Janne Lundén

Persistent Listeria monocytogenes strains show enhanced adherence to food contact surface after short contact times

Adherence of 3 persistent and 14 nonpersistent Listeria monocytogenes strains to stainless steel surfaces after short and long contact times was investigated. L. monocytogenes strains were obtained from poultry plants and an ice cream plant throughout several years. Adherence tests were performed in tryptic soy broth at 25 degrees C for 1, 2, and 72 h. Test surfaces were rinsed after the contact time, and attached cells were stained with acridine orange and enumerated with an epifluorescence microscope. The persistent poultry plant strains showed adherence 2- to 11-fold higher than the nonpersistent strains following 1- and 2-h contact times. The adherence of the persistent ice cream plant strain after 1- and 2-h contact times was higher than most of the nonpersistent strains. Seven of 12 nonpersistent ice cream strains showed an adherence of less than half that of the persistent strain. After 72 h, the differences in adherence were not as marked, since half the nonpersistent strains had reached adherence levels comparable with the persistent strains. In fact, three nonpersistent strains showed even higher adherence than the persistent strains. Thus, results of this study reveal that persistent L. monocytogenes strains show enhanced adherence at short contact times, promoting their survival in food processing facilities and possibly having an effect on initiation of persistent plant contamination.

Transfer of persistent Listeria monocytogenes contamination between food-processing plants associated with a dicing machine.

The possibility of the transfer of persistent Listeria monocytogenes contamination from one plant to another with a dicing machine was evaluated, and possible reasons for persistent contamination were analyzed. A dicing machine that diced cooked meat products was transferred from plant A to plant B and then to plant C. After the transfer of the dicing machine, L. monocytogenes PFGE type I, originally found in plant A, was soon also found in plants B and C. This L. monocytogenes PFGE type I caused persistent contamination of the dicing lines in plants B and C. The persistent L. monocytogenes strain and three nonpersistent L. monocytogenes strains found in the dicing line of plant C were tested for adherence to stainless steel surfaces and minimal inhibitory concentrations of a quaternary ammonium compound and sodium hypochlorite, disinfectants widely used in the dicing lines. The persistent strain showed significantly higher adherence to stainless steel surfaces than did the nonpersistent strains. The minimal inhibitory concentrations of sodium hypochlorite were similar for all strains, and the minimal inhibitory concentrations of the quaternary ammonium compound for three of the L. monocytogenes PFGE types, including the persistent PFGE type, were high. All persistent L. monocytogenes PFGE type I isolates were found in an area with high hygienic standards, with the dicing machine being the first point of contamination. These observations show that the dicing machine sustained the contamination and suggest that the dicing machine transferred the persistent L. monocytogenes PFGE type from one plant to another.

Adaptive and cross-adaptive responses of persistent and non-persistent Listeria monocytogenes strains to disinfectants

Persistent and non-persistent Listeria monocytogenes strains were tested for initial resistance and adaptive and cross-adaptive responses towards two quaternary ammonium compounds, alkyl-benzyl-dimethyl ammonium chloride and n-alkyldimethyl ethylbenzyl ammonium chloride, one tertiary alkylamine, 1,3-propanediamine-N-(3-aminopropyl)N-dodecyl, sodium hypochlorite and potassium persulphate. The initial resistance of two persistent and two non-persistent L. monocytogenes strains was observed to differ. Both types of strains adapted after a 2-h sublethal exposure to the quaternary ammonium compounds and the tertiary alkylamine, the highest increase in the minimum inhibitory concentration (MIC) being 3-fold. Progressively increasing disinfecting concentrations at 10 and 37 degrees C resulted in adaptation of L. monocytogenes to all disinfectants except potassium sulphate. The highest observed increase in MIC was over 15-fold, from 0.63 to 10 microg/ml of n-alkyldimethyl ethylbenzyl ammonium chloride. All strains reached approximately similar MICs. Stability of the increased resistance was tested by measuring MICs every seventh day for 28 days. The increased resistance to sodium hypochlorite disappeared in 1 week, but the quaternary ammonium compounds and the tertiary alkylamine showed increased resistance for 28 days. These results suggest that cellular changes due to adaptive responses continue to have an effect on the resistance some time after the exposure. All disinfectants were shown to cause cross-adaptation of L. monocytogenes, the highest increase in MIC being almost 8-fold. The only agent that L. monocytogenes could not be shown to cross-adapt to was potassium persulphate which did, however, cause cross-adaptation to the other disinfectants. The mechanism behind these adaptive responses seemed to be non-specific as cross-adaptation was observed not only between related but also unrelated disinfectants. These findings suggest that sustaining high disinfectant effectiveness may be unsuccessful by rotation, even when using agents with different mechanisms of action.

Persistent and Nonpersistent Listeria monocytogenes Contamination in Meat and Poultry Processing Plants

Contamination analysis of persistent and nonpersistent Listeria monocytogenes strains in three meat processing plants and one poultry processing plant were performed in order to identify factors predisposing to or sustaining persistent plant contamination. A total of 596 L. monocytogenes isolates were divided into 47 pulsed-field gel electrophoresis (PFGE) types by combining the restriction enzyme patterns of AscI (42 patterns) and ApaI (38 patterns). Persistent and nonpersistent strains were found in all plants. Nonpersistent PFGE types were found mostly at one sampling site, with the processing environment being the most common location, whereas the persistent strains were found at several sampling sites in most cases. The processing machines were frequently contaminated with persistent L. monocytogenes PFGE types, and it was of concern that surfaces having direct contact with the products were contaminated. The role of the processing machines in sustaining contamination and in contaminating the products appeared to be important because the final product of several processing lines was contaminated with the same L. monocytogenes PFGE type as that found in the processing machine. The proportion of persistent PFGE types in heat-treated products was eight times higher than in the raw products, showing the importance of the persistent PFGE types as contaminants of the final heat-treated products. The contamination status of the processing lines and machines appeared to be influenced by the compartmentalization of the processing line, with poor compartmentalization increasing L. monocytogenes contamination. The separation of raw and post-heat treatment areas seemed especially important in the contamination status of post-heat treatment lines.

Similar Listeria monocytogenes pulsotypes detected in several foods originating from different sources.

The purpose of the study was to obtain fingerprinting data of Listeria monocytogenes strains isolated in various foods to determine possible associations of strains with product type, producer, country or isolation time. Two hundred and ninety-five L. monocytogenes strains originating from food items of 41 producers of 10 countries were characterized by pulsed-field gel electrophoresis (PFGE) typing. Combination of AscI and ApaI macrorestriction patterns (MRP) yielded 66 different pulsotypes. Ten pulsotypes were common to two or more product types and 17 pulsotypes were detected in foods of more than one producer having no apparent association with each other. Similar pulsotypes of L. monocytogenes were recovered in products of different countries over several years. Some of the pulsotypes were recurrently recovered from the same product of the same producer, suggesting a possible persistence of these strains in the processing plant. However, some of the recurrently isolated L. monocytogenes pulsotypes were repeatedly found in products of several producers, which may indicate that persistent houseflora strains are not always producer-specific. Furthermore, the similarity of macrorestriction patterns expressed as clusters, based on the numerical analysis of macrorestriction patterns, was not found to correlate with product type, country, producer or year of isolation. Our data suggest a wide geographical and temporal distribution of a number of L. monocytogenes strains isolated in food products. The existence of similar L. monocytogenes strains in various food products of several producers should be considered if food strain fingerprint results are used to help trace the vehicles for infections.

Listeria monocytogenes contamination pattern in pig slaughterhouses.

Ten low-capacity slaughterhouses were examined for Listeria by collecting a total of 373 samples, of which 50, 250, and 73 were taken from carcasses, pluck sets, and the slaughterhouse environment, respectively. Six slaughterhouses and 9% of all samples were positive for Listeria monocytogenes. Of the samples taken from pluck sets, 9% were positive for L. monocytogenes, the highest prevalence occurring in tongue and tonsil samples, at 14% and 12%, respectively. Six of 50 (12%) carcasses were contaminated with L. monocytogenes. In the slaughterhouse environment, L. monocytogenes was detected in two, one, one, and one sample originating from the saws, drain, door, and table, respectively. Carcasses were contaminated with L. monocytogenes in those two slaughterhouses, where the mechanical saws, used for both brisket and back splitting, were also positive for L. monocytogenes. A total of 58 L. monocytogenes isolates were characterized by pulsed-field gel electrophoresis typing. The isolates were divided into 18 pulsotypes, 15 of which were detected in pluck sets. In two slaughterhouses, where the carcasses were contaminated with L. monocytogenes, the same pulsotypes were also recovered from splitting saws. In addition, identical pulsotypes were recovered from pluck sets. Our findings indicate that L. monocytogenes of tongue and tonsil origin may contaminate the slaughtering equipment that may in turn spread the pathogen to carcasses. Thus, it is of the utmost importance to follow good manufacturing practices and to have efficient cleaning and disinfection procedures to prevent equipment being contaminated with L. monocytogenes.

An 8-year surveillance of the diversity and persistence of Listeria monocytogenes in a chilled food processing plant analyzed by amplified fragment length polymorphism.

Contamination routes of Listeria monocytogenes were examined in a chilled food processing plant that produced ready-to-eat and ready-to-reheat meals during an 8-year period by amplified fragment length polymorphism (AFLP) analysis. A total of 319 L. monocytogenes isolates were recovered from raw materials (n = 18), the environment (n = 77), equipment (n = 193), and products (n = 31), and 18 different AFLP types were identified, five of which were repeatedly found to be persistent types. The three compartments (I to III) of the plant showed markedly different contamination statuses. Compartment I, which produced cooked meals, was heavily contaminated with three persistent AFLP types. AFLP type A1 dominated, and it comprised 93% of the isolates of the compartment. Compartment II, which produced uncooked chilled food, was contaminated with four persistent and five nonpersistent AFLP types. The equipment of compartment III, which produced cooked ready-to-reheat meals, was free of contamination. In compartments that produced cooked meals, the cleaning routines, product types, and lack of compartmentalization seemed to predispose production lines to persistent contamination. The most contaminated lines harbored L. monocytogenes in coolers, conveyors, and packing machines. Good compartmentalization limited the flow of L. monocytogenes into the postheat-treatment area and prevented the undesired movement of equipment and personnel, thus protecting the production lines from contamination. In compartment II, grated cheese was shown to cause product contamination. Therefore, special attention should be paid to continuous quality control of raw ingredients when uncooked ready-to-eat foods are produced. In compartment II, reconstruction of the production line resulted in reduced prevalence rates of L. monocytogenes and elimination of two persistent AFLP types.

Typing of Listeria monocytogenes isolates originating from the food processing industry with automated ribotyping and pulsed-field gel electrophoresis

A total of 486 Listeria monocytogenes isolates originating from 17 Finnish food processing plants (representing meat, poultry, fish, and dairy production) were collected and typed by automated ribotyping using EcoRI as the restriction enzyme. The isolates were divided into 16 different ribotypes (RTs). Some of these isolates (121), representing all EcoRI types and 16 food plants, were subjected to ribotyping with the PvuII enzyme, to pulsed-field gel electrophoresis (PFGE) typing with AscI and SmaI restriction enzymes, and to serotyping with O-antigen antisera. Nineteen ribotypes were generated with PvuII, 42 macrorestriction patterns were generated with AscI and 24 with SmaI, and three serotypes were generated with antisera. When the results were combined, the overall number of RTs was 23, and that of the PFGE types was 46. Thus, the overall discrimination power of PFGE was higher (discrimination index [DI] 0.966) than that of ribotyping (DI 0.906). The most common serotype (90.1% of the isolates) was 1/2, and isolates of serotype 4 (3.3%) were rare. There was no connection between food sectors and RTs or PFGE types, but PFGE indicated the single plants (78.3% of the types) better than ribotyping (56.5%). On the basis of its automation and on the availability of identification databases, automated ribotyping had some advantages over PFGE. Overall, automated ribotyping can be considered a practical and rapid tool when Listeria contamination is suspected and when screening a large number of isolates is necessary, e.g., when tracing contamination sources. However, in cases of outbreaks, the identical patterns must be confirmed by PFGE, which is a more discriminatory method.

Characterisation of Persistent and Sporadic Listeria monocytogenes Strains by Pulsed-Field Gel Electrophoresis (PFGE) and Amplified Fragment Length Polymorphism (AFLP)

This study was set up to evaluate the genetic similarity or dissimilarity of persistent and sporadic Listeria monocytogenes strains existing in eleven food processing facilities, including fish, dairy, meat and poultry processing plants. In each plant persistent and sporadic strains were selected on the basis of PFGE typing results. A total of 17 strains representing persistent strains and 38 sporadic strains originating from eleven food processing plants were included in the study. PFGE macrorestriction patterns of persistent and sporadic strains from different processing plants were compared and the strains were further studied by amplified fragment length polymorphism (AFLP), being a characterisation method giving more whole genome based information. The 17 persistent and 38 sporadic strains showed 14 and 35 pulsotypes, 14 and 28 AFLP types, respectively. The combination of PFGE and AFLP typing results yielded a total of 48 genotypes. Thirteen of 15 genotypes presented by persistent strains were only associated with persistent strains and similarly 94% (33/35) of genotypes showed by sporadic strains were recovered among sporadic strains only. Our results showed that L. monocytogenes strains causing persistent contamination differ from sporadic strains. In AFLP analysis persistent strains did not, however, form any specific clusters and neither was there any difference between the known two genomic groups. These results indicate that even though persistent strains differ from sporadic strains there seems not to be any specific evolutional lineage of persistent strains.

Acid and heat tolerance of persistent and nonpersistent Listeria monocytogenes food plant strains

Aims:  Acid and heat tolerance of 17 persistent and 23 nonpersistent Listeria monocytogenes strains, recovered from three meat‐processing plants, were investigated.