Virulence profiles of Shiga toxin-producing Escherichia coli and other potentially diarrheagenic E.coli of bovine origin, in Mendoza, Argentina

This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.

Antibacterial and antioxidant activities of the essential oil of Artemisia echegarayi Hieron. (Asteraceae) / Actividad antibacteriana y antioxidante del aceite esencial extraído de Artemisia echegarayi Hieron. (Asteraceae)

Artemisia echegarayi Hieron. (Asteraceae) is commonly known in Argentina as “ajenjo”. Many studies report high efficacy of essential oils against food-borne pathogenic bacteria. The antimicrobial activity and minimal inhibitory concentration of A. echegarayi essential oil were evaluated against seven bacterial species of significant importance in food hygiene, by using the disc diffusion assay and the micro-well dilution method, respectively. Volatile components of the extract were analyzed by gas chromatography-mass spectrometry and major components were determined. Furthermore, the essential oil was tested for its antioxidant activity. The essential oil inhibited the growth of gram-positive and gram-negative tested bacteria, with the exception of Proteus mirabilis. A. echegarayi essential oil presented the lowest minimal inhibitory concentration against Listeria monocytogenes and Bacillus cereus. Two terpenes, thujone and camphor, were identified from this essential oil as the principal constituents responsible for antibacterial activity. The oil showed a free radical scavenging activity equivalent to 50% of the reference compound. These preliminary studies showed promising results since this essential oil may provide an alternative to promote its use as a natural food additive.

Artemisia echegarayi Hieron. (Asteraceae), conocida como “ajenjo”, es una planta típica de la región de Cuyo (Argentina). En este trabajo se evaluó la actividad antimicrobiana in vitro y la concentración inhibitoria mínima del aceite esencial extraído de sus partes aéreas frente a especies bacterianas que con frecuencia contaminan los alimentos. Se utilizaron las técnicas de difusión con discos en agar y microdilución en placa respectivamente. Además, se determinó la actividad antioxidante de este aceite esencial in vitro por espectrofotometría. En general, tanto las bacterias gram-positivas como las gram-negativas fueron inhibidas por este aceite, con excepción de Proteus mirabilis. Listeria monocytogenes y Bacillus cereus resultaron ser las bacterias más sensibles. El análisis por croma-tografía en fase gaseosa y espectrometría de masa permitió la identificación cualitativa y cuantitativa de los componentes mayoritarios del aceite esencial del ajenjo. Entre ellos, la tuyona y el alcanfor se destacaron como los principales responsables de la actividad antibacteriana observada. Los datos preliminares obtenidos en el presente estudio sugieren que el aceite esencial de Artemisia echegarayi representa una alternativa para promover su empleo como aditivo natural en alimentos.

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.

Listeria spp. en alimentos de origen animal / Listeria spp. in food of animal origin

Se analizaron diferentes alimentos de origen animal para detectar la presencia de Listeria spp. De 208 muestras de leche cruda de tambo, se obtuvieron 5 capas de Listeria innocua, 1 de L. monocytogenes y 1 de L. welshimeri, tipificadas por pruebas bioquímicas y serológicas. El método de enriquecimiento rápido resultó el de elección y tanto el agar Palcam como el agar Oxford permitieron el crecimeitno de las 7 cepas. L. monocytogenes se recuperó de la leche de un animal con mastitis subclínica. Ninguna de las muestras analizadas de leches pasteurizadas o chocolatada ni de quesos contenía listeria, en cambio en las de helados se recuperó una cepa de L. welshimeri. Para los alimentos cárnicos, el empleo de enriquecimiento en 2 etapas facilitó la detección de Listeria spp. Se observó un predominio de L. ivanovii en el 2,5 por ciento de las muestras

Aislamiento de Listeria seeligeri de ciego de vizcacha (Lagostomus maximus maximus) / [Isolation of Listeria seeligery from cecum of vizcacha (Lagostomus maximus maximus)]

Recent food listeriosis outbreaks confirm that more faithful isolation and identification methods for Listeria monocytogenes or other potentially pathogen microorganisms are required. Furthermore, the human and animal reservoir role in the ecology of this disease must be established. Listeria spp. in the vizcacha intestinal content was determined by two isolation procedures, starting from 10 g of homogenized samples in 40 ml of PBS. I)0.1 ml was stripped on phenylethanol agar, selective agar for Listeria and acryflavin ceftazidin agar, then incubated at 37 degrees C for 48 h, suspected colonies were identified by preliminary tests (Gram, hemolysis, catalase, esculin hydrolisis and motility at 22 degrees C) and confirmatory tests (indol, methyl red, Voges Proskauer, nitrate and carbohydrate fermentation) (Table 1). Antibiotic susceptibility, protein profile by PAGE and pathogenic power in mice were determined. II) The remaining homogenate was incubated at 4 degrees C in 100 ml of Donnelly and Baigent enrichment broth, weekly or monthly with subcultures until 30 days or 6-8 months, respectively. The subcultures were followed up as in I). A L. seeligeri strain, susceptible to antibiotics suggested for L. monocytogenes and exhibiting resistance to some second and third generation cephalosporins, was isolated (Table 2). The protein profile of both species was coincident, but L. seeligeri was not virulent for mice. The finding of L. seeligeri in an animal (4.0

) used as human feeding source is of interest due to its potential pathogen power.

Prevalencia de Staphylococcus aureus aislados de mastitis subclínica bovina en tambos de la cuenca lechera de la ciudad de San Luis / [Prevalence of Staphylococcus aureus isolated from subclinical bovine mastitis in dairies of the city of San Luis]

In order to detect subclinical mastitis by means of California Mastitis Test and recounting of somatic cells, 163 cows from the dairies of San Luis city, Argentina, were examined. Seventy six individuals (46.6

) exhibited an inflammatory response ranging > or = 2+ grade and a cellular recounting value of > or = 5 x 10(5), data compatible with those of subclinical mastitis. Staphylococcus aureus was isolated from 39 (51.3

) cultures as estimated by the sum of the two last values listed in Table 1. Organisms were isolated by plating on brain heart infusion agar with 5

of sheep blood and on Baird-Parker media. One hundred and three S. aureus isolates recovered from 51 of 63 cows were characterized by coagulase activity by the tube method using human and bovine plasma; clumping factor; glucose and mannitol fermentation; thermonuclease (TNase), pigment, gelatinase, fibrinolysin, acetoin, hemolysin production; egg yolk, tellurite and catalase reaction and crystal violet types. All isolates were susceptible to cephalothin, clindamycin, methicillin, gentamycin and vancomycin; 94.1

were susceptible to chloramphenicol and 53.8

) were classified according to Hájek and Marsálek scheme as biotype C (bovine and ovine ecovar), 33 isolates (32.0

) were classified as biotype B (swine and poultry ecovar); 1 isolated (0.9

) as intermediate between B and D; 5 isolates (4.8

) as biotype A (human ecovar) and 1 isolated (0.9

) as biotype D (ecovar silvestres spp) (Table 2). Production of enterotoxins A to E and toxic shock syndrome toxin-1 (TSST-1) was determined by the optimal susceptibility plate method on 27 isolates (26.2

) which were coagulase 3+ to 4+ and TNase highly positive. None of them produced enterotoxins including TSST-1. The subclinical mastitis data and the prevalence of S. aureus coincide with those of other authors, both from Argentina and from other countries.

Prevalencia de Staphylococcus aureus aislados de mastitis subclínica bovina en tambos de la cuenca lechera de la ciudad de San Luis / [Prevalence of Staphylococcus aureus isolated from subclinical bovine mastitis in dairies of the city of San Luis]

In order to detect subclinical mastitis by means of California Mastitis Test and recounting of somatic cells, 163 cows from the dairies of San Luis city, Argentina, were examined. Seventy six individuals (46.6

) exhibited an inflammatory response ranging > or = 2+ grade and a cellular recounting value of > or = 5 x 10(5), data compatible with those of subclinical mastitis. Staphylococcus aureus was isolated from 39 (51.3

) cultures as estimated by the sum of the two last values listed in Table 1. Organisms were isolated by plating on brain heart infusion agar with 5

of sheep blood and on Baird-Parker media. One hundred and three S. aureus isolates recovered from 51 of 63 cows were characterized by coagulase activity by the tube method using human and bovine plasma; clumping factor; glucose and mannitol fermentation; thermonuclease (TNase), pigment, gelatinase, fibrinolysin, acetoin, hemolysin production; egg yolk, tellurite and catalase reaction and crystal violet types. All isolates were susceptible to cephalothin, clindamycin, methicillin, gentamycin and vancomycin; 94.1

were susceptible to chloramphenicol and 53.8

) were classified according to Hájek and Marsálek scheme as biotype C (bovine and ovine ecovar), 33 isolates (32.0

) were classified as biotype B (swine and poultry ecovar); 1 isolated (0.9

) as intermediate between B and D; 5 isolates (4.8

) as biotype A (human ecovar) and 1 isolated (0.9

) as biotype D (ecovar silvestres spp) (Table 2). Production of enterotoxins A to E and toxic shock syndrome toxin-1 (TSST-1) was determined by the optimal susceptibility plate method on 27 isolates (26.2

) which were coagulase 3+ to 4+ and TNase highly positive. None of them produced enterotoxins including TSST-1. The subclinical mastitis data and the prevalence of S. aureus coincide with those of other authors, both from Argentina and from other countries.