Juan J. Carramiñana

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).

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.

Antibacterial Efficiency of Spanish Satureja montana Essential Oil against Listeria monocytogenes among Natural Flora in Minced Pork

The purpose of this study was to examine the effectiveness of winter savory (Satureja montana) essential oil (EO) for control of growth and survival of experimentally inoculated Listeria monocytogenes serovar 4b (10(4) CFU/g) among natural flora in minced pork. EOs of French thyme (Thymus vulgaris F) and rosemary (Rosmarinus officinalis) cultivated in the same region of Aragon (northeastern Spain) were used as reference ingredients. The EOs obtained by hydrodistillation were added at concentrations of 0.25, 0.5, 1, and 2.5 microl/g (vol/wt), and the samples were kept at 4 degrees C in air for up to 7 days. The populations of L. monocytogenes and total viable bacteria were determined in the control and treated samples at 0, 1, 3, 5, and 7 days. Moderate activity of S. montana EO against L. monocytogenes was observed (at 2.5 microl/g, reductions of 0.27 log CFU/g by day 3 and 0.61 log CFU/g by day 7), with higher activity against aerobic flora. The greatest reduction in aerobic flora was on day 3 (at 2.5 microl/g) from 1.10 to 1.45 log CFU/g. S. montana EO was comparable to T. vulgaris F EO in listericidal activity, but R. officinalis EO was ineffective against the L. monocytogenes and aerobic flora in the minced meat model. The approximately 3-log reduction in aerobic flora with T. vulgaris F EO at 0.25 to 2.5 microl/g after 5 days of storage was the most significant reduction. Depending on sensory considerations, the addition of active EOs in combination with other preservation techniques for synergistic effects may provide alternatives to synthetic chemical preservatives. Suggestions on relationships between chemical composition and biological activities of EOs are outlined.

Antimicrobial resistance of Salmonella spp. from pigs at slaughter in Spain in 1993 and 2001

Aim:  To compare the incidence of antimicrobial resistance among Salmonella serotypes isolated in a pig slaughterhouse in Zaragoza (Spain) during 1993 and 2001.

Influence of Agricultural Practices on the Contamination of Maize by Fumonisin Mycotoxins

The objective of the present work was to investigate the effect of different agricultural practices on the contamination of maize by fumonisin mycotoxins. Corn samples were collected from 16 maize fields located in Aragón (northeastern Spain) during the 2007 crop year. Corn samples were collected from each field five times at different maturation stages: F1, day 0 (milky corn); F2, day 15; F3, day 30 (yellow corn); F4, day 45; and F5, ripe corn at harvest. The agricultural practices evaluated were type of seed (conventional and transgenic), planting method (dry and wet planting), tillage system (plowing and minimum tillage), type of irrigation (flood and sprinkler), residue management of preceding crop (removal and burial), nitrogen fertilization level (kg N per ha), and harvest date. Mycotoxin analysis was carried out with the ROSA Fumonisin test, which measures both fumonisin B1 and B2 by lateral flow immunoassay. No fumonisins were detected in milky corn (F1 and F2 stages). Only one field had fumonisins in F3 yellow corn (1,037 microg/kg); this field was part of the only farm affected by borer insects. One-third of fields had fumonisins at the F4 stage (363 microg/kg), and 62.5% of the fields were positive for fumonisins at the F5 harvest stage (520 microg/kg). Wet planting and the removal of debris from the previous crop significantly lowered the risk of fumonisin in corn. The use of insect-resistant maize seeds tended to reduce fumonisin levels. However, higher levels of nitrogen fertilizer had a tendency to increase fumonisin levels in corn. Tillage system, type of irrigation, and harvest date had no clear effect on fumonisin levels.

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.

Inhibition of Fusarium Growth and Mycotoxin Production in Culture Medium and in Maize Kernels by Natural Phenolic Acids

The possible role of natural phenolic compounds in inhibiting fungal growth and toxin production has been of recent interest as an alternative strategy to the use of chemical fungicides for the maintenance of food safety. Fusarium is a worldwide fungal genus mainly associated with cereal crops. The most important Fusarium mycotoxins are trichothecenes, zearalenone, and fumonisins. This study was conducted to evaluate the potential of four natural phenolic acids (caffeic, ferulic, p-coumaric, and chlorogenic) for the control of mycelial growth and mycotoxin production by six toxigenic species of Fusarium . The addition of phenolic acids to corn meal agar had a marked inhibitory effect on the radial growth of all Fusarium species at levels of 2.5 to 10 mM in a dose-response pattern, causing total inhibition (100%) in all species except F. sporotrichioides and F. langsethiae . However, the effects of phenolic acids on mycotoxin production in maize kernels were less evident than the effects on growth. The fungal species differed in their responses to the phenolic acid treatments, and significant reductions in toxin concentrations were observed only for T-2 and HT-2 (90% reduction) and zearalenone (48 to 77% reduction). These results provide data that could be used for developing pre- and postharvest strategies for controlling Fusarium infection and subsequent toxin production in cereal grains.