Pulsed field, PCR ribotyping and multiplex PCR analysis of Yersinia enterocolitica strains isolated from meat food in San Luis Argentina

The characterization of phenotypic and genotypic virulence markers of Yersinia enterocolitica strains belonging to biotypes (B) 1A, 2 and 3, mostly isolated from food in San Luis, Argentina, and the assessment of their genotypic diversity using PFGE and PCR ribotyping, were performed in our laboratory for the first time. Thirty five Y. enterocolitica strains, two reference strains and 33 strains isolated in our laboratory were studied. The presence of virF, ail, ystA, and myfA genes was investigated by multiplex PCR. The pathogenic potential of B1A strains, the most predominant biotype of Y. enterocolitica strains isolated from meat in our region, was investigated by simple PCR. Four B1A strains were positive for ystB gene. Four Y. enterocolitica 2/O:9 (bio/serotype) and two 3/O:5 strains isolated in our laboratory showed virulence-related results in the phenotypic tests and multiplex PCR. A good correlation between the expression of virulence markers and their corresponding genotypes was observed for most strains. Sixteen genomic types (GT) and 9 different intergenic spacer region (SR) groups were generated by PFGE and PCR ribotyping, respectively. In both cases the Y. enterocolitica 2/O:9 strains were separately clustered from 1A and 3/O:5 strains. Meat foods might be vehicles of transmission of pathogenic Y. enterocolitica strains in our region.     

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.     

Comparison of DNA extraction methods for pathogenic Yersinia enterocolitica detection from meat food by nested PCR

The objective of this work was to compare three different methods of DNA extraction from meat food, and to determine whether these methods removed inhibitors of nested PCR for pathogenic Yersinia enterocolitica detection. The amplification of the yadA gene from the DNA obtained from a pure Y. enterocolitica culture could be carried out with all the protocols. DNA amplification from the food samples was observed with two of the three tested protocols, which gave highly sensitive amplifications (detection limit 1 CFU/ml). These protocols detected a lower limit of 0.6 fg/μl of DNA extracted from Y. enterocolitica pure culture. We concluded that these protocols were able to eliminate satisfactorily the PCR inhibitors present in the foods. The nested PCR tested could be used satisfactorily in the investigation of pathogenic Y. enterocolitica in foods in the presence of a high background of microflora.     

Evaluation of a Yersinia adhesion gene (yadA) specific PCR for the identification of enteropathogenic Yersinia enterocolitica

A total of 101 Yersinia enterocolitica strains was investigated with a PCR assay [Blais and Phillipe, Food Control, 6 (1995) 211-214] targeting the Yersinia adhesin gene (yadA) responsible for autoagglutination. Compared to the autoagglutination test the PCR assay has a specificity of 100% but a sensitivity of only 70%. This failure might be caused by the sequence heterogeneity of yadA.     

Yersinia enterocolitica biogroup 1A, serotype O:5 in chicken carcasses

To evaluate the presence of Yersinia enterocolitica in raw chicken, 70 samples of chicken carcasses were obtained from a Buenos Aires, Argentina, processing plant. The detection of this psychotropic microorganism was carried out using the whole carcass rinse method and enrichment in phosphate-buffered saline, 0.067 M, pH 7.6, at 4 degrees C, followed by alkaline treatment and isolation on cefsulodin-irgasan-novobiocin nutrient agar. Y. enterocolitica or related species were detected in 7 of 70 samples. From them, 4.3% were identified as Y. enterocolitica, 1.4% as Yersinia intermedia, and 4.3% as Yersinia frederiksenii. The serotype and phagotype of Y. intermedia isolates were O:5(2), 5(3), and Xz and those of Y. frederiksenii were O:4.16 and Xo. All Y. enterocolitica isolates belong to the biogroup 1A, serotype O:5, phagotype Xz, which presents uncertain pathogenic potential. These findings reinforce the worldwide concern on the microbiological quality of food products. Quality assurance programs on the whole poultry process are increasingly being adopted in Argentina.     

Contamination of carcasses, offals, and the environment with yadA-positive Yersinia enterocolitica in a pig slaughterhouse

This study was carried out in order to evaluate the contamination of the pig-slaughtering line with pathogenic Yersinia enterocolitica carrying the yadA gene. A total of 292 samples were collected from the slaughterhouse; 131 swab samples from pig carcasses, ears, livers, kidneys, and hearts; 89 swab samples from the environment; and 72 sedimentation samples from the air. All surface samples were studied with both the polymerase chain reaction (PCR) and culture methods. The contamination rate of edible pig offals was high with both methods. Using PCR, the detection rates of yadA-positive Y. enterocolitica for livers, kidneys, and hearts were 38, 86, and 63%, respectively, and using the culture method, the detection rates were 31, 69, and 50%, respectively. Pathogenic Y. enterocolitica was also detected from different environmental sites in the slaughterhouse. Using PCR, 13% of the surface samples from the environment were contaminated with yadA-positive Y. enterocolitica. PCR-positive samples were found on the brisket saw, the hook from which the pluck set (heart, lungs, esophagus, trachea, diaphragm, liver, kidneys, and tongue with tonsils) hang, the knife used for evisceration, the floors in the eviscerating area and the weighing area, the meat-cutting table, the aprons used by trimming workers, the computer used in the meat-inspection area, and the coffeemaker used by slaughterhouse workers. The respective detection rate (6%) was considerably lower when we used the culture method. Pathogenic Y. enterocolitica was isolated from the air in the bleeding area. Bioserotype 4/O:3 was the only pathogenic bioserotype isolated in this study. A total of 113 isolates of type 4/O:3 were characterized with pulsed-field gel electrophoresis using NotI and XbaI digests. By combining these profiles, nine different pulsotypes were obtained, the most common of which (1a) was found in 19 (61%) of 31 samples from different sites. This is the same type that has dominated in pig tonsils, which suggests that tonsils may be the source of Y. enterocolitica contamination in the slaughterhouse. The four pulsotypes (1a, 4g, 6g, and 19q) found on edible offals were the same as those found in tonsils, which supports our hypothesis that tonsils are the contamination source for the liver, heart, and kidneys.     

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

Syto9 and probe‐based multiplex real‐time PCR assays for simultaneous detection of a group of foodborne pathogens (named SYLC group), targeting Salmonella spp. (invA gene), Yersinia enterocolitica (ystA gene), Listeria monocytogenes (hly gene) and Campylobacter spp. (rrna gene), have been developed. The Syto9 assay generates amplicon DNA melting curve with four peaks of 86.5 ± 0.5, 84 ± 0.5, 81.5 ± 0.5 and 90.5 ± 0.5 °C corresponding Salmonella spp., Y. enterocolitica, L. monocytogenes and Campylobacter spp. targets, respectively. The sensitivities of the Syto9 and TaqMan assays in artificially inoculated chicken wing rinses were in a range of 3.2 × 10² to 3.1 × 10⁴ and 9.8 × 10² to 1.9 × 10⁴ colony‐forming units per millilitre, respectively, depending on the pathogen. All tested target strains (n = 100) were correctly detected by the both assays, whereas nontarget strains (n = 100) demonstrated no cross‐reactivity representing 100% specificity. The assays are suitable for application in qualitative and quantitative detection of SYLC group pathogens in food matrices.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

Summary:  A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.

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Zusammenfassung:  Für den Nachweis von pathogenen Yersinia enterocolitica wurde ein real-time PCR System mit interner Amplifikationskontrolle entwickelt. Das Nachweissystem für pathogene Y. enterocolitica basiert auf dem chromosomal kodierten ail-Gen. Als Zielsequenz der internen Amplifikationskontrolle dient eine Sequenz aus dem Plasmid pUC19. Zur Validierung der Methode wurden sowohl natürlich kontaminierte Proben aus einem Schlachthof als auch künstlich kontaminierte Proben verschiedener Lebensmittelmatrices verwendet. Die Anreicherung der Proben vor der molekularbiologischen Untersuchung erfolgte parallel in Tryptikase Soja-Bouillon (TSB) und in Pepton-Sorbit-Gallensalz-Bouillon (PSB). Die Ergebnisse der molekularbiologischen Untersuchungen wurden anschlie?end kulturell in Anlehnung an das Standardverfahren nach EN ISO 10273:2003 verifiziert. Die real-time PCR mit interner Amplifikationskontrolle weist eine Sensitivit?t von 5 Genomkopien pro Reaktionsansatz auf. Die Nachweisgrenze des Verfahrens, bestimmt anhand künstlich kontaminierter Proben, betr?gt etwa 5 KbE Y. enterocolitica pro 25 g Lebensmittel. Von den natürlich kontaminierten Proben aus einem Schlachthof waren die Tonsillen vom Schwein zu 100% mit pathogenen Y. enterocolitica kontaminiert, Schweinefleischabschnitte aus dem Kopfbereich wiesen einen Kontaminationsgrad von 22% auf. Ein Screening von Proben durch PCR erlaubt in der Routineanalytik die Fokussierung der kulturellen Analyse auf positive Proben. Dies k?nnte zu einer h?heren Nachweisrate durch das kulturelle Verfahren führen.

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Received: March 5. 2008; accepted: March 14. 2008     

Comparison of culture, multiplex, and 5' nuclease polymerase chain reaction assays for the rapid detection of Yersinia enterocolitica in swine and pork products

Bacteriological culture was compared with multiplex and fluorogenic (TaqMan) polymerase chain reaction (PCR) assays for the detection of attachment invasion locus (ail)-bearing Yersinia enterocolitica in market weight swine, chitterlings, and ground pork. The TaqMan assay detected 1 pg of purified Y. enterocolitica DNA, whereas conventional gel-based PCR detected I ng of the same. The presence of ail-bearing Y. enterocolitica was tested in pork and feces artificially inoculated with Y. enterocolitica strain NADC 5561. The sensitivity limits of culture, multiplex, and TaqMan PCR assays were 4 x 10(3), 4 x 10(2), and 0.4 CFU/g, respectively, for the artificially inoculated pork. The sensitivity limits were 4 x 10(2), 4 x 10(2), and 0.4 CFU/g, respectively, for feces after a 48-h enrichment in a Yersinia selective broth. By the culture method, Y. enterocolitica was not detected in any of the swine specimens (n = 2,403) examined. By contrast, it was detected in 48 (2%) of the swine samples screened using the multiplex PCR and in 656 (27.2%) of these samples using the TaqMan assay. Using the culture method, Y. enterocolitica was detected in 8% of chitterling samples (n = 350) and in none of the ground pork samples (n = 350). It was identified in 27% of the chitterling samples using multiplex PCR and in 79% of these samples using the TaqMan assay. Ten percent of the ground pork samples contained Y. enterocolitica, as determined by the multiplex PCR, and 38% based on the TaqMan assay. The results suggest that pork products harbor more ail-bearing Y. enterocolitica than selected organs of freshly slaughtered hogs and that the TaqMan assay is more sensitive than either the multiplex PCR or traditional culture methods.     

Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method

From October 1997 to April 1998, a survey was conducted to assess the occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products, using a traditional culturing method and a PCR assay. A total of 300 pork samples was examined. Five slaughterhouses in the Norwegian Meat Cooperative were represented with 249 samples and another 51 samples were obtained from retail outlets in the city of Oslo. Using the NMKL method, Y. enterocolitica 0:3 was isolated from six (2%) of the samples, while the PCR method indicated presence of pathogenic Y. enterocolitica in 50 (17%) of the samples. The results indicate that a reduction has occurred in the prevalence of pathogenic Y. enterocolitica in Norwegian pork products, as compared to a previous Norwegian study conducted in 1988-1989. The study also highlights the need for further development and improvement of methods applied for the detection of pathogenic Y. enterocolitica in foods.     

Enrichment procedures and plating media for isolation of Yersinia enterocolitica

A shortened enrichment procedure (25 degrees C for 24 h) was compared with cold enrichment procedures (4 degrees C for 1 to 3 weeks) and direct plating for isolation of Yersinia enterocolitica from commercial ground meat samples. The combined data of all recovery procedures showed that this organism was isolated from 34% of the ground beef samples. The highest isolation rate was 32% for the 4 degrees C/3-week enrichment, followed by 28% for the 4 degrees C/2-week enrichment, 26% for the 25 degrees C/24-h enrichment, 22% for the 4 degrees C/1-week enrichment, and 10% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4 degrees C/3-week, 4 degrees C/2-week, 25 degrees C/24-h, and 4 degrees C/1-week enrichments. The combined data of all recovery procedures showed that Y. enterocolitica was isolated from 64% of ground pork samples. The highest isolation rate was 48% for the 4 degrees C/3-week enrichment, followed by 40% for the 25 degrees C/24-h enrichment, 34% for the 4 degrees C/2-week enrichment, 24% for the 4 degrees C/1-week enrichment, and 24% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4 degrees C/3-week, 25 degrees C/24-h, and 4 degrees C/2-week enrichments. During the plating phase of the experiment, the efficiency of a dye-containing, Yersinia-selective medium (KV202) was compared with that of a commercially available cefsulodin-irgasan-novobiocin medium. Recovery rates were similar for both media. However, KV202 agar differentiated Y. enterocolitica from such contaminating bacteria as Enterobacter, Serratia, and Salmonella by colony morphologic characteristics and color.     

Inactivation of Yersinia enterocolitica by nitrite and nitrate in food.

The antimicrobial effects of sodium nitrite and sodium and potassium nitrate against Yersinia enterocolitica were investigated in solution and in treated pork meat. Potassium nitrate and sodium nitrate showed only feeble antimicrobial activity in cultures; no antimicrobial activity was detected with sodium nitrite. Conversely, all three salts displayed apparent antimicrobial activity in pork meat, possibly due to selective effects on competitive flora.     

Evaluation of a combined culture and PCR method (NMKL-163A) for detection of presumptive pathogenic Yersinia enterocolitica in pork products

A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food (NMKL-163A) was evaluated by testing samples of artificially and naturally contaminated pork. The performance of the pre-PCR sample treatment, buoyant density centrifugation, was first compared with two commercially available methods (DNeasy tissue kit and PrepMan). We found that similar sensitivity was reached (i.e., 25 CFU/g of food was detected by single PCR) with the buoyant density centrifugation and the DNeasy Tissue kit when tested on overnight enrichments. However, the DNeasy tissue kit was superior when tested on nonenriched homogenates; the detection limit was 25 CFU/g in minced beef by single PCR and 25 CFU/g in sausage by nested PCR. We then analyzed 100 raw minced pork samples. Thirty-five tested positive for presumptive pathogenic Y. enterocolitica when analyzed by the NMKL-163A method, whereas none tested positive when analyzed in parallel by a standard culture method (ISO 10273). We also analyzed 97 samples of cold-smoked pork sausage, of which approximately 11% tested positive by the NMKL-163A method. This study showed that sensitivities such as those obtained by nested PCR were required for detection of the pathogen in naturally contaminated samples, and therefore the nested PCR primers, which are included in the NMKL-163A method only as an option, need to be validated and applied routinely.     

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.     

A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1–10³ cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10²–10³ cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.     

High prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland

Polymerase chain reaction (PCR) was used to determine the prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland and to confirm the yadA-positive Y. enterocolitica isolates recovered from the same samples using the conventional culture method. A total of 51 pig tongues purchased at 12 retail outlets and 255 minced meat samples purchased at 40 retail outlets in the Helsinki area were studied. The prevalence of Y. enterocolitica carrying the yadA gene was 92% in pig tongues and 25% in minced meat using PCR and 78% in tongues and 2% in minced meat with the culture method. The prevalence of yadA-positive tongues was higher (98%) when both PCR- and culture-positive results were included because Y. enterocolitica carrying the yadA gene could also be isolated in three PCR-negative tongue samples. In the minced meat samples, all PCR-negative samples were also culture-negative. With the culture method, 66 of 80 yadA-positive isolates in 38 tongues and all yadA-positive isolates (4) in four minced meat samples were recovered after selective enrichment. A total of 92 isolates of Y. enterocolitica bioserotype 4/O:3 in tongues and 5 isolates in minced meat were found, of which 13% in tongues and 20% in minced meat did not carry the yadA gene.     

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.     

Inactivation kinetics of Yersinia enterocolitica by citric and lactic acid at different temperatures

Inactivation of Yersinia enterocolitica by citric (1--20% w/v) and lactic (0.3--4.0% v/v) acids at different temperatures (4, 20, 40 degrees C) has been investigated. Inactivation effect of citric and lactic acids was dependent on time and temperature of exposure and acid concentration. Survival curves of Y. enterocolitica suspended in citric acid solutions at 4 and 20 degrees C displayed a shoulder followed by an exponential inactivation, but at 40 degrees C a shoulder was not observed. At all temperatures investigated, survival curves of Y. enterocolitica suspended in lactic acid solutions were linear or slightly concave upwards. A mathematical model based on the Weibull distribution accurately described the kinetics of inactivation of Y. enterocolitica by both acids. The influence of the citric acid concentration on Y. enterocolitica resistance was independent of the treatment temperature. However for lactic acid, the influence of the acid concentration on microbial inactivation depended on the temperature. At any temperature investigated, lactic acid was significantly more effective than citric acid.     

Fate of Yersinia enterocolitica during manufacture, ripening and storage of Lighvan cheese

This study aimed to evaluate the behavior of virulent Yersinia enterocolitica (YE) during the manufacture, ripening and storage of Lighvan cheese with particular reference to strains of YE, initial inoculation level, and storage time. Three strains of YE with low (1 log cfu/ml) and high (3 log cfu/ml) inoculation levels were inoculated to raw whole ewe's milk which was then used for manufacturing of Lighvan cheese. Throughout the manufacturing, ripening and storage periods the number of YE was counted on selective media. Enumerated colonies were then confirmed by duplex PCR using ail and virF genes. Moreover, some microbial and physiochemical characteristics of the cheese samples were examined. According to the results, initial inoculation level and storage time had statistically significant (P<0.01) effects on persistency of YE, while strain type exhibited no statistically significant (P>0.01) impact on survival of the pathogen. Results showed a rapid increase in the number of YE during manufacturing, however, in the ripening and storage periods the number of YE was decreased and eventually it was eliminated in all cheese batches after 4 months of storage.     

Modelling inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation at different temperatures

A detailed study of the inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation was performed at, respectively, 25 and 35 different combinations of process temperature and process pressure covering a range of 5-50 degrees C and 100-300 MPa. It appeared that in the entire studied pressure-temperature domain, S. aureus was more resistant to high-pressure homogenisation than Y. enterocolitica. Furthermore, the effect of the process pressure on the inactivation of S. aureus was considerably smaller than on the inactivation of Y. enterocolitica. Also, temperature between 5 and 40 degrees C did not affect inactivation of S. aureus by high-pressure homogenisation, while Y. enterocolitica inactivation was affected by temperature over a much wider range. Different mathematical models were compared to describe the inactivation of both bacteria under the experimental conditions applied. Such pressure-temperature inactivation models form the engineering basis for design, evaluation and optimisation of high-pressure homogenisation processes as a new preservation technique.