The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods

AIM: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. METHODS AND RESULTS: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. CONCLUSIONS: Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).     

Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus-specific 16S rRNA probes

Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases. The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat. Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested. The probes were also hybridized with nucleic acids from unknown strains of LAB. The identification was consistent with the results of biochemical tests used to characterize the two genera.     

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat

J.L. BROOKS, A.S. MOORE, R.A. PATCHETT, M. D. COLLINS AND R.G. KROLL. 1992. The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (³²P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.     

A TaqMan-based real-time PCR assay for the specific detection and quantification of Leuconostoc mesenteroides in meat products

A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified DNA or 59 CFU per reaction when using calibrated cell suspensions. It performed successfully when tested on an artificially inoculated meat product, with a minimum threshold of 10(4) CFU g(-1) for the accurate quantification of Leuconostoc mesenteroides.     

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.     

Carnobacterium divergens and Carnobacterium maltaromaticum as spoilers or protective cultures in meat and seafood: phenotypic and genotypic characterization

Carnobacterium, a genus of lactic acid bacteria, frequently dominate the microflora of chilled vacuum- or modified atmosphere-packed meat and seafood. In this study Carnobacterium isolates were characterized by phenotypic and molecular methods in order to investigate the association of species and intra-species groups with distinct kinds of meat and seafood. Of 120 test strains, 50 originated from meat (beef and pork products, including 44 strains isolated during this study and 6 strains obtained from culture collections) and 52 from seafoods (cod, halibut, salmon, shrimps and roe products). In addition, 9 reference strains of Carnobacterium spp from other sources than meat and fish and 9 reference strains of lactic acid bacteria belonging to other genera than Carnobacterium were included. Numerical taxonomy relying on classical biochemical reactions, carbohydrate fermentation and inhibition tests (temperature, salt, pH, chemical preservatives, antibiotics, bacteriocins), SDS-PAGE electrophoresis of whole cell proteins, plasmid profiling, intergenic spacer region (ISR) analysis and examination of amplified-fragment length polymorphism (AFLP) were employed to characterize the strains. The numerical taxonomic approach divided the carnobacteria strains into 24 groups that shared less than 89% similarity. These groups were identified as Carnobacterium divergens with one major cluster (40 strains) and 7 branches of one to four strains, Carnobacterium maltaromaticum (previous C. piscicola) with one major cluster (37 strains) and 9 branches of one to four strains and Carnobacterium mobile (three branches consisting in total of 4 strains). Branches consisting of references strains of the remaining Carnobacterium spp. were separated from clusters and branches of C. divergens, C. maltaromaticum and C. mobile. Isolates from the main clusters of C. divergens and C. maltaromaticum were found both in fresh and lightly preserved meat and seafood products. High phenotypic intra-species variability was observed for C. divergens and C. maltaromaticum but despite this heterogeneity in phenotypic traits a reliable identification to species levels was obtained by SDS-PAGE electrophoresis of whole cell proteins and by ISR based on 16S-23S rDNA intergenic spacer region polymorphism. With AFLP, two distinct clusters were observed for C. divergens but only one for C. maltaromaticum. The two C. divergens clusters were not identical to any of the clusters observed by numerical taxonomy. A limited number of C. divergens and C. maltaromaticum isolates possessed a biopreservative potential due to their production of bacteriocins with a wide inhibition spectrum. This study serves as a base-line for further investigations on the potential role of species of Carnobacterium in foods where they predominate the spoilage microflora.     

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat.

The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.     

Specific molecular detection of Carnobacterium piscicola SF668 in cold smoked salmon

AIMS: To establish a rapid and reliable multiplex PCR (mPCR)-based method allowing specific identification of Carnobacterium piscicola SF668 during storage of cold smoked salmon (CSS). METHODS AND RESULTS: CSS was inoculated with C. piscicola SF668 and stored at 4 degrees C. Samples were withdrawn at regular time intervals and analysed by counting the number of viable cells. About 25-100% of colonies grown on Elliker plates were subjected to mPCR amplification. The results show that strains presumably identified as C. piscicola SF668 were predominant over the test period. CONCLUSIONS: mPCR is a powerful tool to study competitiveness of C. piscicola SF668, which inhibits the growth of Listeria monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study demonstrates the importance of molecular methods in studying competitiveness of strains with potential food applications.     

Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Evidence on inhibition of Listeria monocytogenes by divercin V41 action

AIMS: The aim of this study was to investigate the role of divercin V41 in inhibition and prevention of Listeria monocytogenes. METHODS AND RESULTS: Carnobacterium divergens V41 deficient in bacteriocin production was isolated and characterized by enzyme-liked immunosorbent assay, multiplex polymerase chain reaction and bacteriocin diffusion test. Carnobacterium divergens V41 (divercin+) and Carnobacterium divergens V41C9 (divercin-) were grown in the presence of L. monocytogenes in smoked salmon model medium. Carnobacterium divergens V41, but not C. divergens V41C9, was able to inhibit growth of L. monocytogenes. The results indicate that inhibition of L. monocytogenes in the presence of C. divergens V41 is because of the production of divercin V41 and not to a nutritional advantage. CONCLUSIONS: Carnobacterium divergens V41 may be a promising agent in food safety. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates a potential use of a bacteriocin producing lactic acid bacteria in the area food protection.     

Characterization of the bacterial spoilage flora in marinated pork products

Aims: To investigate the microbiota in marinated, vacuum‐packed pork and to characterize isolated bacteria with regard to their spoilage potential. Methods and Results: Laboratory marinated pork meat and commercial products from three Norwegian producers were examined. Lactic acid bacteria dominated in all products at the expiration date. The flora in marinated products was similar only for products from the same plant. Strains of Lactobacillus algidus, Lactobacillus sakei, Lactobacillus curvatus, Carnobacterium divergens, Carnobacterium maltaromaticum, Leuconostoc mesenteroides, Leuconostoc carnosum and Leuconostoc sp. were isolated and tested for their spoilage potential. Samples inoculated with Lact. algidus or Leuc. mesenteroides were rated as most unpleasant by randomly selected people. A sensory panel scored samples with Lact. algidus highest for sour and intense odour. Lactobacillus algidus was found in products from two out of three production plants. Culture‐independent DNA isolation confirmed that cultivation on Blood agar at 20°C yielded a representative picture of the total flora in marinated flintsteak. Conclusions: Lactobacillus algidus may be an important, but underestimated, spoilage organism that needs to be focused on more when spoilage of vacuum‐packed meat is considered. Significance and Impact of the Study: Routine microbial testing may have to be revised in order to detect spoilage LAB that are unable to grow under currently used conditions.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens

Aims: To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results: Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions: This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study: This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.     

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.     

Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in 相似文献   

Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey

AIMS: To investigate the presence of Arcobacter spp. in minced beef meat (n = 97) and rectal faecal samples (n = 200) collected from cattle immediately after slaughter at a local abattoir in Turkey. METHODS AND RESULTS: Meat samples were examined using three different isolation procedures (CAT-supplemented media, de Boer arcobacter isolation method and membrane filtration method), but only one method (CAT-supplemented media) was employed for faecal samples. The isolated Arcobacter strains were identified by genus- and species-(multiplex) specific PCR assays. Arcobacter spp. were isolated from 5 and 9.5% of meat and faecal samples respectively. Although the only Arcobacter sp. found in meat samples was Arcobacter butzleri, all three pathogenic species--A. butzleri, A. cryaerophilus and A. skirrowii--were detected in the rectal swabs. No Arcobacter was isolated when the de Boer method was used for minced meat samples but the same five meat samples were found positive for arcobacters when CAT-supplemented media and membrane filtration method were used. CONCLUSIONS: The membrane filtration method was found to be superior to the CAT-supplemented media, because it led to a reduction in competing microflora. However, the necessity for one filter and medium for each sample makes this method somewhat expensive. The multiplex-PCR (m-PCR) assay shortened significantly the time required for the identification of Arcobacter spp. and also removed the possibility of false positive results due to other campylobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the isolation of Arcobacter spp. in cattle for the first time in Turkey. The m-PCR assay enables the identification and differentiation of all arcobacters simultaneously in one-step PCR.     

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was developed to detect and identify typical Leuconostoc species. This method utilises a set of specific primers for amplification of the 16S rDNA region of typical Leuconostoc species. All Leuconostoc-type strains, all Leuconostoc isolates from kimchi, Korea's traditional, fermented vegetable product, and strains from closely related genera were examined to verify the identification by this method. The primers resulted in amplification only for nine typical Leuconostoc spp., but not for any other genera tested. The size of the amplified products was 976 bp and the amplicons of the different species could be differentiated from each other with MseI, HaeIII and Tsp509I endonucleases, except for the species Leuconostoc argentinum and Leuconostoc lactis, which were indistinguishable. A PCR-RFLP method for the typical Leuconostoc species was optimized to identify a large number of isolates from fermented vegetable product. This PCR-RFLP method enables the rapid and reliable identification of Leuconostoc species and to distinguish them from the other phylogenetically related lactic acid bacteria in food samples.     

A rapid monitoring assay for the detection of Salmonella spp. and Salmonella Senftenberg strain W775 in composts

AIMS: The composting process needs to be validated for its hygienic status in order to ensure that it is free of pathogens. Generally, this is evaluated through temperature monitoring, or additionally through active inoculation and monitoring of indicator organisms. We aimed to develop a monitoring method for the heat-resistant indicator organism Salmonella enterica ssp. enterica serovar Senftenberg strain W775 for detection in composting biowastes. METHODS AND RESULTS: The method development is comprised of: (i) optimization of molecular detection of bacteria belonging to the genus Salmonella; (ii) identification of a DNA marker for Salmonella strain W775; and (iii) development of a multiplex polymerase chain reaction (PCR)-based on both DNA markers. Subsequently, Salmonella strain W775 was inoculated and monitored during composting of biowastes in an industrial composting facility. CONCLUSIONS: A highly sensitive and specific detection of viable cells was obtained by enriching the compost sample prior to multiplex PCR analysis. Complete inactivation of Salmonella strain W775 was obtained within 4 days in an industrial composting facility at temperatures ranging between 41 and 57 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a monitoring method for the simultaneous detection of naturally occurring Salmonella strains and artificially introduced Salmonella strain W775 in composting biowastes that can be applied in routine analysis.