Growth kinetics of Escherichia coli O157: H7 on the epicarp of fresh vegetables and fruits

Despite the increasing reports on the incidence of fresh vegetables and fruits as a possible vehicle for human pathogens, there is currently limited knowledge on the growth potential of Escherichia coli O157:H7 on different plant substrates. This study analyzed the selective adhesion and growth of E. coli O157:H7 on chili habanero (Capsicum chinense L.), cucumber (Cucumis sativus), radish (Raphanus sativus), tomato (Lycopersicon esculentum), beet (Beta vulgaris subsp. vulgaris), and onion (Allium cepa L.) under laboratory conditions. The Gompertz parameters were used to determine the growth kinetics. Scanning electron microscopy was used to visualize the adhesion of E. coli O157:H7 on the epicarp of the samples. Predictive models were constructed to compare the growth of E. coli O157:H7 on the samples with different intrinsic factors and to demonstrate the low selectivity of the pathogen. No significant difference was observed in the lag-phase duration (LPD), generation time (GT), and exponential growth rate (EGR) of the pathogen adhered to the samples. The interaction between the microorganism and the substrate was less supportive to the growth of E. coli O157:H7 for onion, whereas for tomato and cucumber, the time for the microorganism to attain the maximum growth rate (M) was significantly longer than that recorded for other samples.(AU)

Qualidade microbiológica da carcaça bovina durante o processo de abate e a ocorrência de e. Coli o157 h7 na carne / Microbiological quality of cattle carcass during harvest process and the occurrence of e. Coli o157 h7 in meat

A Escherichia coli O157: H7 é uma importante cepa associada a surtos graves de enfermidade em seres humanos, a maioria deles derivada do consumo de carne crua ou mal cozida. É provável que o gado atue como um importante reservatório, sugerindo-se a possibilidade de que a gestão da dieta no confinamento possa influenciar o aparecimento de cepas Shigatoxigênicas. Este estudo teve como objetivo verificar a qualidade microbiológica das carcaças e a ocorrência de E. coli O157: H7, por meio dos resultados obtidos por métodos indicadores (contagem total de microrganismos viáveis, contagem de Coliformes e de E. coli) e por um método automatizado de PCR para detecção de E. coli O157: H7. Foram colhidas amostras de retalhos de carne (carne industrial) e de carcaças de bovinos terminados em pastagem ou em confinamento, permitindo o fornecimento de subsídios necessários para a Análise de Perigos e Pontos Críticos de Controle (HACCP). Desses mesmos animais foram colhidas, também, amostras de swab retal para a detecção experimental de E. coli O157: H7 nas fezes. Um total de 100 swabs retais, 100 amostras de carcaças quentes, além de outras 323 amostras de aparas de carne (retalhos da desossa), foram analisados. Com exceção de uma amostra de retalhos de carne (0,31%), todas as demais, de fezes e de carcaças, foram negativas para a presença de E. coli O157: H7. Não houve diferenças significativas entre os tipos de terminação utilizada para o gado. Os resultados dos métodos indicadores foram considerados aceitáveis em 91%, 85% e 93% das amostras testadas, respectivamente, para a CTV, contagem de Coliformes e de E. coli de carcaças, dando suporte e em acordo com a baixa ocorrência da cepa O157: H7

Escherichia coli O157:H7, an important bacillus strain associated with serious gastroenteritis in humans, is more frequently derived from the consumption of raw or poorly cooked beef. Cattle are important reservoirs suggesting the possibility that feedlot diet management influences the emergence of Shiga-toxigenic strains. This study evaluates the microbiological quality of carcasses and the occurrence of E. coli O157:H7 using the results from general indicator methods (total viable count, coliform rate and E. coli counts) and by an automated PCR method for the detection of E. coli O157:H7. Samples were collected from (industrially processed) meat trimmings and from carcasses of cattle finished on pasture or in feedlots so that sufficient data for the Hazard Analysis and Critical Control Points (HACCP) could be obtained. Samples of rectal swab for experimental detection of E. coli O157:H7 were also collected. One hundred rectal swabs, 100 samples retrieved from warm carcasses and 323 samples of meat trimmings were analyzed. With the exception of one sample of meat trim (0.31%), all the other samples from excreta and carcasses were negative for the O157:H7 E. coli strain. There were no significant differences between the methods used for cattle finishing. Indicator methods results were considered acceptable in 91%, 85% and 93% of tested samples of carcasses respectively for TVC, coliform and E. coli counts. These results agree with statistical data showing the low occurrence of O157:H7 strain

Qualidade microbiológica da carcaça bovina durante o processo de abate e a ocorrência de e. Coli o157 h7 na carne / Microbiological quality of cattle carcass during harvest process and the occurrence of e. Coli o157 h7 in meat

A Escherichia coli O157: H7 é uma importante cepa associada a surtos graves de enfermidade em seres humanos, a maioria deles derivada do consumo de carne crua ou mal cozida. É provável que o gado atue como um importante reservatório, sugerindo-se a possibilidade de que a gestão da dieta no confinamento possa influenciar o aparecimento de cepas Shigatoxigênicas. Este estudo teve como objetivo verificar a qualidade microbiológica das carcaças e a ocorrência de E. coli O157: H7, por meio dos resultados obtidos por métodos indicadores (contagem total de microrganismos viáveis, contagem de Coliformes e de E. coli) e por um método automatizado de PCR para detecção de E. coli O157: H7. Foram colhidas amostras de retalhos de carne (carne industrial) e de carcaças de bovinos terminados em pastagem ou em confinamento, permitindo o fornecimento de subsídios necessários para a Análise de Perigos e Pontos Críticos de Controle (HACCP). Desses mesmos animais foram colhidas, também, amostras de swab retal para a detecção experimental de E. coli O157: H7 nas fezes. Um total de 100 swabs retais, 100 amostras de carcaças quentes, além de outras 323 amostras de aparas de carne (retalhos da desossa), foram analisados. Com exceção de uma amostra de retalhos de carne (0,31%), todas as demais, de fezes e de carcaças, foram negativas para a presença de E. coli O157: H7. Não houve diferenças significativas entre os tipos de terminação utilizada para o gado. Os resultados dos métodos indicadores foram considerados aceitáveis em 91%, 85% e 93% das amostras testadas, respectivamente, para a CTV, contagem de Coliformes e de E. coli de carcaças, dando suporte e em acordo com a baixa ocorrência da cepa O157: H7(AU)

Escherichia coli O157:H7, an important bacillus strain associated with serious gastroenteritis in humans, is more frequently derived from the consumption of raw or poorly cooked beef. Cattle are important reservoirs suggesting the possibility that feedlot diet management influences the emergence of Shiga-toxigenic strains. This study evaluates the microbiological quality of carcasses and the occurrence of E. coli O157:H7 using the results from general indicator methods (total viable count, coliform rate and E. coli counts) and by an automated PCR method for the detection of E. coli O157:H7. Samples were collected from (industrially processed) meat trimmings and from carcasses of cattle finished on pasture or in feedlots so that sufficient data for the Hazard Analysis and Critical Control Points (HACCP) could be obtained. Samples of rectal swab for experimental detection of E. coli O157:H7 were also collected. One hundred rectal swabs, 100 samples retrieved from warm carcasses and 323 samples of meat trimmings were analyzed. With the exception of one sample of meat trim (0.31%), all the other samples from excreta and carcasses were negative for the O157:H7 E. coli strain. There were no significant differences between the methods used for cattle finishing. Indicator methods results were considered acceptable in 91%, 85% and 93% of tested samples of carcasses respectively for TVC, coliform and E. coli counts. These results agree with statistical data showing the low occurrence of O157:H7 strain(AU)

Survival of escherichia coli o157: h7 co-cultured with different levels of pseudomonas fluorescens and lactobacillus plantarum on fresh beef

The purpose of this study was to investigate the effect of different levels of Pseudomonas fluorescens (10² and 10(6)log10 cfu/ml)and Lactobacillus plantarum (10² and 10(4)log10 cfu/ml)on the growth of Escherichia coli O157:H7 on beef loins. Beef loins inoculated with E. coli O157:H7 and P. fluorescens were aerobically stored for 7 days at 4 ºC, while those inoculated with E. coli O157:H7 and L. plantarum were vacuum packaged and stored for 8 weeks at 4 ºC. Aerobic Plate Counts (APC), E. coli O157:H7 and either P. fluorescens or L. plantarum counts were determined at different storage intervals. For the aerobically packaged beef loins, E. coli O157:H7 was detected throughout the 7 day storage period regardless of the P. fluorescens level in the inoculum. For the vacuum packaged beef loins, similar inoculum levels of E. coli O157:H7 and L. plantarum allowed E. coli O157:H7 to survive until week 5 of storage, while a higher inoculum level of L. plantarum inhibited E. coli O157:H7 from week 3. Once fresh beef has been contaminated with E. coli O157:H7, the level of P. fluorescens in the background flora does not inhibit its survival and growth. However, under vacuum storage, the application of L. plantarum as a biopreservative inhibits the survival of E. coli O157:H7 on beef. The higher the level of L. plantarum in the system, the earlier the onset of the inhibition. Farmers and abattoirs have to strengthen preventive strategies to eliminate contamination of beef carcasses with E. coli O157:H7.(AU)

Antimicrobial activity of essential oils against Vancomycin-Resistant enterococci (VRE) and Escherichia coli O157:H7 in feta soft cheese and minced beef meat

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2% (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25% and 0.5%, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2%. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10³ cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1%, 0.5% and 1%) of the EO and stored at 7 ºC for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1% caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 ºC. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

Antimicrobial activity of essential oils against Vancomycin-Resistant enterococci (VRE) and Escherichia coli O157:H7 in feta soft cheese and minced beef meat

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2% (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25% and 0.5%, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2%. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10³ cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1%, 0.5% and 1%) of the EO and stored at 7 ºC for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1% caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 ºC. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

ÓLEO ESSENCIAL DE ALHO, ISOTIOCIANATO DE ALILA E NISINA Z COMO CONSERVANTES NATURAIS EM LINGUIÇA MISTA FRESCAL

O objetivo do presente estudo foi avaliar a atividade antimicrobiana sobre bactérias patogênicas e deteriorantes da associação de compostos naturais (óleo essencial de alho - OA, isotiocianato de alila - ITA e nisina Z pura - NI) in vitro e sua aplicação na estabilidade de linguiça mista frescal. Na avaliação in vitro foram determinadas a Concentração Inibitória Mínima (CIM) e a Concentração Bactericida Mínima (CBM) dos compostos em combinação sobre E. coli O157:H7 e Lactobacillus plantarum. Para avaliação do efeito dos compostos naturais em linguiça foram produzidos 5 lotes divididos nos seguintes tratamentos: T1 = 20 mg/kg NI, T2 = 125 µL/kg OA + 62,5 µL/kg ITA + 20 mg/kg NI e T3 = 62,5 µL/kg OA + 125 µL/kg ITA + 20 mg/kg NI. Nesses tratamentos, as massas de linguiça foram inoculadas com E. coli O157:H7. Além desses, foram produzidos dois lotes controle, sendo um controle positivo (CP), inoculado apenas com E. coli O157:H7 e um controle negativo (CN), sem inoculação bacteriana. As linguiças foram armazenadas a 6ºC durante 20 dias e a cada 5 dias, amostras foram avaliadas quanto a parâmetros microbiológicos (contagem de E. coli O157:H7, E. coli autóctones, aeróbios psicrotróficos e bactérias lácticas) e físico-químicos (pH e cor). Posteriormente, o lote controle negativo e os tratamentos T2 e T3, sem a inoculação de E. coli O 157:H7 foram submetidos à avaliação sensorial de aceitabilidade. Os resultados foram analisados por ANOVA e teste de Tukey (P>0,05). Na associação in vitro de NI com AO e ITA verificou-se redução significativa de E. coli O157:H7 nas concentrações de 0,0125 µL/mL AO + 0,00625 µL/mL ITA + 0,24 mg/mL NI e de L. plantarum nas concentrações de 0,00625 µL/mL AO + 0,0125 µL/mL ITA + 0,24 mg/mL NI. Em linguiça, as reduções na contagem de E. coli O 157:H7, ao final da estocagem, nos T2 e T3 foram de 2,06 e 2,12 log UFC/g, respectivamente, em comparação ao CP. Para a contagem de E. coli autóctone verificou-se que os tratamentos T2 e T3 apresentaram reduções do patógeno a níveis indetectáveis a partir do 10º dia de armazenamento. Em relação à contagem de bactérias lácticas, todos os tratamentos (T1, T2 e T3) apresentaram redução significativa em comparação aos lotes controle (CP e CN), a partir do 5º dia de estocagem (P<0,05). Para os microrganismos psicrotróficos, nenhum dos tratamentos apresentou efeito inibitório significativo. Com relação à cor instrumental, verificaram-se menores valores de chroma (C*) para as linguiças dos T2 e T3 (P<0,05) ao final da estocagem. Contudo, as linguiças adicionadas de antimicrobianos (T1, T2 e T3) apresentaram menor descoloração (hue) e maior valor para cor vermelha (a*) em comparação aos lotes controle. As linguiças tratadas com a combinação dos antimicrobianos (T2 e T3) apresentaram menor pH em comparação aos controles. Quanto à análise sensorial, as linguiças adicionadas dos antimicrobianos naturais apresentaram diferença significativa para o controle para os atributos avaliados. Contudo, apresentaram índice de aceitabilidade acima de 70% para a maioria dos atributos. As combinações de óleos essenciais e nisina Z testadas foram eficientes para a redução significativa da população de E. coli O 157:H7 em linguiça mista frescal em comparação aos grupos controles. As combinações desses antimicrobianos também se mostraram efetivas em manter a estabilidade da linguiça mista frescal pelo controle do crescimento de bactérias lácticas deteriorantes e pela manutenção da cor vermelha e do pH das linguiças, apresentando-se sensorialmente aceitas pelos provadores.

The objective of the present study was to evaluate the antimicrobial activity of pathogenic and spoilage bacteria using the association of natural compounds (garlic essential oil - OA, allyl isothiocyanate - ITA and pure nisin Z - NI) in vitro and its application in stability of fresh sausage. In the evaluation in vitro were determined the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the compounds in combination against E. coli O157: H7 and L. plantarum. To evaluate the effect of natural compounds in sausage, five lots were produced and divided into the following treatments: T1 = 20 mg/kg NI, T2 = 125 µL/kg OA + 62.5 µL/kg ITA + 20 mg/kg NI and T3 = 62.5 µL/kg OA + 125 µL/kg ITA + 20 mg/kg NI. In these treatments, sausage mixtures were inoculated with E. coli O157: H7. In addition, two control sausages were produced - a positive control (CP) inoculated only with E. coli O157: H7 and a negative control (CN) without bacterial inoculation. Sausages were stored at 6 °C for 20 days and samples were evaluated for microbiological (E. coli O157:H7, native E. col, aerobic psychrotrophic and lactic acid bacteria) and physico-chemical parameters (pH and color) every 5 days. Subsequently, the negative control, T2 and T3 without inoculation of E. coli O157:H7 were subjected to sensory evaluation acceptability. Results were analyzed by ANOVA and Tukey's test (P>0.05). In association of NI with AO and ITA, E. coli O157: H7 in vitro was significantly reduced at concentrations of 0.0125 µL/mL AO + 0.00625 µL/mL ITA + 0.24 mg/mL NI and L. plantarum at concentrations of 0.00625 µL / ml AO + 0.0125 µL / mL ITA + 0.24 mg/mL NI. E. coli O157:H7 reduction in sausage, at the end of storage was 2.06 and 2.12 log CFU/g on T2 and T3 when compared to CP. Native E. coli count on T2 and T3 showed undetectable reduction levels from the 10th day of storage. All treatments (T1, T2 and T3) showed significant reduction on lactic acid bacteria levels when compared to the control groups (CP and CN), from the 5th day of storage (P <0.05). None of the treatments had an effect on psychrotrophic bacteria. Smaller C * values for instrumental color were detected on sausages of T2 and T3 (P <0.05) at end of storage. However, the treatments (T1, T2 and T3) showed less discoloration (hue) and larger value for red color (a *) in comparison to the control groups. Sausages treated with combination of antimicrobials (T2 and T3) showed lower pH when compared to controls. On the sensory analysis, the natural antimicrobial added sausages showed a significant difference to the control for the evaluated attributes. However, they showed acceptability index above 70% for most of the attributes. The combinations of essential oils and nisin Z were efficient to reduce significantly the population of E. coli O157:H7 in fresh sausage compared to control groups. The combinations of these antimicrobials were also proven effective in maintaining the stability of the fresh sausage for controlling the growth of spoilage lactic acid bacteria and for maintaining the red color and the pH of the sausages, being wellaccepted by the panelists.

Survival and replication of Escherichia coli O157:H7 inside the mice peritoneal macrophages

The replication of Escherichia coli O157:H7 on the resident peritoneal macrophages of four mice strains (BALB/c, CD1, C57BL, and Swiss) has been investigated. Macrophagial bactericidal killing activity was estimated via studying their ability to internalize (gentamicin-protected) E. coli during 2, 4, 24, and 48 h assays. Host genetic background has been found to show no significant effect on the ability of resident peritoneal macrophages to kill E. coli O157:H7.

A multiplicação de Escherichia coli O157:H7 em macrófagos peritoniais residentes de quatro linhagens de camundongos (BALB/C, CD1, C57BL e Swiss) foi investigada. A capacidade bactericida dos macrófagos foi estimada através da avaliação de sua capacidade de internalizar E.coli (protegidos pela gentamicina) em ensaios de 2, 4, 24 e 48h. Observou-se que as características genéticas do hospedeiro não têm efeito significativo na capacidade dos macrófagos peritoniais eliminarem E. coli O157:H7.

Detecção de Escherichia coli O157: H7 inoculada experimentalmente em amostras de leite cru por método convencional e PCR multiplex / Detection of E. coli O157: H7 experimentally inoculated in raw milk samples by conventional method and multiplex PCR

Padronizou-se um método de reação em cadeia da polimerase (PCR) multiplex para detecção de Escherichia coli O157:H7 e avaliou-se a eficiência da PCR e de um método de cultivo convencional em placas na detecção desse patógeno experimentalmente adicionado em leite estéril e em leite cru com baixa contagem bacteriana total (média de 4,01 x 10³ UFC/ml) e com alta contagem bacteriana (média de 2,10 x 10(6) UFC/ml). Foram padronizadas duas reações de PCR com o uso dos primers: "A" (RfbF; RfbR e FLICh7F/FLICh7R) e "B" (SLT-IF/SLTIR e SLT-IIF/SLT-IIR). A detecção de E. coli O157:H7 (1UFC/ml) a partir do leite estéril e do leite cru com baixa contaminação bacteriana foi possível quando se utilizou o método de contagem em placas e a PCR. A sensibilidade dos dois métodos foi menor quando se testou o leite cru com alta contaminação microbiana, sendo o método convencional mais sensível. Os resultados indicam que a presença de outros microrganismos, em alta quantidade no leite, dificulta a detecção de E. coli O157:H7 pelos métodos utilizados.(AU)

This experiment was carried out in order to evaluate the effect of the raw milk bacterial count on the efficiency of a multiplex polymerase chain reaction and a conventional plate count method for detection of Escherichia coli O157:H7. This pathogen was experimentally inoculated into sterile milk, raw milk with low bacterial count (count mean of 4.01 x 10³ cfu/ml) and, raw milk with high bacterial count (mean 2.10 x 10(6) cfu/ml). Two protocols of PCR were standardized using primers "A" (Rfbf and Rfbr and FLICh7F/FLICh7R) and "B" (SLT-IF/SLTIR and SLT-IIF/SLT-IIR). Both conventional plate count and PCR methods were able to detect the presence of E. coli O157:H7 in either sterile milk or raw milk with low bacterial count initially inoculated with 1cfu of E. coli O157:H7 per ml. The sensibility of both methods for high-contaminated raw milk samples was lower, being the conventional approach more sensitive. These results indicate that high bacterial count in raw milk can affect E. coli O157:H7 detection.(AU)

Detecção de Escherichia coli O157:H7 em leite ARMAZENADO EM DIFERENTES CONDIÇÕES POR PCR do tipo Multiplex

Amostras de leite UHT integral armazenadas sob refrigeração e congelação e inoculadas com cepas de referência da Fundação Oswaldo Cruz foram utilizadas para avaliar a capacidade de detecção da E. coli O157:H7 pela PCR multiplex (mPCR), pela Contagem de Bactérias Heterotróficas Aeróbias Mesófilas (CBHAM) e pelo kit comercial RIDA® COUNT Escherichia coli/Coliformes. Foram utilizadas as seguintes cepas: Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 25923), Salmonella enterica Typhi (ATCC 6539) e E. coli enterohemorrágica (CDC EDL 933). As análises foram realizadas nos 4o e 7o dias de refrigeração e nos 15o, 30o, 60o e 120o dias de congelamento. Foi possível realizar a detecção da E. coli O157:H7 por meio da mPCR em todas as amostras contaminadas artificialmente. Os resultados confirmaram a alta sensibilidade da mPCR na detecção de E. coli O157:H7. Palavras-chave: Escherichia coli enterohemorrágica, Escherichia coli O157:H7, leite, PCR multiplex