Technology Updates Food

Project number: 6697 Date: Jan 2020 Funding source: Teagasc Project dates: Jan 2016-Dec 2019

Technological and

safety evaluation of the food culture

Macrococcus

caseolyticus

Key external stakeholders:  Regulatory bodies  Commercial culture suppliers  Fermented dairy food producers  Flavour ingredient producers  Wider dairy industry  Culture and flavour research communities

Practical implications for stakeholders:

The European Food Safety Authority (EFSA), along with other committees and expert groups, has made recommendations on key scientific criteria for evaluation of microbial food cultures (MFC), but to date, there is no consensus document at European level. The International Dairy Federation maintains an inventory of microbial species with a documented presence in foods. Species are included unless there is scientifically documented evidence that it is undesirable in foods. Macrococcus caseolyticus is an organism known to form part of the secondary microflora of certain cheese types (Ragusano, Fortina) and some fermented meats but it also reported as being deliberately inoculated as part of the starter blend in the production of certain hard cheeses. This work, through an extensive genomic and metabolic analysis, demonstrates that while this organism has a role to play in flavour development in fermented dairy products, the presence of antimicrobial resistance genes and some virulence factors suggests it is at the boundary of what is acceptable in a food-grade culture.

Main results:  Dairy-derived strains of the species Macrococcus caseolyticus drive flavour formation through the action of a highly active cell-envelope proteinase but lack significant peptidolytic activity.  Species of the Macrococcus genus are characterised by genetic and metabolic diversity with evidence of extensive horizontal gene transfer between species and other genera and the presence of antimicrobial resistance and virulence genes.  A novel species, Macrococcus linguae sp.nov., and two novel subspecies, Macrococcus goetzii subsp. corkensis and Macrocococcus canis subsp. bovinus, were isolated from bovine tongue using a PCR-based detection method designed in this study.

Opportunity / Benefit: Polyphasic approaches such as this, combining genomics, metabolomics and other analysis, can be undertaken in collaboration with interested groups to assess whether the presence of an organism in foods is beneficial, fortuitous or undesirable.

Collaborating Institutions: UCC, UCD, AgResearch.

1 Contact Olivia McAuliffe Email: [email protected]

http://www.teagasc.ie/publications/ Technology Updates Food

Teagasc project team: Dr. Olivia McAuliffe (PI) Dr. Shahneela Mazhar Dr. Kieran Kilcawley

External collaborators: Prof. Colin Hill, UCC. Prof. Séamus Fanning, UCD Dr. Scott Nyugen, UCD Dr. Eric Altermann, AgResearch

1. Project background: For the generation of flavour and aroma in cheese, it is widely agreed that the proteolytic system and amino acid-converting enzymes possessed by the starter cultures are of pivotal importance. To date, research in the field has focused primarily on members of the lactic acid (LAB), a group of organisms commonly associated with dairy fermentations. However, examination of the metabolic diversity which exists naturally in strains from outside the dairy environment may provide a means to identify novel cultures with enhanced flavour and aroma-forming activities for use in food fermentations, and this has attracted increased interest in recent times. Macrococus caesolyticus is not typically associated with dairy foods but the species does have a documented presence in fermented foods. In different varieties of soft and hard cheeses, strains of this species appear to be a component of the secondary microflora, playing a role in ripening and development of the final organoleptic qualities. The deliberate use of Macrococus caesolyticus as an adjunct for flavour formation has also been reported. Certain strains have demonstrated significant proteolytic activity towards milk proteins and a positive influence on flavour development in cheese. This aim of this project was to examine Macrococcus caseolyticus from a technological (flavour and aroma) and safety perspective by employing a polyphasic approach to examine the biology of the organism, its evolutionary relationship with , and its potential for use in dairy fermentations as a driver of flavor formation.

2. Questions addressed by the project:  What is the extent of the genetic and metabolic diversity within the Macrococcus caseolyticus species?

 What is the relationship of this species to other members of the Macrococcus genus and to its sister genus Staphylococcus?

 What does the proteolytic system of the Macrococcus caseolyticus species look like?

 What is the potential of strains from this group for the production of flavour compounds?

 Is there evidence that this organism may be undesirable for use in foods?

3. The experimental studies:  Establishment of a Macrococcus strain bank. A rapid PCR-based method was developed to isolate and establish a strain collection of M. caseolyticus and M. canis from diverse sources, including raw milk and bovine tongue swabs. The PCR method was based on the partial amplification of a region within the cytochrome c oxidase (COX) subunit II (ctaC) gene which is present in M. caseolyticus and M. canis but absent in most Staphylococcus species, including S. aureus.

 Genome analysis of novel strains isolated. Whole genome sequencing and extensive comparative analysis (phylogeny based on WGS and 16S rRNA, ANI and dDDH) was undertaken to examine the pangenome of the Macrococcus genus to establish the relationship to closely related species.

 Phenotype microarrays. The Biolog system was used extensively in this study for two specific purposes. Firstly, the GEN-III plate system was used for the purposes of microbial identification for the novel isolates recovered and secondly, the PM plate system was used to characterize the strains in terms of nitrogen metabolism and resistance to antimicrobials.

2 Contact Olivia McAuliffe Email: [email protected]

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 Enzymatic and metabolomic analysis. The flavor forming capacity of strains of Macrococcus caseolyticus were studied using assays for enzymes of the proteolytic pathway (CEP, peptidase, AraT and GDH) and those of the lipolytic pathway (lipases and esterases). Gas chromatography- mass spectrometry was used to identify volatile flavor compounds and HPLC was employed to examine casein degradation.

4. Main results:  A rapid PCR-based method to discriminate Macrococcus caseolyticus and from closely-related Staphylococcus species based on the ctaC gene sequence was developed. This work demonstrated that a PCR-based method designed for partial amplification of the ctaC gene, encoding the cytochrome c oxidase (COX) subunit II (ctaC), is capable of discriminating M. caseolyticus and M. canis species from a background of Staphylococcus. Screening of raw milk and bovine tongue samples with the developed method resulted in the isolation of 13 distinct strains of the targeted species.

 A novel bacterial species, Macrococcus linguae sp. nov., was isolated from bovine tongue. The taxonomic affiliation of strain DPC7161T, isolated with the newly-developed PCR method, with a morphology similar to the one described for the Macrococcus genus was delineated on the basis of a polyphasic approach. Phylogenetic analysis of the 16S rRNA gene sequence determined that the strain DPC7161T shared the highest sequence identity with Macrococcus caseolyticus subsp. caseolyticus DSM 20597T (98.5 %) and formed an independent branch from other macrococci in the phylogenetic tree. Average nucleotide identity (ANI) < 74 % similarity, in silico DNA-DNA hybridisation (DDH) < 21 % similarity and genome-based relatedness values between DPC7161T and type strain of all of the known Macrococcus species suggested that the isolate should be assigned to a new species within the genus Macrococcus. Strain DPC7161T displayed differential phenotypic characteristics from M. caseolyticus and the other characterised Macrococcus species in terms of its ability to ferment D-mannitol and D-sorbitol and its inability to produce acid from lactose. Based on genomic, genotypic, and phenotypic characteristics, strain DPC7161T represents a novel species of the genus Macrococcus, for which the name Macrococcus linguae sp. nov. is proposed.

 Two new subspecies, Macrcoccus canis subsp. bovinus subsp. nov. and Macrocccus goetzii subsp. corkensis subsp. nov, were proposed on the basis of whole-genome analysis. During the screening programme, ten Gram-positive cocci were isolated from bovine tongue samples. The 16S rRNA gene sequences of these strains identified these isolates as Macrococcus, belonging to two species, namely Macrococcus canis and Macrococcus goetzii. Strains DPC 7158T, DPC 7160, DPC 7162, DPC 7163, DPC 7165, DPC 7168, and DPC 7169 shared ≥ 99.9 % sequence identity with the 16S rRNA gene sequence of M. canis KM 45013T. The other three strains, DPC 7159, DPC 7164, and DPC 7166T, shared 100 % identity with the 16S rRNA gene sequence of M. goetzii CCM 4927T. ANI and dDDH values obtained for these strains were slightly higher than the threshold generally accepted for delineating different species (95-96 % and 70 %, respectively). In addition, differential phenotypic characteristics of these strains from their respective type strains further supported the taxonomic characterisation of these strains as a novel subspecies of M. canis and M. goetzii. Macrococcus canis subsp. bovinus subsp. nov. type strain DPC 7158T and Macrococcus goetzii subsp. corkensis subsp. nov. type strain DPC 7166T were proposed.

 A systems-wide analysis of proteolytic and lipolytic pathways uncovers the flavour-forming potential of Macrococcus caseolyticus subsp. caseolyticus A bank of M. caseolyticus subsp. caseolyticus strains derived from dairy and non-dairy sources were examined. Enzyme activities analysed comprised those of the proteolytic and lipolytic cascades. Strain to strain variation was observed, often associated with niche. All strains, except those isolated from non-dairy sources, demonstrated high CEP activity. Such high CEP activity associated with dairy strains implies the importance of this characteristic in the adaptation of these strains to a dairy- specific niche. HPLC profiles confirmed the extensive hydrolysis of milk proteins induced by CEP- active strains. However, limited downstream peptidolytic activity, in addition to a limited ability to generate free amino acids was observed across all strains, indicating weak ability of this organism to generate amino-acid derived flavor compounds. Interestingly, the strains with high CEP activity also demonstrated high esterase activity and gas chromatography-mass spectrometry analysis of the volatile compounds produced when these strains were grown in lactose-free milk demonstrated

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differences in the range and types of volatiles produced. Hierarchical clustering of the total volatile compounds identified illustrated the separation of high CEP and esterase active strains from those relatively less active.

 Species of the Macrococcus genus harbour a large open pangenome encoding antimicrobial resistance genes and some virulence factors. Genus-wide distributions of genomic islands (GIEs) carrying clusters of carbohydrate metabolism genes, resistance determinants and mobile genetic elements amongst others, were the source of genomic differences between strains of the same species. The presence of large numbers of transposases genes and prophages in a number of strains indicated multiple encounters with foreign DNA. The genus-wide distribution of com operon suggests a likely contributor for the high genomic diversity. All of the antimicrobial resistance (AMR) genes identified were associated with mobile genetic elements (MGE) harboured on GIEs. Furthermore, a number of notable AMR genes in M. bohemicus DPC7215 were identified. These include a resistance island carrying the novel methicillin resistance homolog mecD, which was previously defined in M. caseolyticus IMD0819. The presence of the plasmid pS0385-2, carrying streptomycin resistance, in M. bohemicus DPC7215 suggests that horizontal gene transfer (HGT) is a key driver in the spread of AMR resistance across the members of the Macrococcus genus and highlights the potential for further spread of these AMR determinants to related veterinary pathogenic bacteria.

5. Opportunity/Benefit: The granting of QPS (qualified presumption of safety) status to any microbial culture used in food production is based on scientific rationale, and the polyphasic approach used in this study, combining genomic and metabolomics, can be offered to food companies, culture suppliers and others, to develop a portfolio of information on strains of interest for safety evaluation.

6. Dissemination: The work outlined in this project has been disseminated by means of peer-reviewed publications (see below) and has been presented at a number of conferences, both nationally and internationally, including the 47th Annual Food Research Conference, Dublin, 2018; the American Dairy Science Association Annual Meeting 2018, Knoxville, TN, USA; the 26h International ICFMH Conference (Food Micro 2018), Germany, 2018; the 6th World Congress and Exhibition on Antibiotics and Antibiotic Resistance, UK, 2019; the 9th International Conference of the Swedish South Asian Network (SASNET) on Fermented Foods, India, 2019.

Main publications:  Mazhar, S., Kilcawley, K. N., Hill, C. and McAuliffe, O. A Systems-wide analysis of Proteolytic and Lipolytic pathways uncovers the Flavour-forming Potential of the Gram-positive bacterium Macrococcus caseolyticus subsp. caseolyticus. Frontiers in Microbiology. In press.  Mazhar S, Altermann E, Hill C, McAuliffe O. 2019. Draft Genome Sequences of the Type Strains of Six Macrococcus Species. Microbiol Resour Announc. 2019 May 9;8(19). pii: e00344-19. doi: 10.1128/MRA.00344-19.  Mazhar S, Altermann E, Hill C, McAuliffe O. 2019. Draft genome sequences of Macrococcus caseolyticus, Macrococcus canis, Macrococcus bohemicus, and Macrococcus goetzii. Microbiol Resour Announc. 2019 May 9;8(19). pii: e00343-19. doi: 10.1128/MRA.00343-19.  Mazhar S, Hill C, McAuliffe O. 2018. The Genus Macrococcus: An Insight Into Its Biology, Evolution, and Relationship With Staphylococcus. Adv Appl Microbiol. 2018;105:1-50. doi: 10.1016/bs.aambs.2018.05.002. Epub 2018 Jun 13.  Mazhar S, Hill C, McAuliffe O. 2018. A rapid PCR-based method to discriminate Macrococcus caseolyticus and Macrococcus canis from closely-related Staphylococcus species based on the ctaC gene sequence. J Microbiol Methods. 2018 Sep;152:36-38. doi: 10.1016/j.mimet.2018.07.008. Epub 2018 Jul 17.

7. Compiled by: Olivia McAuliffe

4 Contact Olivia McAuliffe Email: [email protected]

http://www.teagasc.ie/publications/