Traceback identification of an ingredient (pork dewlap) as the possible source of Listeria monocytogenes serotype 4b contamination in raw chicken products

In surveys conducted on finished product samples from a single poultry processing plant in Spain, Listeria monocytogenes was found in 14 different uncooked products. To track contamination patterns, 77 L. monocytogenes isolates were characterized by PCR-based serotyping, pulsed-field gel electrophoresis (PFGE) restriction analysis, and PCR-based allelic analysis of the virulence gene actA. Serotyping revealed that 12 isolates (15.6%) were of the L. monocytogenes serotype 4b complex (serotype 4b or the closely related serotypes 4d and 4e). A combination of endonucleases AscI and ApaI PFGE patterns yielded 15 different pulsotypes among all 77 tested isolates. All the serotype 4b isolates belonged to one pulsotype. Sequencing of the actA gene confirmed that all serotype 4b isolates corresponded to the same allelic subtype. The subtype was recovered from five product types, but its presence was not correlated with the production line or the date of isolation, suggesting a possible association of this strain with a common ingredient. This traceback investigation established that pork dewlap, an ingredient common to all the products contaminated with this strain, was the most probable source of L. monocytogenes 4b. The same 4b strain was isolated from four samples of pork dewlap from one specific supplier. After replacement of this contaminated ingredient in the fresh products, this strain of L. monocytogenes serotype 4b was not detected. This study confirms the effectiveness of molecular subtyping to control contamination by specific strains of L. monocytogenes and the importance of testing the different ingredients added to the food products.     

Development of a multilocus variable-number of tandem repeat typing method for Listeria monocytogenes serotype 4b strains

Listeria monocytogenes serotype 4b strains have been identified as the causative agent in many human listeriosis epidemics as well as in a considerable number of sporadic cases. Due to the genetic homogeneity of serotype 4b isolates, development of rapid subtyping methods with high discriminatory power for serotype 4b isolates is required to allow for improved outbreak detection and source tracking. In this study, multilocus variable-number tandem repeat analysis (MLVA) was developed and used to characterize 60 serotype 4b isolates from various sources. All isolates were also characterized by automated EcoRI ribotyping, single enzyme pulsed-field gel electrophoresis (PFGE) with ApaI, and a multilocus sequence typing (MLST) scheme targeting six virulence and virulence-associated genes. Discriminatory power of MLVA (as determined by Simpson Index of Discrimination) was higher than the discriminatory power of any of the other three methods. MLVA markers targeted were found to be stable and did not change when three isolates were passaged daily for 70 days. Cluster analyses of MLVA, PFGE and MLST consistently grouped the same isolates into three major clusters, each of which includes one of the three major L. monocytogenes epidemic clones (i.e., ECI, ECIa and ECII). We conclude that the MLVA method described here (i) provides for more discriminatory subtyping of L. monocytogenes serotype 4b strains than the other three methods, (ii) identifies three major groups within the serotype 4b, which are consistent with the groups identified by other subtyping methods, and (iii) is easy to interpret. Use of MLVA may thus be recommended for subtyping of serotype 4b isolates, including as a secondary more discriminatory subtyping method that could be used after initial isolate characterization by PFGE or ribotyping.     

Genetic homogeneity among Listeria monocytogenes strains from infected patients and meat products from two geographic locations determined by phenotyping,ribotyping and PCR analysis of virulence genes

Thirty Listeria monocytogenes isolates from human patients and foods originated from two different geographic locations without any epidemiological relations were analyzed for their genotypic and phenotypic virulence gene expressions and genetic relatedness. All strains contained virulence genes, inlA, inlB, actA, hlyA, plcA and plcB, with expected product size in PCR assay except for the actA gene. Some strains produced actA gene product of 268 and others 385 bp. Phenotypically, all were hemolytic but showed variable expressions of phospholipase activity. Ribotyping classified isolates into 12 different groups based on the similarity to DuPont Identification numbers (DID), which consisted primarily of clinical or food isolates or both. Cluster analysis also indicated possible existence of clones of L. monocytogenes that are found in food or human hosts or are evenly distributed between these two. Two isolates (F1 from food and CHL1250 from patient) had unique ribotype patterns that were not previously reported in the RiboPrinter database. This study indicates distribution of diverse L. monocytogenes strains in clinical and food environments. The isolates showed 92-99% genetic homogeneity, in spite of their origins from two different geographic locations and environments.     

Detection of multiple virulence-associated genes in Listeria monocytogenes isolated from bovine mastitis cases

Clinical samples (n=725) were collected from bovines (n=243) which were positive for mastitis using the California mastitis test (CMT) and somatic cell count (SCC). The clinical samples comprising blood (n=239), milk (n=243), and faecal swabs (n=243) were examined for the presence of pathogenic Listeria spp. Isolation of the pathogen was done using selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Confirmation of the isolates was based on biochemical tests and Christie, Atkins, Munch-Petersen (CAMP) test followed by pathogenicity testing. Pathogenicity of the isolates was tested by phosphatidylinositol-specific phospholipase C (PI-PLC) assay as well as in vivo tests namely, chick embryo and mice inoculation tests. The isolates were subjected to PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. Listeria spp. were isolated from 12 (1.66%) samples. Of these 4 (0.55%) and 1 (0.14%) were confirmed as Listeria monocytogenes and Listeria ivanovii, respectively. L. monocytogenes and L. ivanovii were recovered from milk samples (2) and faecal (3) of mastitic cattle (3) and buffaloes (2). L. monocytogenes recovered from the milk of mastitic cattle and L. ivanovii from the faecal swab of buffalo turned out to be pathogenic. However, the remaining three hemolytic isolates exhibiting positive CAMP test turned out to be negative in PI-PLC assay, chick embryo and mice inoculation. L. monocytogenes and L. ivanovii isolates characterized as pathogenic by PI-PLC assay and in vivo pathogenicity tests were found to possess all the five virulence-associated genes and three genes, plcA, prfA and actA respectively. The remaining three hemolytic but non-pathogenic L. monocytogenes isolates were negative for plcA by PCR. It seems that the plcA gene and its expression (in the PI-PLC assay) have an important role as virulence determinants in pathogenic Listeria spp. In conclusion, the PI-PLC assay and virulence genes targeted PCR (plcA, prfA and hlyA genes for L. monocytogenes and plcA, prfA and actA genes for L. ivanovii) hold a good promise as rapid and reliable in vitro alternatives to in vivo pathogenicity tests.     

Characterization of Listeria monocytogenes isolates in import food products of China from 8 provinces between 2005 and 2007

A total of 48 Listeria monocytogenes isolates of different import food products from 8 provinces between 2005 and 2008 were characterized. The serotype and virulence were confirmed for each strain and molecular subtyping were analyzed by multilocus sequence typing (MLST). Twenty five strains were assigned to serotype 1/2a, and 11 isolates to serotype 1/2b, serotype 4b were found in 7 isolate, and the remaining 5 strains were grouped into serotypes 1/2c, 4a, and 4e. Molecular subtyping schemes found thirty two sequence types (STs) among these isolates and the majority of L. monocytogenes strains belonged to lineage II (56%), followed by lineage I (38%), lineage III (6%). Two molecular subtype clusters, cluster A included all isolates of lineage II, while cluster B contained the isolates of lineages I and lineages III. Two L. monocytogenes strains were not grouped in either of the two clusters. Fifty three isolates were as virulent as L. monocytogenes reference strain EGD in mouse virulence assay, while the isolates 22213 and 22265 had low pathogenicity. These results provide the first molecular insight into the L. monocytogenes strains isolated from import food products of 8 provinces in China and indicate the potential risk to cause human disease if intake by contaminated foods. MLST could be used as a routine subtyping method of L. monocytogenes isolates. In China, inspection and quarantine strategies of imported foods should be strengthened. PRACTICAL APPLICATION: There is a potential risk of listeriosis in China and routine subtyping of L. monocytogenes isolates is important. It is necessary for food hygiene management to strengthen the supervision of imported foods.     

Development of multilocus single strand conformation polymorphism (MLSSCP) analysis of virulence genes of Listeria monocytogenes and comparison with existing DNA typing methods

Development of rapid and simple typing methods is required for analyzing the distribution and contamination routes of food-borne pathogens. We established a simple typing method for Listeria monocytogenes using MLSSCP (Multilocus Single Strand Conformation Polymorphism) analysis. Four virulence genes, hlyA, iap, actA and inlB were amplified by PCR, digested with endonucleases and applied to gels for SSCP. As banding patterns have been shown to reflect even a single nucleotide difference, this method has a potential discriminatory power comparable to that of sequencing analysis. The 64 strains isolated from five meat processing plants were divided into 18 groups by this MLSSCP. Additionally, clustering obtained with this method showed strong correspondence with phylogenetic lineages I and II, and was achieved with much less expenditure in time and cost than is required for other methods, such as MLST. The validity of the MLSSCP lineage classification was confirmed by PFGE, AFLP and ribotyping results. This newly developed MLSSCP method is suitable when obtaining accurate results quickly and simply is crucial.     

台州市食源性单核细胞增生李斯特菌分子特征研究

目的了解台州市食品中分离的单核细胞增生李斯特菌的血清型、毒力基因以及基因分型情况,建立食源性单核细胞增生李斯特菌的分子特征本底信息,为食源性疾病的防治提供技术支持。方法对近几年从食品中分离的37株单核细胞增生李斯特菌进行多重PCR血清分型、9种毒力基因(prf A、inl A、inl B、iap、fla A、hly A、plc B、mpl和act A)PCR检测、PFGE基因分型,用Bio Numerics 6.6软件对分型数据进行聚类分析。结果 37株食源性单核细胞增生李斯特菌的血清型以1/2a或3a型别为主;所有菌株均检出4种以上毒力基因,有15株菌携带所有9种毒力基因;37株菌经Apa I酶切PFGE分型后,共得到22种带型,每种带型包含1~5株不等,相似度区间为67%~100%。结论台州市食源性单核细胞增生李斯特菌存在致病流行风险,建立的指纹图谱数据库可为食源性疾病的防治提供技术支持。     

Molecular Subtyping and Tracking of Listeria monocytogenes in Latin-Style Fresh-Cheese Processing Plants

Latin-style fresh cheeses, which have been linked to at least 2 human listeriosis outbreaks in the United States, are considered to be high-risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in 3 Latin-style fresh-cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-mo period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n = 111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. Listeria monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains, and floor samples showed the highest contamination rates, with 55.6, 30.0, and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the United States, was the most commonly identified subtype (20/36 isolates) and was isolated from 2 plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination.     

Prevalence of Listeria monocytogenes in broilers at the abattoir, processing plant, and retail level.

The environment and products from two broiler abattoirs and processing plants and raw broiler pieces at the retail level were sampled for Listeria monocytogenes in order to evaluate the contamination level of the broiler carcasses and products. Sampling started in the slaughtering process and finished with raw broiler meat or ready-to-eat cooked product. Sampling sites positive for L. monocytogenes at the broiler abattoir were the air chiller, the skin-removing machine, and the conveyor belt leading to the packaging area. The L monocytogenes contamination rate varied from 1 to 19% between the two plants studied. Furthermore, 62% (38 of 61) of the raw broiler pieces, bought from retail stores, were positive for L. monocytogenes. Altogether, 136 L. monocytogenes isolates were obtained for serotyping and pulsed-field gel electrophoresis (PFGE) characterization performed with two rare-cutting enzymes (ApaI and AscI). Altogether three serotypes (1/2a, 1/2c, and 4b) and 14 different PFGE types were obtained using information provided from both ApaI and AscI patterns for discrimination basis. The two broiler abattoirs studied did not share the same PFGE types. However, the same PFGE types found in the raw broiler pieces at the retail level were also found in the broiler abattoirs where the broilers had been slaughtered.     

Comparative characterization of Listeria monocytogenes isolated from Portuguese farmhouse ewe's cheese and from humans

In order to investigate the possible relationships between Listeria monocytogenes strains isolated from farmhouse ewe's cheese and clinical strains collected, in partially overlapping dates, from the same geographical area in Portugal, a total of 109 isolates from seven ewe's cheese manufactures (n=94) and from humans (n=15) were characterized by serotyping, RAPD, PFGE and allelic analysis of the virulent actA gene. Serotyping indicated the presence of four different serovars: 1/2a, 1/2b, 1/2c and 4b. The 15 clinical isolates were either serovar 4b (86.7%) or serovar 1/2b (13.3%). Among the 94 isolates from cheese and related environments the serovars prevalence was 1/2a (1.1%), 1/2b (17.0%), 1/2c (12.8%) and, unexpectedly, 4b (69.1%). Based on results obtained with PFGE typing of the strains, 25 genotypes were identified, 10 from farmhouses and 15 from human cases. Isolates from serovars 1/2a and 1/2c were assigned to single genotypes, respectively. Within serovars 1/2b and 4b three and 20 genotypes were established, respectively. RAPD typing of the isolates rendered 18 types indicating the lack of accuracy of the primers used in strain differentiation within serovar 4b. The actA gene typing of the strains showed a prevalence of actA gene type I (90.4%) compared with the rest of the strains that were all actA gene type II (9.6%). In spite of the fact that all the farmhouses were completely independent, the distribution of L. monocytogenes genotypes, intra and inter cheese manufactures, was relatively homogeneous, suggesting the existence of resident strains. In contrast, among human isolates there was a great genetic diversity. There was no common genotype between L. monocytogenes implicated in the cases of listeriosis and these cheese-related isolates, suggesting the absence of a causal relationship.