Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant.

Molecular typing of bacteria has been widely used in epidemiological studies but not as extensively for tracing the transmission of pathogenic bacteria in food plants. This study was conducted to examine the potential use of two molecular typing methods, random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE), to trace Listeria monocytogenes contamination in a shrimp processing plant. Ribotyping and phase typing were also performed on a select number of strains. One hundred fifteen strains of L. monocytogenes collected in different areas of a shrimp processing plant were first serotyped and then subtyped by molecular typing. RAPD and PFGE showed great promise for typing L. monocytogenes isolates since distinguishable and reproducible DNA polymorphisms were obtained. When the composite profile from both (RAPD and PFGE) methods was generated, there was an increase in the discriminatory power to discern differences between strains of L. monocytogenes. The results indicated that environmental strains all fell into composite profile groupings unique to the environment, while strains from both water and utensils shared another composite profile group. L. monocytogenes fresh shrimp isolates belonging to one profile group were found in different areas of the processing line. This same profile group was also present in food handlers from the processing and packaging areas of the plant.     

Detection and identification ofListeria monocytogenes from milk and cheese by a single-step PCR

Primers ofiap gene were used as a target to develop a PCR technique for detectingListeria monocytogenes in milk and cheese. The PCR technique gives good results in the detection ofListeria monocytogenes either in artificially or naturally contaminated foodstuffs and has a high sensitivity and specificity. Application of this rapid diagnostic tool could provide further information about the spread ofL. monocytogenes in milk and cheese.     

Enumeration of Listeria monocytogenes in raw milk

Enumeration of Listeria monocytogenes in raw milk was compared by direct plating and Most Probable Number (MPN) techniques involving both direct and two-stage enrichments. Direct plating was found to lack sensitivity for the enumeration of low numbers of listerias found in milk but an MPN technique with direct selective enrichment was found to be more suitable than one with two-stage enrichment.     

Isolation of Listeria monocytogenes from raw milk and its environment at dairy farms in Japan

The incidence of Listeria monocytogenes contamination in raw milk from farm bulk tanks and silage was investigated monthly at 20 dairy farms in Aomori prefecture, Japan, from January to July in 1989. Listeria sp. was isolated from one sample (5%) of raw milk collected in May, but L. monocytogenes was not isolated from raw milk. L. monocytogenes was isolated from two samples of silage (10%) collected in June. Silage, cow feces, sawdust bedding, dust and soil in these farms were tested for listerias, and we confirmed listerias contamination of the environment in the dairy farms.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Use of PCR methods for identification of Listeria monocytogenes in milk

The aim of this work was to estimate the limit of Listeria monocytogenes cfu in polymerase chain reaction (PCR) for a DNA fragment of listeriolysine O (hly A) gene. The PCR method, with used primers selected in areas of the listeriolysin O gene, allows to differentiate L. monocytogenes strains from other Listeria species. The amplified fragment (456 bp) of hly A gene was obtained for all strains L. monocytogenes and no other Listeria species. The PCR method with the selected primers allowed to detect 50-500 cfu L. monocytogenes/ml suspended in water or milk. Among 20 samples of raw milk from cows, 10 samples contained > 50 cfu L. monocytogenes/ml. Obtained results indicate that the PCR assay of L. monocytogenes identification is technically simple and may be conduct with minimal time. So, it could be recommended as quick diagnostic method in identification L. monocytogenes in milk.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.     

A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese

Aims: In this study, we compare seven different methods which have been designed or modified to extract total DNA from raw milk and raw milk cheese with a view to its subsequent use for the PCR of bacterial DNA. Materials and Results: Seven extraction methods were employed to extract total DNA from these foods, and their relative success with respect to the yield and purity of the DNA isolated, and its quality as a template for downstream PCR, was compared. Although all of the methods were successful with respect to the extraction of DNA naturally present in cheese, they varied in their relative ability to extract DNA from milk. However, when milk was spiked with a representative Gram‐positive (Listeria monocytogenes EGDe) or Gram‐negative (Salmonella enterica serovar Typhimurium LT2) bacterium, it was established that all methods successfully extracted DNA which was suitable for subsequent detection by PCR. Conclusions: Of the seven approaches, the PowerFood? Microbial DNA Isolation kit (MoBio Laboratories Inc.) was found to most consistently extract highly concentrated and pure DNA with a view to its subsequent use for PCR‐based amplification and also facilitated accurate detection by real‐time quantitative PCR. Significance and Impact of the Study: Accurately assessing the bacterial composition of milk and cheese is of great importance to the dairy industry. Increasingly, DNA‐based technologies are being employed to provide an accurate assessment of this microbiota. However, these approaches are dependent on our ability to extract DNA of sufficient yield and purity. This study compares a number of different options and highlights the relative success of these approaches. We also highlight the success of one method to extract DNA from different microbial populations as well as DNA which is suitable for real‐time PCR of microbes of interest, a challenge often encountered by the food industry.     

Genetic diversity and molecular typing of Listeria monocytogenes in China

ABSTRACT: BACKGROUND: Listeria monocytogenes can cause invasive diseases in humans and farm animals and is frequently isolated from dairy products and poultry. Listeriosis is uncommon in China but L. monocytogenes has been isolated from foods and food processing environments in China. However little is known of genetic diversity of Chinese L. monocytogenes isolates and their relationships with global isolates. RESULTS: Two hundred and twelve isolates of L. monocytogenes from food sources from 12 provinces/cities in China were analysed by serotyping, Pulsed Field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST). The predominant serotypes are 1/2a, 1/2b and 1/2c accounting for 90.1% of the isolations. PFGE divided the isolates into 61 pulse types (PTs). Twenty nine PTs were represented by more than one isolates with PT GX6A16.0004 containing the most number of isolates. MLST differentiated the isolates into 36 STs, among which 15 were novel. The most common 3 STs were ST9 (29.1%), ST8 (10.7%) and ST87 (9.2%), accounting for 49.0% of the isolates. CONCLUSIONS: STs prevalent in other parts of the world are also prevalent in China including 7 STs (ST1-ST3, ST5, ST6, ST8, ST9) which caused maternal fetal infections or outbreaks, suggesting that these STs potentially can also cause severe human infections or outbreaks in China. Surveillance of these STs will provide important information for prevention of listeriosis. This study also enhances our understanding of genetic diversity of L. monocytogenes in China.     

Direct polymerase chain reaction detection of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products.

A polymerase chain reaction (PCR) method designed to sensitively detect and identify Campylobacter jejuni and Campylobacter coli without the need for isolating and culturing strains is described. The intergenic sequence between the flagellin genes flaA and flaB was amplified and characterized with a triple primer or seminested primer approach. A total of 50 bacterial strains, 27 of C. jejuni and C. coli and 23 of other species, were tested, giving no false-positive or false-negative results. The detection limit as determined by ethidium bromide staining of amplification products on agarose gels was 10 bacteria or less in artificially contaminated water, milk, and soft cheese samples with the seminested primer PCR assay. As an application of the PCR system, a set of 93 samples of milk and other dairy products was screened for the presence of C. jejuni and C. coli. We identified six positive samples (6.5%), while none were found with a conventional culture method.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69 degrees to 73 degrees C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73 degrees C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Diversity of Staphylococcus aureus enterotoxin types within single samples of raw milk and raw milk products

AIM: To find out if testing of up to 10 Staphylococcus aureus isolates from each sample from raw milk and raw milk products for staphylococcal enterotoxin (SE) might increase the chances of identifying potential sources of food intoxication. METHODS AND RESULTS: Altogether 386 S. aureus isolates were tested for the presence of SE by reversed passive latex agglutination (SET-RPLA), and SE genes (se) by a multiplex polymerase chain reaction (PCR). In 18 of 34 (53%) S. aureus positive samples a mixture of SE and/or se positive and negative isolates were identified. Multiplex PCR increased the number of potential SE producing strains, i.e. isolates that harboured se, with 51% among the product and 48% among the raw bovine milk isolates. Examination by pulsed-field gel electrophoresis mostly confirmed clonal similarity among isolates sharing SE/se profile, but did not further differentiate between them. CONCLUSIONS: Isolates of S. aureus collected from one sample may show great diversity in SE production and different plating media seem to suppress or favour different strains of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Several isolates of S. aureus from each sample should be tested for enterotoxin production in cases with typical SE intoxication symptoms with methods that are able to reveal new SE/se.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69° to 73°C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73°C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml(-1). L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.