Italian Journal of Food Safety 2018; volume 7:6923 Microbiological profile of chick- en carcasses: A comparative Introduction Correspondence: Alessandra De Cesare, Techniques and technologies used for Dipartimento di Scienze e Tecnologie Agro- analysis using shotgun Alimentari, Università di Bologna, via del detection and characterization of foodborne metagenomic sequencing Florio 2, 40064 Ozzano dell’Emilia (BO), pathogens in food products have evolved Italy. tremendously over the past several decades Tel: +39.051.2097853 - Fax: +39.051.2097852. Alessandra De Cesare, Federica Palma, (Gracias and McKillip, 2004; Nugen and E-mail: [email protected] Alex Lucchi, Frederique Pasquali, Baeumner, 2008; Valderrama et al., 2015). Gerardo Manfreda Traditional methods for pathogen detection, Key words: Shotgun Metagenomic including microscopy and culture-based Sequencing, microbial profile, chicken carcas- Department of Agriculture and Food ses. analyses, are biased according to the specif- Sciences, Alma Mater Studiorum ic culture requirements for most genera and University of Bologna, Italy Contributions: AD, FeP, FrP, AL data collec- species. Moreover, they do not assess the tion; AD, GM experimental plan and data ana- microbiome at ecological level. More mod- lysis; AD, GM manuscript writing, reviewing ern approaches, including immunoassays and references search. Abstract and/or nucleic acid amplification, only allow for detection of single or a few specif- Conflict of interests: the authors declare no In the last few years metagenomic and ic pathogen(s) at a time. However, it is well potential conflict of interests. 16S rRNA sequencing have completly known that changes in the surrounding changed the microbiological investigations Funding: the work was supported by the EU environment cause stresses on bacterial of food products. In this preliminary study, founded project COMPARE (Grant populations, leading to reorganization of the microbiological profile of chicken car- Agreement N° 643476). microbial communities, which potentially casses collected from animals fed with dif- affects the persistence of foodborne Received for publication: 15 July 2017. ferent diets were tested by using shotgun pathogens in the food production chain Revision received: 5 December 2017. metagenomic sequencing. A total of 15 car- (Larsen et al., 2014; Pricope et al., 2013). Accepted for publication: 5 December 2017. casses have been collected at the slaughetr- Therefore, the real challenge is to assess the This work is licensed under a Creative house at the end of the refrigeration tunnel influence that the entire microbial commu- from chickens reared for 35 days and fed Commons Attribution-NonCommercial 4.0 nities have on presence of pathogens in International License (CC BY-NC 4.0). with a control diet (n=5), a diet supplemen- food products. ted with 1500 FTU/kg of commercial Within this framework, shotgun ©Copyright A. De Cesare et al., 2018 phytase (n=5) and a diet supplemented with metagenomic, which is the study of whole- Licensee PAGEPress, Italy 1500 FTU/kg of commercial phytase and community DNA extracted directly from Italian Journal of Food Safety 2018; 7:6923 3g/kg of inositol (n=5). Ten grams of neck samples, has been increasingly used in mul- doi:10.4081/ijfs.2018.6923 and breast skin were obtained from each tiple disciplines, particularly as sequencing carcass and submited to total DNA extrac- costs decrease and output increases tion by using the DNeasy Blood & Tissue (Manichanh et al., 2008). When compared most meta-genomics studies on the detec- Kit (Qiagen). Sequencing libraries have to target amplicon metagenomics (e.g., 16S tion of microbes in foods have focused on been prepared by using the Nextera XT rRNA gene sequencing), shotgun metage- characterizing the microbial ecology and DNA Library Preparation Kit (Illumina) nomics provides the potential for both high- micro-bial successions during fermenta- and sequenced in a HiScanSQ (Illumina) at er resolution identification of organisms tions (van Hijum et al., 2013). The opportu- 100 bp in paired ends. A number of (i.e., to the strain level), as well as study of nities for metagenomics approaches to sequences ranging between 5 and 9 million microbial communities without introduc- improve foodborne pathogen detection are was obtained for each sample. Sequence tion of sequencing bias due to unequal illustrated by a study that used metage- analysis showed that Proteobacteria and amplification of the target gene (Shah et al., nomics approaches to characterize the Firmicutes represented more than 98% of 2011). Although obtaining a complete indi- whole bacterial populations associated to vidual genome from metagenomic species composition associated with the carcass skin in all groups but their abun- sequences is still challenging, it is sufficient tomato phyllosphere – both on the native dances were different between groups. to characterize the major functions of the plant and in the pre-enrichment and enrich- Moraxellaceae and other degradative bacte- microbial communities, as well as to identi- ment media used to isolate Salmonella ria showed a significantly higher abundance fy their taxon by assigning to public (Ottersen et al., 2013). This study was con- in the control compared to the treated genome reference databases (Li et al., ducted because isolation of Salmonella groups. Furthermore, Clostridium perfrin- 2014). Overall, the goals for metagenomic from the tomato phyllosphere has previous- gens showed a relative frequency of abun- analysis are to understand i) community ly proven challenging despite the fact that dance significantly higher in the group fed composition/structure, including the taxo- tomatoes have been implicated as the with phytase and Salmonella enterica in the nomic breakdown and relative abundance source of several human salmonellosis out- group fed with phytase plus inositol. The of the various species; ii) genic contribution breaks. Interestingly, this metagenomic results of this preliminary study showed of each member of the community, includ- study identified considerable growth of that metagenome sequencing is suitable to ing number and functional capacity; iii) Paenibacillus spp. during enrichment; this is investigate and monitor carcass microbiota intra-species or intra-population hetero- important because this organism may out- in order to detect specific pathogenic and/or geneity of the genes (Scholz et al., 2012). compete or even kill Salmonella during degradative populations. Although there are many opportunities enrichment. In addition, sequences match- to use metage-nomics tools to support ing different Salmonella serovars were detection of foodborne pathogens from identified both from the uncultured samples foods and food-associated environments, as well as from different enrichments, sug- [page 62] [Italian Journal of Food Safety 2018; 7:6923] Short Communication gesting the presence of Salmonella despite for the host, the supplementation of phytase end of the refrigeration tunnel. All carcasses the fact that these samples were negative by can avoid the anti-nutritional effect of phyt- were obtained from birds belonging to the both bacteriological methods and real-time ic acid reducing endogenous losses and same breeder flock and hatching session, PCR. Although these findings do support increasing protein digestibility. housed in the same poultry house at the the possibility that Paenibacillus may have Since the effect of phytase supplemen- stocking density of about 10 chicks/m2 and outcompeted Salmonella during enrich- tation on microbiological profile of poultry fed with three different diets up to 35 days. ment, it is also possible that the detection of meat has been never investigated, in this A total of 5 carcasses were obtained from Salmonella DNA sequences is due to pres- preliminary study shotgun metagenomics birds fed with a basal diet (group A); 5 car- ence of dead Salmonella cells (Bergholz et has been applied to compare the microbial casses from birds fed with the basal diet al., 2014). compositions of 15 chicken carcasses col- supplemented with phytase at the concen- The administration of feed additives in lected at the end of the rearing period (i.e., tration of 1500 FTU/kg feed (group B); 5 chickens has been linked to changes in the 35 days) from animals fed with a control carcasses from birds fed with the basal diet animal gut influencing meat safety. diet and diets supplemented with 1500 supplemented with phytase at the concen- Lactobacillus administration has been FTU/kg of commercial phytase and 1500 tration of 1500 FTU/kg feed and 3g/kg shown to reduce colonization by foodborne FTU/kg of commercial phytase plus 3g/kg inositol (group C). According to the official pathogens like Campylobacter (Ghareeb et of inositol. sampling procedures to verify the hygienic al., 2012; Neal-McKinnet et al., 2012), quality of broiler carcasses, ten grams of Clostridium (La Ragione et al., 2004) and neck and breast skin were collected from Salmonella (Chen et al., 2012) improving each carcass and placed in a sterile bag with the microbial food safety of poultry meat. 40 mL of sterile saline solution. After Beside probiotics, broiler diet can be sup- Materials and Methods homogenization for 1 minute using the plemented with enzymes like phytase. A total of 15 poultry carcasses were ran- Pulsifier® (Microgen Bioproducts Ltd, Other than making the phosphorus available domly collected at the slaughterhouse at the Cambrige, UK) the whole