Javier Yangüela

Salmonella incidence and distribution of serotypes throughout processing in a Spanish poultry slaughterhouse

A survey of contamination with Salmonella spp. was done at 11 sampling sites in a poultry slaughter establishment in Spain for a total of 192 samples. Samples included fecal material, utensils, water, and poultry carcasses and livers at several stages of processing. Salmonella incidence rates increased from 30% in fecal material collected from incoming birds to 60% in air-chilled carcasses and 80% in cold-stored livers, indicating that cross-contamination occurred. The rate of incidence of Salmonella organisms on carcasses averaged 56.7% through post-picking to post-air chilling and reached a maximum of 70% on carcasses at the post-spray wash site. Poultry livers were more heavily contaminated with salmonellae, as 55% and 80% samples after evisceration and cold storage, respectively, were positive for those pathogenic bacteria. From a total of 112 strains isolated, 87 (77.6%) were S. enteritidis , 7 (6.2%) Salmonella serotype 4,5,12:b:-(II), and 6 (5.4%) Salmonella serotype 4,12:b:-(II), and the remaining 12 strains were equally distributed among S. typhimurium . S. virchow , and S. blockley (3.6% each). Serotypes isolated from feces were later detected in matched carcasses and livers indicating a cross-contamination of carcasses by endogenous microflora in bird feces. The incidence of Salmonella serotype 4,5, 12:b:-(II) and that of S. typhimurium were significantly higher (P < 0.05) in samples obtained prior to evisceration than in those collected after that particular step. The situation with S. enteritidis was quite the reverse, since this serotype was more frequently detected in samples taken after the evisceration step (P < 0.01).

Antioxidant and antibacterial effects of Lavandula and Mentha essential oils in minced beef inoculated with E. coli O157:H7 and S. aureus during storage at abuse refrigeration temperature

The essential oils (EOs) of Lavandula angustifolia L. and Mentha piperita L. were analyzed by gas chromatography mass spectrometry (GC/MS). The major constituents were linalool (22.35%), linalyl acetate (21.80%), trans-ocimene (6.16%) and 4-terpineol (5.19%) for L. angustifolia and menthol (33.28%), menthone (22.03%), and menthyl acetate (6.40%) for M. piperita. In vitro antibacterial activity of both EOs against Escherichia coli O157:H7 and Staphylococcus aureus CECT 4459 showed high inhibition against S. aureus. The lowest minimal inhibitory concentrations (MIC) were obtained with L. angustifolia (0.25 μL/mL) against S. aureus; M. piperita exhibited a MIC of 0.50 μL/mL against both microorganisms. Both EOs caused a significant decrease of bacterial growth in minced beef (p<0.05) stored at 9±1 °C. Minced beef treated with EOs showed the lowest TBARS values (lipid oxidation). Moreover, the results showed that the addition of EOs significantly extended fresh meat odor even at abuse temperature.

High Prevalence of Multiple Resistance to Antibiotics in 144 Listeria Isolates from Spanish Dairy and Meat Products

The resistance to 12 commonly used antimicrobial agents of 144 foodborne isolates belonging to the genus Listeria (23 L. monocytogenes , 54 L. innocua , 66 L. seeligeri , and 1 L. welshimeri ) was tested by using the agar disc-diffusion assay. The Listeria strains were isolated from dairy products (different varieties of unripened fresh and bacteria-ripened hard cheeses made from ewes, cows, and goats milk) and meat products ( longaniza , a type of pork sausage). A total of 84 (93%) and 54 (100%) Listeria strains isolated from cheese and pork sausage, respectively, were resistant to multiple antimicrobial agents. More than 80% of the Listeria strains of both food origins were found to be susceptible to penicillin G and ampicillin, whereas the proportion of isolates resistant to the cephalosporins cefotaxime and cefoxitin was nearly 100%. The prevalence of resistance was much higher for isolates from pork sausage (73.8% on average) than for isolates from cheese (20.9%). This marked difference was statistically significant (P < 0.05; chi-square test) for all antibiotics except ampicillin, cefotaxime, and cefoxitin. The strains of the foodborne pathogen L. monocytogenes isolated from cheese were all susceptible to 9 of the 12 antimicrobial agents evaluated. In contrast, more than 80% of the L. monocytogenes strains isolated from pork sausage exhibited resistance to cefotaxime, cefoxitin, tobramycin, amikacin, chloramphenicol, tetracycline, and erythromycin. The appearance of substantial resistance to antibiotics in foodborne Listeria isolates suggests the need for more prudent use of antibiotics by farmers, veterinarians, and physicians.

Effect of lactic acid bacteria on extention of shelf life and growth of Listeria monocytogenes in beef steaks stored in CO2- rich atmosphere

Beef steaks were inoculated with one or other of two protective strains of lactic acid bacteria (bacteriocinogenic Lactobacillus sakei CTC 372 or the uncharacterized Lactobacillus CTC 711), and stored under modified atmospheres (20-40% CO2). Inoculation of meat with LAB inhibited growth of the spoilage bacteria. Neither CO2 in the pack atmosphere or inoculation with protective strains, nor a combination of both, affected formation of metmyoglobin or the development of off-odours. The formation of metmyoglobin in meat pigments and the sensory odour scores were compatible to those of fresh meat which had not undergone either oxidative deterioration or microbial spoilage. Listeria monocytogenes was inhibited in broth by meat surface microbiota containing the protective strains. With initial numbers of 5.6 log cfu mL-1, after 7 days incubation at 3oC, L. monocytogenes were recovered at log mean numbers of 2.8 log cfu mL-1 when no protective strain was present. At 8oC, the numbers of L. monocytogenes were reduced by about 2.5 or 1.5 log mL-1 in the presence of Lb. sakei CTC 372 or Lb. CTC 711, respectively. At 25oC, the numbers of L. monocytogenes recovered from broth containing either protective strain were about 5 log lower than the numbers recovered from broth containing L. monocytogenes only.

Occurrence of Listeria monocytogenes in Ready-to-Eat Meat Products and Meat Processing Plants in Spain

The aim of this work was to study the occurrence of Listeria monocytogenes in several types of ready-to-eat (RTE) meat products and in the environment of meat processing plants. A total of 129 samples of RTE meat products and 110 samples from work surfaces and equipment were analyzed. L. monocytogenes was detected in 6 out of 35 cooked products (17.14%), 21 out of 57 raw-cured products (36.84%), and 9 out of 37 dry-cured, salted products (24.32%). The number of sample units that exceeded the food safety limit of 100 cfu/g decreased from the manufacture date to half shelf life, and then it was further reduced at the end of shelf life. L. monocytogenes was detected in 25 out of 110 (22.72%) food contact surfaces. The number of positive and negative results from both food and environmental samples were cross-tabulated and the calculated Cohen’s kappa coefficient (κ) was 0.3233, indicating a fair agreement in terms of Listeria contamination. L. monocytogenes was recovered after cleaning and disinfection procedures in four plants, highlighting the importance of thorough cleaning and disinfection.

Comparison of sampling procedures for recovery of Listeria monocytogenes from stainless steel food contact surfaces.

A number of techniques exist for microbiological sampling of food processing environments in food industries. In the present study the efficacies of nine sampling procedures for the recovery of Listeria monocytogenes from food contact surfaces, including a new sampling device consisting of a miniroller, were evaluated and compared. A stainless steel table was inoculated with L. monocytogenes strain 935 (serovar 4b, human origin) and L. monocytogenes strain 437/07 (serovar 1/2b, food origin), at 10(5) CFU/100 cm(2). L. monocytogenes strain 935 was best recovered with the minirollers (recovery of up to 6.27%), while poor recoveries (<0.30%) were obtained with the towel (one-ply composite tissue), alginate swab, metallic swab, and Petrifilm methods. In the case of L. monocytogenes strain 437/07 the replicate organism detection and counting (RODAC) ALOA contact plates yielded the best recoveries (4.15%), followed by the minirollers (up to 1.52%). Overall, recovery percentages with the minirollers were higher with stomacher homogenization than with Vibromatic agitation. The recovery percentages obtained for the Listeria strain of human origin were higher than those obtained with the food strain for all sampling procedures except Petrifilm and RODAC ALOA. With the miniroller device coated with wool fiber, the recovery of L. monocytogenes can be improved from 2 to 17 times over recoveries obtained with the sponge and cotton swab. This is the first report of a miniroller device for microbiological sampling in the available literature. The novel sampling procedure is convenient to apply on surfaces, is cost-effective, and results in better recovery of L. monocytogenes than do the conventional methods.

Potential virulence determinants of Salmonella serovars from poultry and human sources in Spain.

A total of 173 Salmonella strains of different serovars isolated during 1992 from poultry and human sources in Zaragoza (NE Spain) were investigated for potential virulence factors. Parameters studied included production of aerobactin, enterobactin, colicin (including colicin V) and hemolysin, serum resistance against serum from man, sheep, cattle and chicken, binding of Congo red and crystal violet, auto-agglutination and calcium dependency at 37 degrees C and double colony morphology. Preliminary studies showed tests with completely negative results (colicins and hemolysin production, double colony morphology, auto-agglutination and calcium dependency at 37 degrees C) and tests with completely positive results (enterobactin production and binding of Congo red). The tests with variability of results were production of aerobactin, serum resistance and crystal violet binding. Aerobactin production was detected in 80% of Salmonella strains of clinical human origin and in 30% of Salmonella strains isolated from healthy slaughtered chickens. Sixty-five per cent of patient isolates were human serum resistant, 73% were ovine serum resistant, 85% were bovine serum resistant and 98% were chicken serum resistant. The percentages of poultry isolates serum resistant were 61, 86, 60 and 89% in human, ovine, bovine and chicken serum, respectively. Crystal violet binding was detected in 22.5% of Salmonella isolates from human origin and in 32.3% of Salmonella strains from poultry origin. This study can form a marker for the prevalence of strains with various characteristics (production of aerobactin, serum resistance and crystal violet binding) for comparison in future epidemiological studies. Furthermore, the data of this work suggested that strains causing enteric salmonellosis in man are partially identical to strains isolated from carrier broilers.

Composition chimique et activité anti-Salmonella enteritidis CECT 4300 des huiles essentielles d’Eucalyptus globulus, de Lavandula angustifolia et de Satureja hortensis. Tests in vitro et efficacité sur les œufs entiers liquides conservés à 7 ± 1 °C

RésuméLes huiles essentielles (HE) extraites par entranement à la vapeur de trois plantes aromatiques de la région kabyle en Algérie (Eucalyptus globulus, Lavandula angustifolia et Satureja hortensis) ont été analysées par chromatographie en phase gazeuse couplée à la spectrométrie de masse (CG/SM). Les composants majoritaires des HE sont 1,8-cinéole (81,70 %) pour Eucalyptus globulus; 1,8-cinéole (37,80 %) et β-Caryophyllène (20,90 %) pour Lavandula angustifolia et finalement Carvacrol (46,10 %), p-cymène (12,04 %) et γ-terpinène (11,43 %) pour Satureja hortensis. Pour les tests antibactériens, les méthodes de diffusion sur gélose et celle de microdilution ont été utilisées vis-à-vis de Salmonella enterica sérotype Enteritidis CECT 4300. Les résultats ont révélé une activité antisalmonelle significative exercée par l’ensemble des HE. Des diamètres d’inhibition de 41,30 et 35,26 mm ont été rapportés respectivement pour Lavandula angustifolia et Eucalyptus globulus. Cependant, l’HE de Satureja hortensis a démontré une activité antisalmonelle supérieure par rapport aux deux autres HE (51,15 mm). Les valeurs des concentrations minimales inhibitrices (CMI) rapportées dans cette étude sont comprises entre 1–8 μl/ml. Lavandula angustifolia fut l’HE dont la valeur CMI était la plus basse (1 μl/ml). De plus, l’activité antisalmonelle des HE additionnées à différentes concentrations dans les oeufs entiers liquides a été évaluée. Les résultats ont montré que l’effet antibactérien est proportionnel à la quantité d’HE additionnée au produit. Les résultats obtenus permettent de dire que les HE testées possèdent une activité antisalmonelle et par conséquent constituent une alternative naturelle prometteuse pour être utilisées dans la préservation des oeufs entiers liquides.AbstractEssential oils (EO) extracted by steam distillation of three aromatic plants of Kabyle region in Algeria (Eucalyptus globulus, Lavandula angustifolia and Satureja hortensis) were analyzed by gas chromatography coupled with mass spectrometry (GC/MS). The main components of the EOs are 1.8 cineole (81.70 %) for Eucalyptus globulus; 1.8 cineole (37.80%) and β-Caryophyllene (20.90 %) for Lavandula angustifolia and finally Carvacrol (46.10%), p-cymene (12.04%) and γ-terpinene (11.43%) for Satureja hortensis. For antibacterial testing, the agar diffusion and the microdilution methods have been used against Salmonella enterica serovar Enteritidis CECT 4300. The results showed a significant antisalmonelle activity exerted by all HEs. The diameters inhibition of 41.30 and 35.26 mm were reported respectively for Lavandula angustifolia and Eucalyptus globulus. However, the Satureja hortensis EO demonstrated an antisalmonelle activity superior to the other EO (51.15 mm). The values of minimum inhibitory concentrations (MIC) reported in this study were between 1–8 μl/ml. Lavandula angustifolia was the EO whose MIC value was the lowest (1 μl/ml). In addition, the activity antisalmonelle of the EO added at different concentrations in liquid whole egg was also evaluated. The results showed that the antibacterial effect is proportional to the amount of EO added to the product. The antisalmonelle activity and therefore constitute a promising natural alternative for use in the preservation of liquid whole egg.

Olive tree leaf extract; in vitro tests on Staphylococcus aureus, Salmonella enteritidis and Pseudomonas aeruginosa; application in turkey meat

RésuméDans cette étude, un extrait brut aqueux et l’oleuropéine ont été obtenus à partir de feuilles d’olivier de la variété azerradj. L’effet antimicrobien de ces composés a été déterminé par la méthode de diffusion sur gélose vis-à-vis de trois souches bactériennes pathogènes communes: Staphylococcus aureus, Salmonella Enteritidis et Pseudomonas aeruginosa. Ces composés se sont révélés très actifs contre l’ensemble des bactéries testées. Cependant, l’oleuropéine a montré une très forte activité vis-à-vis de Staphylococcus aureus (diamètre d’inhibition = 30,18 mm). L’activité antimicrobienne de ces deux composés a été confirmée par la méthode de microdilution (concentrations minimales inhibitrices: CMI). Les mêmes composés ont été additionnés à des escalopes fraîches de dinde expérimentalement inoculées avec les mêmes bactéries cibles à une charge initiale approximativement de 2 × 105 ufc/g. Tous les échantillons de viande ont été stockés à l’air libre à 8 ± 1 °C pendant sept jours, simulant les conditions réelles algériennes de conservation. Les résultats ont montré que les deux composés exercent une remarquable activité antimicrobienne durant toute la phase de stockage. Cependant, l’oleuropéine a montré une activité bactéricide sur Staphylococcus aureus. L’analyse sensorielle a révélé que l’odeur de la viande additionnée de ces composés reste acceptable au seuil de concentration antimicrobienne utilisée. Les résultats obtenus sont très encourageants et permettent de suggérer que les feuilles d’olivier possèdent des composés ayant des propriétés antibactériennes importantes, ce qui laisse prévoir leur application en industrie de la viande. De ce fait, nous avons essayé d’ouvrir une voie pour l’exploitation des feuilles d’olivier pour lutter contre des bactéries pathogènes dans la viande fraîche de dinde.AbstractIn this study, an aqueous crude extract and oleuropein were obtained from the leaves of the “azerradj” variety of olive tree. The antimicrobial effect of these compounds has been determined via the diffusion method on agar, against three common pathogenic bacterial strains: Staphylococcus aureus, Salmonella Enteritidis and Pseudomonas aeruginosa. These compounds have proven to be very active against all of the bacteria tested. However, oleuropein has demonstrated a much greater activity against Staphylococcus aureus (inhibition diameter = 30.18 mm). The antimicrobial activity of these two compounds was confirmed by the microdilution method (Minimum Inhibitory Concentrations: MICs). The same compounds have been added to fresh turkey escalopes that have been experimentally inoculated with the same target bacteria, at an initial load of approximately 2 × 105 cfu/g. All samples of meat were stored in the open air at 8 ± 1 °C for 7 days, simulating the real-life storage conditions in Algeria. The results demonstrated that the two compounds exhibited remarkable antimicrobial activity throughout the whole storage phase. However, oleuropein showed bactericidal activity against Staphylococcus aureus. Sensory analysis revealed that the smell of the meat with these compounds added remained acceptable at the threshold antimicrobial concentration used. The results obtained are very encouraging and it can be suggested that the leaves from the olive tree contain compounds that have significant antimicrobial properties, which can be applied to use in the meat industry. Thus, we have proposed that the leaves of the olive tree can be exploited in order to control pathogenic bacteria in fresh turkey meat.

Extrait de feuilles d’olivier; tests in vitro vis-à-vis de Staphylococcus aureus, Salmonella Enteritidis et Pseudomonas aeruginosa ; application sur la viande de dinde

RésuméDans cette étude, un extrait brut aqueux et l’oleuropéine ont été obtenus à partir de feuilles d’olivier de la variété azerradj. L’effet antimicrobien de ces composés a été déterminé par la méthode de diffusion sur gélose vis-à-vis de trois souches bactériennes pathogènes communes: Staphylococcus aureus, Salmonella Enteritidis et Pseudomonas aeruginosa. Ces composés se sont révélés très actifs contre l’ensemble des bactéries testées. Cependant, l’oleuropéine a montré une très forte activité vis-à-vis de Staphylococcus aureus (diamètre d’inhibition = 30,18 mm). L’activité antimicrobienne de ces deux composés a été confirmée par la méthode de microdilution (concentrations minimales inhibitrices: CMI). Les mêmes composés ont été additionnés à des escalopes fraîches de dinde expérimentalement inoculées avec les mêmes bactéries cibles à une charge initiale approximativement de 2 × 105 ufc/g. Tous les échantillons de viande ont été stockés à l’air libre à 8 ± 1 °C pendant sept jours, simulant les conditions réelles algériennes de conservation. Les résultats ont montré que les deux composés exercent une remarquable activité antimicrobienne durant toute la phase de stockage. Cependant, l’oleuropéine a montré une activité bactéricide sur Staphylococcus aureus. L’analyse sensorielle a révélé que l’odeur de la viande additionnée de ces composés reste acceptable au seuil de concentration antimicrobienne utilisée. Les résultats obtenus sont très encourageants et permettent de suggérer que les feuilles d’olivier possèdent des composés ayant des propriétés antibactériennes importantes, ce qui laisse prévoir leur application en industrie de la viande. De ce fait, nous avons essayé d’ouvrir une voie pour l’exploitation des feuilles d’olivier pour lutter contre des bactéries pathogènes dans la viande fraîche de dinde.AbstractIn this study, an aqueous crude extract and oleuropein were obtained from the leaves of the “azerradj” variety of olive tree. The antimicrobial effect of these compounds has been determined via the diffusion method on agar, against three common pathogenic bacterial strains: Staphylococcus aureus, Salmonella Enteritidis and Pseudomonas aeruginosa. These compounds have proven to be very active against all of the bacteria tested. However, oleuropein has demonstrated a much greater activity against Staphylococcus aureus (inhibition diameter = 30.18 mm). The antimicrobial activity of these two compounds was confirmed by the microdilution method (Minimum Inhibitory Concentrations: MICs). The same compounds have been added to fresh turkey escalopes that have been experimentally inoculated with the same target bacteria, at an initial load of approximately 2 × 105 cfu/g. All samples of meat were stored in the open air at 8 ± 1 °C for 7 days, simulating the real-life storage conditions in Algeria. The results demonstrated that the two compounds exhibited remarkable antimicrobial activity throughout the whole storage phase. However, oleuropein showed bactericidal activity against Staphylococcus aureus. Sensory analysis revealed that the smell of the meat with these compounds added remained acceptable at the threshold antimicrobial concentration used. The results obtained are very encouraging and it can be suggested that the leaves from the olive tree contain compounds that have significant antimicrobial properties, which can be applied to use in the meat industry. Thus, we have proposed that the leaves of the olive tree can be exploited in order to control pathogenic bacteria in fresh turkey meat.