Performance of two alternative methods for Listeria detection throughout Serro Minas cheese ripening

ABSTRACT The ability of pathogens to survive cheese ripening is a food-security concern. Therefore, this study aimed to evaluate the performance of two alternative methods of analysis of Listeria during the ripening of artisanal Minas cheese. These methods were tested and compared with the conventional method: Lateral Flow System™, in cheeses produced on laboratory scale using raw milk collected from different farms and inoculated with Listeria innocua; and VIDAS®-LMO, in cheese samples collected from different manufacturers in Serro, Minas Gerais, Brazil. These samples were also characterized in terms of lactic acid bacteria, coliforms and physical-chemical analysis. In the inoculated samples, L. innocua was detected by Lateral Flow System™ method with 33% false-negative and 68% accuracy results. L. innocua was only detected in the inoculated samples by the conventional method at 60-days of cheese ripening. L. monocytogenes was not detected by the conventional and the VIDAS®-LMO methods in cheese samples collected from different manufacturers, which impairs evaluating the performance of this alternative method. We concluded that the conventional method provided a better recovery of L. innocua throughout cheese ripening, being able to detect L. innocua at 60-day, aging period which is required by the current legislation.

Chemical composition and antibacterial activities of seven Eucalyptus species essential oils leaves

BACKGROUND: In this paper, we have studied the essential oils chemical composition of the leaves of seven Eucalyptus species developed in Tunisia. Eucalyptus leaves were picked from trees growing in different arboretums in Tunisia. Choucha and Mrifeg arboretums located in Sedjnene, region of Bizerte (Choucha: E. maideni, E. astrengens et E. cinerea; Mrifeg : E. leucoxylon), Korbous arboretums located in the region of Nabeul, North East Tunisia with sub-humid bioclimate, (E. lehmani), Souiniet-Ain Drahem arboretum located in region of Jendouba (E. sideroxylon, E. bicostata). Essential oils were individually tested against a large panel of microorganisms includingStaphylococcus aureus (ATCC 6539), Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC29212), Listeria ivanovii (RBL 30), Bacillus cereus (ATCC11778). RESULTS: The yield of essential oils ranged from 1.2% to 3% (w/w) for the different Eucalyptus species. All essential oils contain α-pinene, 1,8-cineol and pinocarveol-trans for all Eucalyptus species studied. The 1,8-cineol was the major compound in all species (49.07 to 83.59%). Diameter of inhibition zone of essential oils of Eucalyptus species varied from 10 to 29 mm. The largest zone of inhibition was obtained for Bacillus cereus (E. astrengens) and the lowest for Staphylococcus aureus (E. cinerea). The essential oils from E. maideni, E. astrengens, E. cinerea (arboretum of Bizerte), E. bicostata(arboretum of Aindraham) showed the highest antibacterial activity against Listeria ivanovii and Bacillus cereus. CONCLUSION: The major constituents of Eucalyptus leaves essential oils are 1,8-cineol (49.07 to 83.59%) and α-pinene (1.27 to 26.35%). The essential oils from E. maideni, E. astrengens, E. cinerea, E. bicostatashowed the highest antibacterial activity against Listeria ivanovii and Bacillus cereus, they may have potential applications in food and pharmaceutical products.

Electroforesis en Gel de Campo Pulsado (PFGE) para la diferenciación molecular de Listeria monocytogenes / Pulsed-field gel electrophoresis (PFGE) for the molecular differentiation of Listeria monocytogenes

En Colombia no es obligatoria la notificación deL. monocytogenes en alimentos, pero se vigilan los alimentos dealto riesgo. Clínicamente se reportan como microorganismoGram-positivo sólo cuando causan meningitis. L. monocytogeneses un patógeno intracelular, transmitido por alimentos, letalpara humanos y animales, que causa Listeriosis; enfermedadque genera varios brotes en el mundo, con pérdidashumanas y económicas. Pocos trabajos en Colombia hanlogrado identificar y serotipificar molecularmente losaislamientos, lo que sólo permite distribuir teóricamentelos serotipos en linajes. Esta revisión se limita a mostrarcaracterísticas del patógeno, su importancia en salud públicay en la industria de alimentos, generalidades de la PFGECHEF;identificando el protocolo estandarizado de trabajoy las enzimas de restricción adecuadas para cortar el ADN.Se encontró que la combinación de enzimas XbaI-AscI,seguida de ApaI es la que ofrece mejores resultados en ladiferenciación de los aislamientos; agrupándolos por linajes;mostrando variaciones intra-serotipo y que en varios paíseslatinoamericanos se analizan los resultados a través dePulseNet, lo que garantiza la comparación de los patronesde PFGE en igualdad de condiciones...

The reporting of L. monocytogenes in food in Colombia is not a mandatory; however, foods consideredhigh-risk are monitored, and the organism is only reported clinically as Gram-positive when it causesmeningitis. L. monocytogenes is a foodborne, intracellular, pathogen which causes listeriosis, a disease lethalto humans and animals. Outbreaks of this disease worldwide can bring about human and economiclosses. Only a few studies in Colombia have been able to identify and molecularly serotype isolatesallowing only the theoretical distribution of serotypes by lineage. This review explains the characteristicsof the pathogen, its importance in public health and in the food industry, and provides an overview ofPFGE-CHEF; identifying the standard work protocol and the appropriate restriction enzymes to cutDNA. We found that the enzyme combination, XbaI-AscI, followed by ApaI offers the best results todifferentiate isolates, by grouping them by lineages, and displaying intra-serotype variations. Additionally,we found that in several Latin American countries the results are analyzed using PulseNet; this ensuresthe comparison of PFGE patterns in equivalent conditions...

Na Colômbia não há uma notificação compulsóriade L. monocytogenes em alimentos, mas alimentos de altorisco são monitorados. Clinicamente, são relatados comoorganismos Gram-positivos apenas quando eles causammeningite. L. monocytogenes é um patógeno intracelularde origem alimentar, letal para seres humanos e animais,que causa a listeriose, que gera surtos em todo o mundo,com perdas humanas e econômicas. Poucos trabalhos naColômbia identificaram e sorotipificaram molecularmenteos isolados, que só permite a distribuição de sorotiposteoricamente em linhagens. Esta avaliação é limitada amostrar características do patógeno, sua importância nasaúde pública e na indústria de alimentos, e uma visãogeral do PFGE-CHEF; identificar o protocolo-padrão detrabalho e enzimas de restrição apropriadas para cortar oADN. Verificou-se que a combinação de enzimas XbaIAscI,seguido por ApaI representa a combinação de enzimasque ofereceu melhores resultados na diferenciação dosisolados, agrupando-a por linhagens, mostrando a variaçãointra-serotipo e que, em muitos países da América Latina,os resultados são analisados através PulseNet, que asseguraa comparação de padrões de PFGE em igualdade decondições...

Comparison of selective agars recommended by method ISO 11290-1 and chromogenic agars for the isolation of Listeria sp. in refrigerated sausages

The aim of this study was to determine the prevalence of Listeria sp. in refrigerated sausages, and to compare the performance of the selective plating media employed in the ISO 11290-1 method (PALCAM and Oxford agars) with chromogenic agars (Chromogenic Listeria agars CM 1080 (OCLA) and CM 1084). The prevalence of Listeria sp. detected was 52.9%, comprising 13.7% L. monocytogenes strains. The efficacy of the four agars for the isolation of L. monocytogenes proved to be satisfactory. Despite differences in composition of the chromogenic media assessed, these disparities did not affect concordance among results. However, PALCAM agar was shown to suppress other microorganisms more effectively, being more applicable for detecting Listeria strains present in lower quantities. Based on these results, the use of PALCAM agar, in combination with a chromogenic media, is recommended for enhanced isolation of atypical Listeria sp. strains in meat products.

Este estudo teve como objetivo a análise da prevalência de Listeria sp. em linguiças resfriadas e a comparação dos meios seletivos utilizados no plaqueamento do método ISO 11290-1 (Ágar PALCAM e Ágar Oxford), e ágares cromogênicos (Ágares Listeria Cromogênico CM 1080 (OCLA) e CM 1084 (ISO)). A frequência de Listeria sp. foi de 52,9%, sendo que destas, 13,7% corresponderam à L. monocytogenes. A eficácia dos quatro ágares para o isolamento de L. monocytogenes demonstrou-se satisfatória. Apesar de haver algumas diferenças nas composições dos meios cromogênicos analisados, estas não pareceram influenciar nas concordâncias entre os resultados expressos. Contudo, o ágar PALCAM mostrou-se mais eficaz na supressão de outros micro-organismos, aumentando, assim, a possibilidade de detecção de espécies de Listeria presentes em número reduzido. Através deste trabalho sugere-se a utilização do ágar PALCAM associado a um meio cromogênico para aumentar a chance de isolamento de cepas atípicas de Listeria sp. em produtos cárneos.

DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis: Argentina / Caracterización molecular por ERIC-PCR de cepas de Listeria spp. aisladas de diversos orígenes en San Luis: Argentina

In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina) from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR). Repetitive intergenic consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.

En este estudio se analizaron 24 cepas de Listeria spp. De ellas, 22 fueron obtenidas en San Luis (Argentina), a partir de muestras humanas, de animales y alimentos. Se incluyeron 2 cepas de referencia Listeria monocytogenes CLIP 22762 y Listeria innocua CLIP 74915. Todos los aislamientos fueron identificados bioquímicamente y caracterizados por serotipificación, fagotipificación y detección del gen flaA por reacción en cadena de la polimerasa (PCR). Se generaron perfiles de bandas de ADN mediante la amplificación de secuencias repetitivas de consenso intergénico de enterobacterias (ERIC-PCR). De acuerdo a los resultados obtenidos por ERIC-PCR, las cepas de Listeria spp. fueron divididas en 3 grupos según su origen. Los perfiles de los aislamientos humanos y animales fueron distintos de los correspondientes a alimentos. Por otra parte, dentro de los grupos I y II se incluyeron 10 cepas de L. monocytogenes y solamente una de Listeria seeligeri. Dentro del grupo III no sólo estuvieron incluidas las 9 cepas de L. innocua sino también 4 de L. monocytogenes. La evaluación de los perfiles de bandas obtenidos por ERIC-PCR permitió la discriminación entre los serotipos ensayados 1/2b, 4b, 6a y 6b dentro de cada grupo. El índice de discriminación calculado para ERIC-PCR fue de 0,94. Los resultados sugieren que la técnica de ERIC-PCR provee un método alternativo válido para la identificación de especies de Listeria y, asimismo, permite la diferenciación de cepas dentro de una misma especie.

Development of a sandwich ELISA for the detection of Listeria spp. using specific flagella antibodies

Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x105cells/0.1 ml at 22degrees C and 1x106 cells/0.1 ml at 30degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 104cells/0.1 ml at 22degrees C and 30degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.

Prevalence of Listeria in soil.

One hundred thirty soil samples from agricultural fields and animal-inhabited areas were examined for the presence of Listeria. The microorganism was identified in 23 (17.7%) samples. L. monocytogenes was detected in 7 samples (5.4%), L. ivanovii in 2 (1.5%), L. innocua in 10 (7.7%) and L. welshimeri in 4 samples (3.1%). Prevalence of Listeria in soil from agricultural fields (17.5%) did not differ considerably from that in the soil and animal-inhabited area (18.0%), but L. ivanovii was isolated only from the latter source. Frequency of occurrence of different species of Listeria differed from place to place.

Prevalence of Listeria spp and Listeria monocytogenes in meat and fermented fish in Malaysia.

Fermented fish and meat samples were purchased from supermarket and wet market for microbiological analysis of Listeria species and Listeria monocytogenes contamination. Listeria species were isolated from 17 (73.9%) of 23 samples of imported frozen beef, 10 (43.5%) of the 23 samples of local beef and 14 (56%) of the 25 samples of fermented fish from wet market. Listeria monocytogenes occurred in 15 (75%) of the frozen beef samples, 6 (30.4%) of the 23 samples of local meat and 3 (12%) of the 25 samples from fermented fish. Listeria species was not isolated from any of the 23 samples of imported frozen beef from supermarket and from the 5 samples of buffalo meat examined. This highlights the possibility of Listeria spp or L. monocytogenes to persist in meat and fermented fish in wet market and raises the problem of illness due to the handling and consumption of Listeria-contaminated meat or fermented fish are likely as evidence by the high contamination rates of samples sold at the wet market.

Species and serovars of the genus Listeria isolated from different sources in Brazil from 1971 to 1997

Using phenotype techniques, characterization was made to species and serovar of 3,112 strains of Listeria, isolated from different sources of infection such as human (247-7.9 percent) and animals (239-7.6 percent), as well as from various routes of infection, including food (2,330-74.8 percent) and environmental constituents (296-9.5 percent), all coming from different regions of the country and collected during the period 1971-1997. The following species were recovered in the cultures analysed: L. monocytogenes (774-24.8 percent), L. innocua (2,269-72.9 percent), L. seeligeri (37-1.1 percent), L. welshimeri (22-0.7 percent), L. grayi (9-0.2 percent), and L. ivanovii (1-0.03 percent). L. monocytogenes was represented by ten serovars, the most prevalent being 4b (352-11.3 percent), 1/2a (162-5.2 percent), and 1/2b (148-4.7 percent). The predominant serovar in L. innocua was 6a (2,093-67.2 percent). Considerations about laboratory methods for diagnosis and epidemiological aspects are presented on the basis of the results obtained.

Caracteres mínimos para la identificación de Listeria Monocytogenes "sensu stricto" / Minimum characters for the identification of Listeria Monocytogenes \"sensu stricto\"

Se seleccionó el mínimo número de ensayos necesarios para la caracterización de microorganismos pertenecientes al género Listeria. Con el propósito de establecer el grado de correlación o independencia de esos caracteres se aplicó la Técnica R, método numérico de agrupamiento. Esta técnica permite deducir el origen, valor selectivo y patrones de variación de los mismos. Se estudiaron 10 cepas de referencia pertenecientes a las diferentes especies propuestas para integrar el género Listeria, empleando 76 caracteres diferenciales. El código de los estados de los caracteres se hace asignando igual peso a todos ellos. La matriz básica de datos considera a los caracteres como OTUs (Unidades Taxonómicas Operacionales). Los caracteres son agrupados a partir de la matriz de distancia, obtenida por aplicación del coeficiente de Manhattan Distance. Se empleó el método de los pares no pesados usando promedios aritméticos (U.P.G.M.A.). La proximidad en el espacio delimitado por los componentes establece la relación entre los caracteres: cuanto más relacionados están, más cercanos se ubican. Los ensayos seleccionados permiten una correcta identificación de las diferentes especies propuestas para el género Listeria. Para la caracterización de estos microorganismos, a partir de una muestra de la cual se aíslen Bacilos regulares Gram positivos no formadores de esporos, morfológicamente compatibles con Listeria, deben realizarse como mínimo las siguientes pruebas: catalasa, hipurato, esculina, rojo de metilo, Voges Proskawer y arginina dehidrolasa. La Técnica-R permitió seleccionar el mínimo número de ensayos para la caracterización de las diferentes especies propuestas actualmente para el género Listeria. Ellos son: producción ácido/gas a partir de: adonitol, dulcitol, ramnosa, xilosa, sacarosa y alfa-metil-D-manósido, beta-hemólisis en sangre de conejo, equina y ovina (método convencional y del calor-frío), hemólisis en medio líquido (con y sin activación con 2-mercaptoetanol)

Ocorrência de espécies de Listeria em camaräo industrializado / Occurrence of Listeria species in frozen shrimp

Em 45 amostras de camarão capturado na costa norte do Estado do Rio de Janeiro e posteriormente, industrializado, foram constatadas as seguintes espécies e sorotipos: Listeria monocytogenes 1/2a (3 amostras), L. monocytogenes 4b (1) e L. innocua 6a (3) e uma não tipável.