterianas inicialmente classificadas como pertencentes ao gênero Listeria foram observadas as características relativas à morfologia colonial em meios seletivos e Ágar-sangue, morfologia e coloração dos microrganismos, fermentação de açúcares e reação no CAMP teste. Os resultados obtidos mostraram que a espécie Listeria innocua foi a mais prevalente, tendo sido isolada de 10 amostras, seguida de L. monocytogenes, isolada em duas amostras.