Victoria López

A 3-year surveillance of the genetic diversity and persistence of Listeria monocytogenes in an Iberian pig slaughterhouse and processing plant.

Contamination routes of Listeria monocytogenes were examined for 3 years in an Iberian pork-processing plant that produced high-quality ready-to-eat meat products. Molecular subtypes of L. monocytogenes were determined by polymerase chain reaction-based serotyping and pulsed-field gel electrophoresis (PFGE) restriction analysis. A total of 541 L. monocytogenes isolates were recovered from the environment and equipment (n = 165), carcasses (n = 28), raw products (n = 321), and dry-cured products (n = 27). Only 29 different PFGE types were identified, 3 of which were repeatedly found to be persistent types and accounted for 73% of the isolates. One PFGE type dominated (45% of the isolates) and was mostly recovered from intermediate manufactured products and the environment of the manufacturing area. L. monocytogenes persistence appeared strongly linked to the manufacture of products and not to its sustained entrance with the raw material. Some clones were found to survive in the manufacturing area for 3 years. Controlling the contamination of raw ingredients, improving the compartmentalization, and changing the cleaning protocols resulted in reduced prevalence rates of L. monocytogenes on products; two persistent PFGE types were eliminated from the processing plant, although eradication of other adapted strains has not been achieved.

Molecular tracking of Listeria monocytogenes in an Iberian pig abattoir and processing plant

The environment and products from an Iberian pig abattoir and processing plant were sampled to investigate the prevalence and genetic diversity of Listeria monocytogenes. The organism was not detected in the pig carcasses prior to processing. Fresh and dry-cured pork did show detectable levels, always ranging below 100CFU per gram. A total of 163 L. monocytogenes isolates collected during one year were characterized by PCR-based serotyping and pulsed-field gel electrophoresis (PFGE) restriction analysis. Three predominant PFGE types or pulsotypes seemed to persist in the plant. The pulsotype S1 (serotype group 1/2a, 53% of the isolates) was mostly recovered from whole pieces of meat and environmental sites, while pulsotypes S2 (1/2a, 17%) and S4 (1/2b, 21%) were more frequently associated with ground pork products. The pulsotype S4 was also found in a grinding machine, suggesting a possible association of this machine with the contamination of the ground meat products.

The influence of subminimal inhibitory concentrations of benzalkonium chloride on biofilm formation by Listeria monocytogenes.

Disinfectants, such as benzalkonium chloride (BAC), are commonly used to control Listeria monocytogenes and other pathogens in food processing plants. Prior studies have demonstrated that the resistance to BAC of L. monocytogenes was associated with the prolonged survival of three strains of molecular serotype 1/2a in an Iberian pork processing plant. Because survival in such environments is related to biofilm formation, we hypothesised that the influence of BAC on the biofilm formation potential of L. monocytogenes might differ between BAC-resistant strains (BAC-R, MIC≥10mg/L) and BAC-sensitive strains (BAC-S, MIC≤2.5mg/L). To evaluate this possibility, three BAC-R strains and eight BAC-S strains, which represented all of the molecular serotype 1/2a strains detected in the sampled plant, were compared. Biofilm production was measured using the crystal violet staining method in 96-well microtitre plates. The BAC-R strains produced significantly (p<0.05) less biofilm than the BAC-S in the absence of BAC, independent of the rate of planktonic growth. In contrast, when the biofilm values were measured in the presence of BAC, one BAC-R strain (S10-1) was able to form biofilm at 5mg/L of BAC, which prevented biofilm formation among the rest of the strains. A genetic determinant of BAC resistance recently described in L. monocytogenes (Tn6188) was detected in S10-1. When a BAC-S strain and its spontaneous mutant BAC-R derivative were compared, resistance to BAC led to biofilm formation at 5mg/L of BAC and to a significant (p<0.05) stimulation of biofilm formation at 1.25mg/L of BAC, which significantly (p<0.05) reduced the biofilm level in the parent BAC-S strain. Our results suggest that the effect of subminimal inhibitory concentrations of BAC on biofilm production by L. monocytogenes might differ between strains with different MICs and even between resistant strains with similar MICs but different genetic determinants of BAC resistance. For BAC-R strains similar to S10-1, subminimal inhibitory BAC may represent an advantage, compensating for the weak biofilm formation level that might be associated with resistance. Biofilm formation in the presence of increased subminimal inhibitory concentrations of the disinfectant may represent an important attribute among certain resistant and persistent strains of L. monocytogenes.

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.

Different Enrichment Procedures for Recovery of Listeria monocytogenes from Raw Chicken Samples Can Affect the Results of Detection (by Chromogenic Plating or Real-Time PCR) and Lineage or Strain Identification

This study aimed to evaluate the effect of different enrichment procedures on the detection of Listeria monocytogenes in food, by a comparison of subculture onto chromogenic agar with real-time PCR. Two different culture media, the primary and secondary enrichment broths of the U.S. Food Safety and Inspection Service (FSIS) method used for PCR detection of L. monocytogenes, were compared for the primary enrichment of retail ground chicken samples. L. monocytogenes was detected after the completion of each enrichment procedure in 63% (complete FSIS procedure) and 60% (plain FSIS secondary enrichment broth incubated for 48 h) of the samples by both culture and PCR, whereas a combination of the results for the two enrichment procedures revealed 86% of the samples to be positive. Most of the samples analyzed contained a mixture of lineage I and II strains, and their ratio varied for each enrichment procedure. This mixture could have a significant effect on the result of detection of L. monocytogenes for each individual sample, explaining the increase in positive samples when the results of the two enrichment procedures were combined. The use of different isolation procedures can affect the specific samples identified as positive and the specific strains isolated.

Low potential virulence associated with mutations in the inlA and prfA genes in Listeria monocytogenes isolated from raw retail poultry meat.

Packaged raw foods can represent a potential source of Listeria monocytogenes contamination when opened at home, and listeriosis is associated with the consumption of undercooked raw foods. The aim of this study was to characterize a group of L. monocytogenes strains isolated from 56 packages of raw chicken meat from a single brand in order to determine the diversity of the strains that dominate in a particular food over time, as well as their pathogenic potential. Forty (71%) samples were found to be positive for L. monocytogenes, and three isolates per sample were subjected to PCR molecular serotyping. Subtyping of 45 isolates from different manufacturing dates (n = 40) or different molecular serotype within the same sample (n = 5) identified 11 different L. monocytogenes subtypes as defined by pulsed-field gel electrophoresis and sequencing of virulence genes actA and inlA. Two of the subtypes accounted for 51% of the isolates. About 40% of isolates (three subtypes) were found to potentially present attenuated virulence because of the presence of mutations in the prfA and inlA genes.