Beatriz Melero

Tracing Campylobacter jejuni strains along the poultry meat production chain from farm to retail by pulsed-field gel electrophoresis, and the antimicrobial resistance of isolates

In this study Campylobacter jejuni isolates were recovered from birds, carcasses and carcass portions from two broiler chicken flocks and from equipment used for carcass and meat processing along the production chain from farms to retail stores. Isolates were subjected to pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes and their antimicrobial susceptibilities were determined. C. jejuni was recovered from product and equipment used with both flocks at each point in the production chain. The prevalence of C. jejuni in poultry products at retail stores was 58.97% (flock 1) and 69.23% (flock 2). SmaI divided 122 C. jejuni strains from flock 1 and 106 from flock 2 into 17 and 13 PFGE types, respectively. PFGE types H and F were present at all steps along the chain, from farms to retail products. Similarly, for both flocks PFGE type D was detected in crates, slaughterhouse and retail stores. Moreover, the PFGE types were highly diverse at the processing and retail steps. Most PFGE types were resistant to ciprofloxacin (95.45%) and tetracycline (81.82%); and multidrug resistant PFGE types were found in the final products. Our study showed that there were several points of cross-contamination of product along the chain, and a high diversity of PFGE types with antimicrobial resistance to ciprofloxacin and tetracycline in the retail products.

Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1.

Characterization by culture-dependent and culture-independent methods of the bacterial population of suckling-lamb packaged in different atmospheres.

This study offers insight into the dynamics of bacterial populations in fresh cuts of suckling lamb under four different atmospheric conditions: air (A), and three Modified Atmosphere Packaging (MAP) environments, 15%O2/30%CO2/55%N2 (C, commercial), 70%O2/30%CO2 (O), and 15%O2/85%CO2 (H) for 18 days. Microbial analyses by both conventional methods and PCR-DGGE were performed. Controversial and surprising results emerged from comparing both methods in relation to the genus Pseudomonas. Thus, conventional methods detected the presence of high numbers of Pseudomonas colonies, although PCR-DGGE only detected this genus in air-packaged samples. PCR-DGGE detected higher microbial diversity in the control samples (A) than in the modified atmospheres (C, O, H), having atmosphere H the fewest number of species. Brochothrix thermosphacta, LAB (Carnobacterium divergens and Lactobacillus sakei), and Escherichia spp. were detected in all the atmospheres throughout storage. Moreover, previously undescribed bacteria from lamb meat such as Enterobacter hormaechei, Staphylococcus equorum and Jeotgalicoccus spp. were also isolated in this study by DGGE. Additionally, qPCR analysis was used to detect and characterize strains of Escherichia coli. Virulence genes (stx1, stx2 and eae) were detected throughout storage in 97% of the samples. A high CO2 atmosphere was the most effective packaging combination doubling storage time in comparison with commercial atmosphere.

Lactic acid bacteria as protective cultures in fermented pork meat to prevent Clostridium spp. growth.

In meat fermented foods, Clostridium spp. growth is kept under control by the addition of nitrite. The growing request of consumers for safer products has led to consider alternative bio-based approaches, the use of protective cultures being one of them. This work is aimed at checking the possibility of using two Lactobacillus spp. strains as protective cultures against Clostridium spp. in pork ground meat for fermented salami preparation. Both Lactobacillus strains displayed anti-clostridia activity in vitro using the spot agar test and after co-culturing them in liquid medium with each Clostridium strain. Only one of them, however, namely L. plantarum PCS20, was capable of effectively surviving in ground meat and of performing anti-microbial activity in carnis in a challenge test where meat was inoculated with the Clostridium strain. Therefore, this work pointed out that protective cultures can be a feasible approach for nitrite reduction in fermented meat products.

Listeria monocytogenes colonization in a newly established dairy processing facility

The presence and colonization of Listeria monocytogenes were investigated in a newly established dairy processing plant during a one-year period. A total of 250 non-food contact surfaces, 163 food contact surfaces, 46 personnel and 77 food samples were analyzed in two different buildings according to the cheese production chain. Initial steps, including salting, are performed in building I (old facility), while the final steps, including ripening, cutting and packaging, are performed in building II (new facility). Overall, 218 samples were collected from building I and 318 from building II. L. monocytogenes isolates were subtyped by PFGE and MLST, and a questionnaire about quality measures was completed. The overall prevalence of L. monocytogenes was 8.40%, and while the presence of the pathogen was observed just during the first sampling in building I, L. monocytogenes was found in building II at the third sampling event. The salting area in building I had the highest proportion of positive samples with the highest diversity of PFGE types. Moreover, L. monocytogenes PFGE type 3 (sequence type -ST- 204) was first detected in building II in the third visit, and spread through this building until the end of the study. The answers to the questionnaire implied that lack of hygienic barriers in specific parts of the facilities and uncontrolled personnel flow were the critical factors for the spread of L. monocytogenes within and between buildings. Knowledge of the patterns of L. monocytogenes colonization can help a more rational design of new cheesemaking facilities, and improve the food safety within current facilities.

Protective Cultures for the Safety of Animal-Derived Foods

The objective of this chapter is to explain the state of the art, about the real application of protective cultures in food preservation, and to answer in a simple way some questions related to this issue: What are protective cultures? Are they really different from other microbiological food cultures like starter or probiotics? Which bacteria can be used as protective culture? How do they work? Are they really useful? Are they effective against foodborne pathogen? Which are their limitations? Can they really improve by themselves the shelf life or safety of a food product? Can they be used commercially? In order to get this aim, we split this chapter in several sections: definitions, source of protective cultures, mode of action, and applications in animal-derived foods. We hope the reader will be able to find out, after reading this chapter, all the answers to the former questions. However, if the reader wants to focus on a specific topic, she/he will be addressed to more specialized literature recommended along the chapter.

Campylobacter in the Food Chain

Currently Campylobacter is the most commonly reported zoonosis in developed and developing countries. In the European Union, the number of reported confirmed cases of human campylobacteriosis was 246,307 in 2016, which represents 66.3 cases per 100,000 population. The genus Campylobacter includes 31 species with 10 subspecies. Within the genus Campylobacter, C. jejuni subsp. jejuni and C. coli are most frequently associated with human illness. Mainly, the infection is sporadic and self-limiting, although some cases of outbreaks have been also reported and some complications such as Guillain-Barré syndrome might appear sporadically. Although campylobacters are fastidious microaerophilic, unable to multiply outside the host and generally very sensitive, they can adapt and survive in the environment, exhibiting aerotolerance and resistance to starvation. Many mechanisms are involved in this, including pathogenicity, biofilm formation, and antibiotic resistant pathways. This chapter reviews the sources, transmission routes, the mechanisms, and strategies used by Campylobacter to persist in the whole food chain, from farm to fork. Additionally, different strategies are recommended for application along the poultry food chain to avoid the public health risk associated with this pathogen.

Characterization of Campylobacter species in Spanish retail from different fresh chicken products and their antimicrobial resistance

Contaminated chicken products have been recognized as the primary vehicles of Campylobacter transmission to human. Pulsed-field gel electrophoresis (PFGE) and antimicrobial resistance of Campylobacter isolates from fresh chicken products at retail were studied. A total of 512 samples including: thigh, breast, marinated and minced chicken were purchased from different retail stores. Half of the samples were packed and the other half were unpacked. The 39.4% of the samples were Campylobacter positive; being unpacked chicken products (45.3%) more contaminated than packed chicken (33.6%). PFGE typing showed a high diversity among isolates; clustering 204 isolates into 76 PFGE types: 55 clusters of C. jejuni, 19 of C. coli and 2 of C. lari. C. coli genotypes showed higher resistance than other Campylobacter species. Although modified atmosphere packaging can reduce the prevalence of Campylobacter spp., it does not avoid their presence in at least 33.6% of packed chicken products analyzed. Some pulsotypes might persist in the processing plant or butcher shops environment for longer than previously thought. More stringent control measures are needed in previous steps of the chicken food chain, in order to avoid the presence of Campylobacter spp. strains at retail that can compromise consumers safety.