Effect of heat exposure on invasiveness of Listeria monocytogenes strains.

The effect of a 54°C heat exposure lasting 20 and 60 minutes on the invasiveness of 10 Listeria monocytogenes strains in the exponential and stationary phases was investigated. It was shown that heat exposure significantly reduced the L. monocytogenes count. A maximal reduction of 5 log took place after 60 minutes of heating. No significant differences in survival between exponential and stationary phases were observed for most strains. Twenty minutes of heat exposure resulted in a reduction of the invasiveness of L. monocytogenes, which was more pronounced in the exponential-phase bacteria. Prolongation of heating to 60 minutes was shown to have a significant impact on the invasiveness of five stationary-phase strains. It was demonstrated that heat exposure influences the survival of L. monocytogenes strains as expected. Also, the invasiveness was significantly changed in a time- and growth phase-dependent manner. Low reductions of bacterial counts in milder conditions, that is, 20 minutes of heat, were accompanied by a decrease of the invasiveness of all L. monocytogenes strains. Prolongation of heating time to 60 minutes resulted in significant reduction of bacterial numbers. However, bacteria, especially those in the stationary phase, which survived this treatment can become more invasive.

Effect of osmotic stress and culture density on invasiveness of Listeria monocytogenes strains.

The effect of osmotic stress on its capacity to invade the human enterocytic cell line HT-29 was studied in the early log through the stationary phase in 10 L. monocytogenes strains representing three genetically independent lineages. The results demonstrate that the transition of the bacteria from the log to the stationary phase results in a stepwise reduction of invasiveness. This effect was heterogeneous in the studied L. monocytogenes population, as the range of invasiveness reduction between the log and stationary phases varied from 10- to 380-fold depending on the strain. Ten-minute exposure to 0.3 M NaCl was sufficient to generate invasiveness alteration. No significant change in invasiveness induction caused by osmotic stress was found between the different points of the log phase (OD600 0.4-1.2), being significantly different in the early log phase (OD600 0.2-0.3) and in the stationary phase after 18 h of culture. The level of internalins and opuCA transcripts in response to osmotic stress did not correlate with invasiveness alteration in most L. monocytogenes strains. Prolongation of stress exposure to 1 h and an increase in NaCl concentration from 0.3 to 1.8 M had no significant effect on a further increase in invasiveness. Short exposure times and low NaCl concentrations were sufficient for the generation of maximal invasiveness response of L. monocytogenes. It appears that although stationary-phase bacteria exhibit lower invasiveness than log-phase bacteria, they have a greater capacity to enhance their pathogenicity in response to stress.

Characterization of borderline oxacillin-resistant Staphylococcus aureus isolated from food of animal origin.

In this study, the molecular characteristics of food-derived oxacillin-resistant Staphylococcus aureus were determined. Eight borderline oxacillin-resistant strains with MICs of 2 to 4 microg/ml were identified from 132 S. aureus isolates of food origin. One of the two isolates with a MIC of 4 microg/ml was methicillin-resistant determinant (mecA) gene positive, and the other six with MICs of 2 microg/ml were mecA negative. The mecA-positive isolate was classified as sequence type (ST)228, staphylococcal protein A (spa) type t041, and carried the staphylococcal cassette chromosome mec type I element. Two borderline oxacillin-resistant strains were classified as spa t008 and ST8, and the remaining five as spa t164 and ST20. The mecA-positive strain and four borderline oxacillin-resistant strains were found enterotoxigenic. The enterotoxin genes detected in these strains included selp, egc1, and sed-sej-selr. The borderline-resistant S. aureus isolates from a manually handled product, i.e., minced pork, were shown genetically related to strains associated with human infections. This suggests that humans can be considered as a source of contamination of this food with oxacillin-resistant S. aureus strains. The genotypes of the investigated milk borderline-resistant isolates were shown to occur not only in cows, but also in humans. Since manual handling is reduced in raw milk production, a human origin of S. aureus seems unlikely. Because knowledge of the genotypes of animal staphylococci is limited, more research is needed to address the question of the origin of antibiotic-resistant S. aureus strains in food.

Genotypes and enterotoxin gene content of S. aureus isolates from poultry.

Little is still known about the genotypes and prevalence of enterotoxin genes in animal-derived Staphylococcus aureus strains. In this study, spa type, the presence of known enterotoxin genes, and mecA gene was determined in 42 S. aureus isolates from poultry. All these isolates were classified as mecA-negative. t002, found in 19 staphylococci, was the most prevalent spa type in the studied population. t034 was found in 11 isolates. MLST performed on three t034 isolates confirmed its attachment to ST398. A strong association between the CC5 genetic background and the egc gene grouping in staphylococci of animal origin was confirmed, since all 23 isolates with t002, t214, and t010, belonging to CC5, contained egc1. No enterotoxin genes were found in 15 S. aureus isolates. In this population the most prevalent genotype was t034, found in 11 isolates. It was demonstrated that MSSA strains with the t034 ST398 genetic background also occur in poultry. This may imply, that ST398-type strains occur in a wider range of livestock species than previously believed.

Enterotoxin gene content in Staphylococcus aureus from the human intestinal tract.

Enterotoxigenic Staphylococcus aureus has been associated with staphylococcal food poisoning, which in a number of patients is accompanied by gastroenteritis. It has also been found to persist asymptomatically in the human intestinal tract, being considered one of the sources of pathogen transmission to manually handled food. However, very little is known about the incidence and enterotoxigenicity of intestinal S. aureus not associated with enteritis. There are practically no data on the frequency of some enterotoxin genes in intestinal S. aureus. Six thousand six hundred and twenty-one fecal swabs from 6-month- to 8-year-old children were analyzed for S. aureus. Growth of S. aureus was found in 347 samples. Two hundred and eight S. aureus isolates (4.2% of 4900 swabs) were from patients with sporadic enteritis and 139 isolates (8% of 1721 swabs) from patients who did not develop diarrhea during hospitalization. The genes encoding 16 members of the enterotoxin family (sea-see, seg-selp, and selu) and tst were present in 174 (83%) S. aureus isolates accompanying enteritis and in 101 (72%) isolates derived from patients with no enteritis symptoms. The genes of the classical enterotoxins (sea-see) and tst were present in 56% and 59% of the enteritis-associated and nonenteritic isolates, respectively.

Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food.

Extensive analysis of the Staphylococcus aureus genome has allowed the identification of new genes encoding enterotoxin-like superantigens (SEls). Some of these are thought to be involved in staphylococcal food poisoning, while others do not elicit any emetic effect. The potential impact of these members of the enterotoxin-like family on the human organism seems to rely mainly on their superantigenic activity. In this paper the distribution of the genes coding for enterotoxin-like superantigens in S. aureus isolated from food was studied. Fifty isolates of S. aureus were examined and 27 were shown to be enterotoxigenic. Only 9 of the 27 strains carried genes encoding enterotoxins SEA-SEE. In 18 SEA-SEE-negative strains the presence of newly described enterotoxin genes was detected. All SEA-SEE-positive strains simultaneously carried genes of new SEls. We show that the gene encoding SElH (staphylococcal enterotoxin-like enterotoxin H) was the most frequently detected (n=14), while genes encoding SElI together with SElG accompanied by the other genes of the egc locus were detected in three strains. We also detected the presence of three less investigated genes: sep, sel, and sek. These genes were present in eight, two, and one isolate, respectively. In one strain, sep was accompanied by genes of other SEls, while in the remaining seven it was the only enterotoxin-like gene detected. The high prevalence of newly discovered enterotoxin genes, including the genes encoding emetic toxins, was demonstrated in food-derived strains. This supports the need for additional work on its role in food poisoning and, alternatively, to monitor its presence in S. aureus isolated from food. Our results suggest that yet unknown genetic elements encoding enterotoxin genes may exist.