Biotyping and antibiotic susceptibility of animal streptococci from raw/thermized milks of native Epirus sheep breeds

Abstract

Aim: Animal streptococci surviving thermization of raw milk prior to traditional Greek cheese processing may include both harmless and potentially harmful strains with multiple antibiotic resistances. Therefore, the aims of this study were to biotype and evaluate the antibiotic resistance and primary technological and safety properties of wild streptococci occurring in Greek sheep milk before and after mild thermization. Methods: Sixteen Streptococcus isolates previously isolated from two raw/thermized (65^oC; 30 s) counterpart milks of native Epirus sheep breeds and identified by 16S ribosomal RNA (rRNA) gene sequencing were biochemically characterized using conventional methods (phenotypic tests, sugar fermentation patterns, API 20 STREP and API ZYM profiles) and assessed for susceptibility to eight antibiotics by the disk diffusion method, and for their milk acidification capacity, bacteriocin activity, enzymatic activity profiles, and biogenic amine (BA) formation in vitro. Results: Tetracycline-resistant (57% of the total 7 isolates) Streptococcus parauberis and ciprofloxacin-resistant Streptococcus gallolyticus subsp. pasteurianus (1 isolate) and Streptococcus lutetiensis (2 isolates) strains occurring in raw milk were not recovered post-thermally. Instead, thermization selected for a tetracycline-resistant (50% of the total 6 isolates) thermophilic group previously genotyped as Streptococcus equinus, herein biotyped as Streptococcus pneumoniae. None of the Streptococcus isolates was resistant to ampicillin, chloramphenicol, erythromycin, gentamycin, penicillin, or vancomycin, or β-hemolytic, or produced histamine and tyramine. The milk acidifying activity rate (final skimmed milk pH 4.79 to 5.47 after growth at 37^oC for 6 h, gradually decreasing to 22^oC for a total of 48 h) of the isolates was species-dependent and increased in the order S. equinus > S. gallolyticus > S. lutetiensis > S. parauberis. Conclusions: Except for the two bacteriocin-producing and α-galactosidase-positive S. lutetiensis antilisterial strains (KFM 55 and KFM 60), which show promise, all animal streptococci derived from Epirus sheep milk should be considered a priori unsafe for inclusion in complex natural cheese starter cultures because they belong to pathogenic, mastitis-causing species or subspecies. The two promisingS. lutetiensis strains require further safety evaluations for streptococcal virulence and antibiotic resistance genes, mainly to ciprofloxacin, before their commercial use as natural cheese starters.

Keywords

sheep milk / thermization / antibiotic resistance / animal streptococci / Streptococcus lutetiensis / S. parauberis / S. equinus / S. gallolyticus

References

[1] Tilocca B, Costanzo N, Morittu VM, Spina AA, Soggiu A, Britti D, et al. Milk microbiota: Characterization methods and role in cheese production. J Proteom. 2020; 210: 103534.

[2] Stepaniak L, Rukke EO. Thermization of milk. In: Roginski H, Fuquay JW, Fox P, editors. Encyclopedia of Dairy Sciences, London: Elsevier Science Ltd. Academic Press; 2003. pp. 2619—23.

[3] Eugster E, Jakob E. Pre—treatments of milk and their effect on the food safety of cheese. Milk Sci Int. 2019; 72: 45-52.

[4] Lindsay D, Robertson R, Fraser R, Engstrom S, Jordan K. Heat induced inactivation of microorganisms in milk and dairy products. Int Dairy J. 2021; 121: 105096.

[5] Samelis J, Lianou A, Kakouri A, Delbès C, Rogelj I, Bogovic—Matijasić B, et al. Changes in the Microbial Composition of Raw Milk Induced by Thermization Treatments Applied Prior to Traditional Greek Hard Cheese Processing. J Food Prot. 2009; 72: 783-90.

[6] Samelis J, Bosnea L, Kakouri A. Microbiological Quality and Safety of Raw Sheep Milks from Native Epirus Breeds: Selective Effects of Thermization on the Microbiota Surviving in Resultant Thermized Milks Intended for Traditional Greek Hard Cheese Production. Appl Microbiol. 2025; 5: 11.

[7] Coelho MC, Malcata FX, Silva CCG. Lactic Acid Bacteria in Raw—Milk Cheeses: From Starter Cultures to Probiotic Functions. Foods. 2022; 11: 2276.

[8] Chessa L, Paba A, Dupré I, Daga E, Fozzi MC, Comunian R. A Strategy for the Recovery of Raw Ewe’s Milk Microbiodiversity to Develop Natural Starter Cultures for Traditional Foods. Microorganisms. 2023; 11: 823.

[9] Chessa L, Daga E, Dupré I, Paba A, Fozzi MC, Dedola DG, et al. Biodiversity and Safety: Cohabitation Experimentation in Undefined Starter Cultures for Traditional Dairy Products. Fermentation. 2024; 10: 29.

[10] Švec P, Devriese LA. Genus I Enterococcus (ex Thiercelin and Jouhaud 1903) Schleifer and Klipper—Bälz 1984, 32VP. In: Whitman WB, editor. Bergey’s Manual of Systematic Bacteriology, The Firmicutes. Vol. 3. 2nd ed. New York: Springer; 2009. pp. 594-607.

[11] Whiley RA, Hardie JM. Genus I Streptococcus Rosenbach 1884, 22AL. In: Whitman WB, editor. Bergey’s Manual of Systematic Bacteriology, The Firmicutes, Vol. 3. 2nd ed. New York: Springer; 2009. pp. 655-711.

[12] Dapkevicius MLE, Sgardioli B, Câmara SPA, Poeta P, Malcata FX. Current Trends of Enterococci in Dairy Products: A Comprehensive Review of Their Multiple Roles. Foods. 2021; 10: 821.

[13] Glajzner P, Szewczyk EM, Szemraj M. Pathogenicity and drug resistance of animal streptococci responsible for human infections. J Med Microbiol. 2021; 70: 001339.

[14] Dimov SG. The Controversial Nature of Some Non—Starter Lactic Acid Bacteria Actively Participating in Cheese Ripening. BioTech. 2023; 12: 63.

[15] Zhang H, Yang F, Li X, Luo J, Wang L, Zhou Y, et al. Detection of antimicrobial resistance and virulence—related genes in Streptococcus uberis and Streptococcus parauberis isolated from clinical bovine mastitis cases in northwestern China . J Integr Agric. 2020; 19: 2784—91.

[16] Chen P, Qiu Y, Liu G, Li X, Cheng J, Liu K, et al. Characterization of Streptococcus lutetiensis isolated from clinical mastitis of dairy cows . J Dairy Sci. 2021; 104: 702—14.

[17] Rosa NM, Penati M, Fusar—Poli S, Addis MF, Tola S. Species identification by MALDI—TOF MS and gap PCR—RFLP of non—aureus Staphylococcus, Mammaliicoccus, and Streptococcus spp. associated with sheep and goat mastitis . Vet Res. 2022; 53: 84.

[18] De Vuyst L, Tsakalidou E. Streptococcus macedonicus, a multi—functional and promising species for dairy fermentations . Int Dairy J. 2008; 18: 476-85.

[19] Jans C, Meile L, Kaindi DWM, Kogi—Makau W, Lamuka P, Renault P, et al. African fermented dairy products — Overview of predominant technologically important microorganisms focusing on African Streptococcus infantarius variants and potential future applications for enhanced food safety and security . Int J Food Microbiol. 2017; 250: 27-36.

[20] Özkan ER, Öztürk Hİ, Demirci T, Akın N. Detection of biofilm formation, virulence factor genes, antibiotic—resistance, adherence properties, and some beneficial properties of cheese origin S. infantarius, S. gallolyticus, and S. lutetiensis strains belonging to the S. bovis/S. equinus complex . LWT. 2021; 150: 112077.

[21] Güley Z, Fallico V, Cabrera—Rubio R, Cotter PD, Beresford T. Identification of Streptococcus infantarius subsp. infantarius as the species primarily responsible for acid production in Izmir Brined Tulum Cheese from the Aegean Region of Türkiye . Food Res Int. 2022; 160: 111707.

[22] EFSA Panel on Biological Hazards (BIOHAZ), Allende A, Alvarez—Ordóñez A, Bortolaia V, Bover—Cid S, De Cesare A, et al. Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 21: Suitability of taxonomic units notified to EFSA until September 2024. EFSA J. 2025; 23: e9169.

[23] Sioziou E, Kakouri A, Bosnea L, Samelis J. Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture—dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota. Curr Res Microb Sci. 2024; 6: 100209.

[24] Samelis J, Kakouri A. Antibiotic Susceptibility of Autochthonous Enterococcus Strain Biotypes Prevailing in Sheep Milk from Native Epirus Breeds Before and After Mild Thermization in View of Their Inclusion in a Complex Natural Cheese Starter Culture . Appl Microbiol. 2025; 5: 125.

[25] Kim EB, Kopit LM, Harris LJ, Marco ML. Draft Genome Sequence of the Quality Control Strain Enterococcus faecalis ATCC 29212 . J Bacteriol. 2012; 194: 6006—7.

[26] CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 26th ed. Supplement M100S. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

[27] Tsanasidou C, Asimakoula S, Sameli N, Fanitsios C, Vandera E, Bosnea L, et al. Safety Evaluation, Biogenic Amine Formation, and Enzymatic Activity Profiles of Autochthonous Enterocin—Producing Greek Cheese Isolates of the Enterococcus faecium/durans Group . Microorganisms. 2021; 9: 777.

[28] Samelis J, Tsanasidou C, Bosnea L, Ntziadima C, Gatzias I, Kakouri A, et al. Pilot—Scale Production of Traditional Galotyri PDO Cheese from Boiled Ewes’ Milk Fermented with the Aid of Greek Indigenous Lactococcus lactis subsp. cremoris Starter and Lactiplantibacillus plantarum Adjunct Strains . Fermentation. 2023; 9: 345.

[29] Parapouli M, Delbès—Paus C, Kakouri A, Koukkou AI, Montel MC, Samelis J. Characterization of a Wild, Novel Nisin A—Producing Lactococcus Strain with an L. lactis subsp. cremoris Genotype and an L. lactis subsp. lactis Phenotype, Isolated from Greek Raw Milk . Appl Environ Microbiol. 2013; 79: 3476-84.

[30] Bover—Cid S, Holzapfel WH. Improved screening procedure for biogenic amine production by lactic acid bacteria. Int J Food Microbiol. 1999; 53: 33-41.

[31] Schlegel L, Grimont F, Ageron E, Grimont PAD, Bouvet A. Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. gallolyticus subsp. pasteurianus subsp. nov. Int J Syst Evol Microbiol. 2003; 53: 631—45.

[32] Schlegel L, Grimont F, Collins MD, Régnault B, Grimont PA, Bouvet A. Streptococcus infantarius sp. nov., Streptococcus infantarius subsp. infantarius subsp. nov. and Streptococcus infantarius subsp. coli subsp. nov., isolated from humans and food . Int J Syst Evol Microbiol. 2000; 50 Pt 4: 1425—34.

[33] Poyart C, Quesne G, Trieu—Cuot P. Taxonomic dissection of the Streptococcus bovis group by analysis of manganese—dependent superoxide dismutase gene (sodA) sequences: reclassification of ' Streptococcus infantarius subsp. coli' as Streptococcus lutetiensis sp. nov. and of Streptococcus bovis biotype 11.2 as Streptococcus pasteurianus sp. nov. Int J Syst Evol Microbiol. 2002; 52: 1247—55.

[34] Morandi S, Silvetti T, Miranda Lopez J, Brasca M. Antimicrobial Activity, Antibiotic Resistance and the Safety of Lactic Acid Bacteria in Raw Milk Valtellina Casera Cheese. J Food Saf. 2014; 35: 193-205.

[35] Pitkälä A, Koort J, Björkroth J. Identification and Antimicrobial Resistance of Streptococcus uberis and Streptococcus parauberis Isolated from Bovine Milk Samples . J Dairy Sci. 2008; 91: 4075—81.

[36] McDonald WL, Fry BN, Deighton MA. Identification of Streptococcus spp. causing bovine mastitis by PCR—RFLP of 16S—23S ribosomal DNA . Vet Microbiol. 2005; 111: 241—6.

[37] Alnakip MEA, Rhouma NR, Abd—Elfatah EN, Quintela—Baluja M, Böhme K, Fernández—No I, et al. Discrimination of major and minor streptococci incriminated in bovine mastitis by MALDI—TOF MS fingerprinting and 16S rRNA gene sequencing. Res Vet Sci. 2020; 132: 426—38.

[38] Quigley L, O’Sullivan O, Stanton C, Beresford TP, Ross RP, Fitzgerald GF, et al. The complex microbiota of raw milk. FEMS Microbiol Rev. 2013; 37: 664-98.

[39] Krishnamoorthy P, Suresh KP, Jayamma KS, Shome BR, Patil SS, Amachawadi RG. An Understanding of the Global Status of Major Bacterial Pathogens of Milk Concerning Bovine Mastitis: A Systematic Review and Meta—Analysis (Scientometrics). Pathogens. 2021; 10: 545.

[40] Meral Aktaş H, Erdoğan A. Characterization of technological properties of lactic acid bacteria isolated from Turkish Beyaz (white) cheese. J Food Process Preserv. 2022; 46: e16837.

[41] Demirci T, Akin N, Sözeri Atik D, Rabia Özkan E, Dertli E, Akyol İ. Lactic acid bacteria diversity and dynamics during ripening of traditional Turkish goatskin Tulum cheese produced in Mut region assessed by culturing and PCR—DGGE. LWT. 2021; 138: 110701.

[42] Tsuda H, Kodama K. Evaluating the technological properties of lactic acid bacteria in Wagyu cattle milk. J Dairy Res. 2021; 88: 210—6.

[43] de Oliveira IMF, Godoy—Santos F, Oyama LB, Moreira SM, Dias RG, Huws SA, et al. Whole—Genome Sequencing and Comparative Genomic Analysis of Antimicrobial Producing Streptococcus lutetiensis from the Rumen . Microorganisms. 2022; 10: 551.

[44] Nowak A, Śliżewska K. β—Glucuronidase and β—glucosidase activity and human fecal water genotoxicity in the presence of probiotic lactobacilli and the heterocyclic aromatic amine IQ in vitro. Environ Toxicol Pharmacol. 2014; 37: 66-73.

[45] Domingos—Lopes MFP, Stanton C, Ross PR, Dapkevicius MLE, Silva CCG. Genetic diversity, safety and technological characterization of lactic acid bacteria isolated from artisanal Pico cheese. Food Microbiol. 2017; 63: 178-90.

[46] Paventi G, Di Martino C, Coppola F, Iorizzo M. β—Glucosidase Activity of Lactiplantibacillus plantarum: A Key Player in Food Fermentation and Human Health . Foods. 2025; 14: 1451.

[47] Sameli N, Skandamis PN, Samelis J. Application of Enterococcus faecium KE82, an Enterocin A—B—P—Producing Strain, as an Adjunct Culture Enhances Inactivation of Listeria monocytogenes during Traditional Protected Designation of Origin Galotyri Processing . J Food Prot. 2021; 84: 87-98.

[48] Zoumpopoulou G, Georgalaki M, Anastasiou R, Sakoulaki G, Vass A, Gkitsaki I, et al. Lesvos breed sheep milk: An unexplored source of lactic acid bacteria with probiotic potential. Int Dairy J. 2026; 180: 106676.