Prevalence of Listeria monocytogenes in pork-meat and other processed products from the Colombian swine industry Prevalencia de Listeria monocytogenes en carnes y derivados de la industria porcícola colombiana

Objective. To determine the prevalence of L. monocytogenes in pork carcasses, meat cuts, and meat products (“chorizo”, sausage and ham). Materials and methods. Stratified sampling was implemented in meat-processed products. We analyzed 566 (37%) carcasses, 472 (31%) meat cuts, and 481, (32%) meat-processed products, distributed as follows: 169 (11%) sausage, 163 (11%) ham, and 149 (10%) “chorizo”, for a total of 1519 (100%) samples in a period of 18 months. The samples were processed using the ISO-17604, ISO-11290-1 and the USDA/FSIS (MLG-8.03) methods. Genus and species were confirmed by multiplex-PCR. Results. We obtained isolates of L. monocytogenes from 21 carcasses (10%), 160 (76%) from meat deboning, 10 (5%) from ham, 6 (3%) from “chorizo”, and 13 (6%) from sausage. The prevalence found was 3.7% and 33.9% in carcasses and meat deboning respectively. The prevalence in the meat-processed products was 4.03% in “chorizo”, 6.13% in ham and 7.69% in sausage. The overall prevalence of L. monocytogenes in the study was 13.82%. Conclusions. We found L. monocytogenes in different products analyzed, with particular interest in ham and sausage since both are consumed without previous heat treatment.


INTRODUCTION
L. monocytogenes is a Gram-positive non-spore forming bacilli that can be found individually, in pairs or as short chains of 3 to 5 microorganisms with a V or Y shape.It lacks a capsule and mobilizes by means of peritrichous flagella with an optimum mobilization temperature between 20 and 25°C, with little or no mobility at 37°C (1).Although they are Gram-positive bacilli, old cultures can express pleomorphism.As a zoonotic and emerging microorganism in the food industry, L. monocytogenes causes listeriosis in humans and animals (1,2).This ubiquitous bacterium can contaminate water, concentrates and especially farm silos, resulting in dissemination of L. monocytogenes during animal breeding.It is known for swines to excrete the bacteria thorugh their feces, thus acting as reservoirs (3).
L. monocytogenes has caused several food outbreaks, especially through a group of readyto-eat-food (REF), where meat-and dairyproducts are the most affected (4,5).In several international reports pork galantine and ham, among other contaminated foods, has been declared as an important source of outbreaks in France and Canada (1,(6)(7)(8).
Presently in Colombia there are no outbreak reports by meat product consumption.However, a recent study conducted by the National Institute of Surveillance and Control of Beverages, Medicines and Food (INVIMA) stated that meat products may be contaminated with this microorganism and could constitute a potential listeriosis' vehicle (9).Recently, the INVIMA in its control and surveillance program reported the withdrawal of a batch of imported ham ("Serrano" type) due to the presence of L. monocytogenes.The Colombian legislation does not require searching for L. monocytogenes in meat derivates.However, the Institute of Technical Norms (Icontec), in its standard 1325 (sixth update), establishes the rejection of a product if 25 grams of the sample contain L. monocytogenes.
According to the national policy for safety and health regarding the pork chain (10), the Colombian porcine sector has experienced a significant growth with a considerable improvement in productivity.However, there are no reports determining the minimum permitted for this pathogen in the pork chain.One of the objectives stated in this work was to determine the prevalence of L. monocytogenes in meat derivatives, to help improve the porcine national chain health status.In Colombia prevalence reports of L. monocytogenes in meat derivates are scarce.This is most likely due to the fact that studies related with this microorganism have been focused on milk derivates.The few existing reports show prevalence of about 14.3% in meat derivates (11).
The main objective of this work was to determine the prevalence of L. monocytogenes in pork carcasses, pork-meat and pork-meat processed products from the domestic pork industry.

MATERIALS AND METHODS
Pork processing plant selection.Plants were selected according to the following criteria: 1) pork-only abbatoirs 2) plants that comply to regulations with Decree 1500, i.e. pay a fee for development 3) plants located in areas determined by Fedegan (10).At the time of this study 42 processing plants were throughout the country, this study included 23.

Pork meat selection.
As the inclusion criteria we considered: 1.) markets that sell pork and 2.) points located in the pork-industry areas.
The number of samples to study was 472, distributed in 58 lots.
Selection of meat derivatives.For the selection of meat-processed products we took into account 1.) use of pork-meat as raw material and 2.) high consumption of pork-meat processed products.The products included were: cooked ham (sandwich type), low-cost sausage (cooked) and pre-cooked "chorizo".The number of included samples were 481, distributed as follows: 169 (35%) sausages, 163 (34%) ham, and 149 (31%) "chorizo".

Sample collection.
For pork carcasses, samples were collected using the nondestructive method described in standard ISO 17604 (12).Meat cuts were taken directly from market sale-points in different areas of the country, with a sample size of 500 g.Meatprocessed products were taken directly from the sale point in commercial presentations with an approximate size of 250 to 500 g.

Microbiological analysis.
The samples were transported to the Laboratory of Food Microbiology at the Pontificia Universidad Javeriana, under aseptic conditions to be processed immediately.The method used for the processing of pork carcasses was described by the Department of Agriculture of United States (USDA/FSIS) (13).ISO11290-1 method was used for meat cuts and meat-processed products.Briefly, 25 g of sample was taken and homogenized in 225 ml of Fraser-semi broth and incubated at 30°C for 48 hours.One hundred μl suspension was incubated in 10 ml Fraser broth and incubated for 40 hours at 35°C.L. monocytogenes was isolated in Ottaviani-Agosti and Palcam agar.Presumptive colonies were purified in TSAYE agar.Catalase test, sugar fermentation (mannitol, Xylose, rhamnose), hemolysis test and Camp test were carried-out.Isolates that biochemically corresponded to L. monocytogenes were subjected to molecular identification (genus and species).

Master Cell Bank (MCB) development.
A colony of each isolate was inoculated in 20 ml of BHI broth, and incubated at 35°C for 24 hours at 120 rpm.The culture was then mixed at a proportion of 1:1 with glycerol at 40% (v/v) and mixed thoroughly.One ml was placed in a cryopreservation vial, and stored at -20°C.Each MCB was periodically studied to verify the microbial purity and viability (14).

Genomic DNA extraction (gDNA).
For genomic DNA extraction, a frozen vial of each isolate was inoculated in 5 ml BHI broth supplemented with 0.5% (p/v) of glucose.Cells were cultured during 18 hours at 35°C and 120 rpm.One ml culture was centrifuged during 10 minutes at 5000 x g.Cells were washed with 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0±0.2).DNA was extracted by using the "Wizard Genomic DNA Purification Kit" (Promega, Madison WI, USA).Reactions were carried out in a Gene CyclerTM (BioRad, Philadelphia PA, USA) programmed in the following way: 1 minute at 95oC, followed by 40 cycles (30 s at 94°C, 20 s at 51°C, 30 s at 72°C), and a final extension step of 8 minutes at 72°C.L. monocytogenes ATCC 19115 was used as positive control (16).

Prevalence of L. monocytogenes.
The following formula was used to calculate prevalence (17).

Molecular identification of L. monocytogenes by Multiplex-PCR.
All the isolates previously identified as L. monocytogenes amplified the two expected products of 938 bp and 750 bp, for genus and the species respectively (16). Figure 1 shows representational results.

Meat cuts.
Fifty eight lots of fresh pork meat were analyzed and L. monocytogenes was isolated in 40 lots (69%).Out of the 40 lots thirteen samples were contaminated with L. monocytogenes (22.4%).The bacterium was not isolated in the remaining 31% (18/58) of pork meat samples.There was an overall high prevalence (33.9%, 160/472), (Figure 2

DISCUSSION
L. monocytogenes is a food-transmitted pathogen with implications in the food industry due to its ubiquitous nature.This feature allows the bacterium to be disseminated in the environment, facilitating accession to meat-processing plants, in particular pork-meat processing product plants, favoring food contamination (18)(19)(20).
In the present study the prevalence of L. monocytogenes in meat carcassess was 3.7%.These values are within the range of those published by other investigations: 2.0% in Finland (21), 4.1% in United States (22).Our results are lower than those presented by Gil and Jones who detected 8.3% in a Canadian plant.However, the Gil and Jones study only had 24 samples, which is not representative.Prevalence data from this research are consistent with several authors who have pointed out that processing plants are not a significant source of L. monocytogenes contamination.(23)(24)(25)(26)(27).

Pork meat derivates
The presence of the pathogen in swines is associated to the fact that these animals are carriers of L. monocytogenes.For example it has been shown that pork lodge this bacterium in tonsils and intestine (3,21,22,28).For this reason if during evisceration there is organ rupture, the carcass can be contaminated with L. monocytogenes.Although it is frequent for L. monocytogenes to contaminate carcasses and processing plants, subsequent washing and disinfecting procedures should get rid of the contamination.Pathogen-free product depends on the effectiveness of these processes (21).
In this study meat cuts had a prevalence of 33.9%.This percentage was the highest among all samples assayed.Our results agree with those reported by authors in different countries.(8).The low prevalence in these countries can be associated to control measures implementation during the stages of cleaning and disinfection.It has been determined during deboning there might be cross-contamination with utensils.In particular if this process is carried out at room temperature below 10°C.This favors the presence of L. monocytogenes due to its psychrotrophic nature (20,28).The conditions of contact surfaces and equipment affect the persistence of L. monocytogenes.Stainless steel surfaces are used in the food industry to prevent contamination (26).
In the present study prevalence of L. monocytogenes in sausage was 7.69%, similar to that found by Vera et al (11) (33).These studies show that there is variability in the prevalence of L. monocytogenes in sausage, which may be related to preparation process and storage (29).It is noteworthy that L. monocytogenes' prevalence is dependent on the country and the nature of the product-processing.As a case in point, raw fermented sausage or products without proper packaging can have a higher prevalence.In contrast, the samples evaluated in this study were sausages cooked and vacuum packed at the production plant.Sausages are regional products that vary depending on the country with regards to ingredients and production processes.In some cases they are considered a type of fermented sausage, making it difficult to compare porkmeat processed products from different origins, in particular with the international literature.
The prevalence of L. monocytogenes in "chorizo" was 4.0%, less than what was stated by Vera et al (11).They reported in Bogotá (2004) a prevalence of 18.1% with two positive samples out of 11 assessed.Their results might be due to a low sampling number and sampling of precooked sausage.
In the case of the ham the prevalence in the present research was 6.13%.This prevalence was higher than what was reported by Martins and Leal (0.8%) (20), but lower than that reported by Cabedo et al (33) (12.5%).Vera et al ( 11) also reported higher prevalence (16.8%) with 67 positive samples out of 398 analyzed.
Ham contamination may occur during slicing and/or packaging probably due to the presence of the pathogen in equipment, when hygiene conditions are not appropriate (20,26).
Chasseignaux et al. pointed-out to contamination of meat products with L. monocytogenes associated to cross-contamination with raw meat, equipment, processing areas (34), cooling areas, or even staff of the establishment.Therefore the incorporation of the microorganism in REF occurs after having subjected the product to thermal processes.Lastly, it is important to note that L. monocytogenes was found in different samples analyzed.This demonstrates the importance of pork meat products as a transmission vehicle for this bacterium.

Figure 2 .
Figure 2. Prevalence of L. monocytogenes in carcasses, meat cuts and meat products from the Colombian swine industry.