foods Article Susceptibility to Bacteriocins in Biofilm-Forming, Variable Staphylococci Isolated from Local Slovak Ewes’ Milk Lump Cheeses Andrea Lauková 1,* , Monika Pogány Simonová 1 , Valentína Focková 1, Miroslav Kološta 2, Martin Tomáška 2 and Emília Dvorož ˇnáková 3 1 Institute of Animal Physiology, Centre of Biosciences of the Slovak Academy of Sciences, Šoltésovej 4–6, 040 01 Košice, Slovakia; [email protected] (M.P.S.); [email protected] (V.F.) 2 Dairy Research Institute, a.s. Dlhá 95, 010 01 Žilina, Slovakia; [email protected] (M.K.); [email protected] (M.T.) 3 Parasitological Institute of the Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice, Slovakia; [email protected] * Correspondence: [email protected] Received: 25 August 2020; Accepted: 18 September 2020; Published: 22 September 2020 Abstract: Seventeen staphylococci isolated from 54 Slovak local lump cheeses made from ewes’ milk were taxonomically allotted to five species and three clusters/groups involving the following species: Staphylococcus aureus (5 strains), Staphylococcus xylosus (3 strains), Staphylococcus equorum (one strain) Staphylococcus succinus (5 strains) and Staphylococcus simulans (3 strains). Five different species were determined. The aim of the study follows two lines: basic research in connection with staphylococci, and further possible application of the bacteriocins. Identified staphylococci were mostly susceptible to antibiotics (10 out of 14 antibiotics). Strains showed γ-hemolysis (meaning they did not form hemolysis) except for S. aureus SAOS1/1 strain, which formed β-hemolysis. S. aureus SAOS1/1 strain was also DNase positive as did S. aureus SAOS5/2 and SAOS51/3. The other staphylococci were DNase negative. S. aureus SAOS1/1 and SAOS51/3 showed biofilm formation on Congo red agar. However, using quantitative plate assay, 12 strains out of 17 showed low-grade biofilm formation (0.1 A < 1), ≤ 570 while five strains did not form biofilm (A570 < 0.1). The growth of all strains, including those strains resistant to enterocins, was inhibited by nisin and gallidermin, with high inhibition activity resulting in the inhibition zone in size from 1600 up to 102,400 AU/mL (arbitrary unit per milliliter). This study contributes to microbiota colonization associated with raw ewe’s milk lump cheeses; it also indicates bacteriocin treatment benefit, which can be used in prevention and/or elimination of staphylococci. Keywords: bacteriocins; inhibition; ewes’ milk cheese; staphylococci 1. Introduction Milk from farm animals is a good source of nutrients. Consumers increasingly demand traditional/local products, to which those made from ewes’ milk belong. Ewes’ milk is rich in dry matter, minerals (calcium, zinc, and phosphorus) and B group vitamins. The principal protein in ewes’ milk, casein, is tolerated by people who are allergic, for example to cows’ milk [1]. Products made from ewes’ milk are even more suitable for persons suffering from lactose intolerance. In ewes’ milk lump cheese, lactose is mostly removed into the whey during processing, which means that consumption of such cheese should be safe for lactose-intolerant people. Despite modernization and centralization of the dairy industry, many European cheeses are produced traditionally from raw or heat-treated milk with or without the use of natural undefined starters in farms [2]. This is also the case of local Slovak ewes’ milk lump cheese. It is a traditional Slovak cheese produced Foods 2020, 9, 1335; doi:10.3390/foods9091335 www.mdpi.com/journal/foods Foods 2020, 9, 1335 2 of 12 from unpasteurized ewes’ milk without any added culture. However, the microbiota in ewes’ milk comes from various sources. Of course, the predominant microorganisms are beneficial lactic acid bacteria [3]. The ratio of beneficial to other bacteria in milk can depend on various factors, such as the environment itself or sheepfold hygiene. It is necessary therefore to maintain suitable safety conditions during processing. Ewe’s milk lump cheese should contain at least 47% dry matter and 52% fat in dry matter [1]. Processing of ewe’s milk consists of the following phases: making cheese curd, treating of raw material, lump forming, and dripping [4]. During the application of rennet, the milk temperature is optimized according to the season between 28 ◦C and 30 ◦C. When the raw material is ready after rennet addition, it is turned and cut, and then it is left to settle. All these processes last for 15 min. The settled raw material is then put in a form and the whey is allowed to drain out. Each lump is left dripping at a temperature of 18–20 ◦C for 10–24 h and is then placed on a shelf at 13–15 ◦C with sufficient ventilation. A slight skin covering forms on the cheese during the course of three days, and small grain-sized holes develop inside the cheese; the taste of the final cheese is slightly acidic [4]. As previously mentioned, in spite of efforts to maintain hygiene conditions, bacteria such as staphylococci can contaminate ewes’ milk. Staphylococci belong in the phylum Firmicutes, the order Coccus, the family Staphylococcaceae, and the genus Staphylococcus. The taxonomy is based on 16S rRNA sequences and most of the staphylococcal species fall into 11 clusters [5]. Staphylococci belong also in a group of bacteria, which are able to form biofilm [6]. Biofilms are an aggregation of microorganisms attached to and growing on a surface, and any type of bacteria with the ability to form biofilm can play a key role in contamination and infection [7]. However, some antimicrobial substances such as bacteriocins can inhibit those biofilm-forming bacteria. Bacteriocins are antimicrobial proteinaceous substances with inhibition activity against more or less relative bacteria [8,9]. A special group among them consists of enterocins produced mostly by enterococci [8,9] and also lantibiotic bacteriocins such as nisin and gallidermin [10]. While enterocins are known to inhibit Gram-positive and Gram-negative bacteria under in vitro and in vivo conditions [11,12], nisin and gallidermin act mostly against Gram-positive bacteria [10], although under in vivo conditions their inhibition effect against Gram-negative species has also been reported [13,14]. To know occurrence of microbiota which can influence character and consumption of products is beneficial for both consumers and producers. However, considering that only limited information exists regarding the characterization of bacteria detected in ewes’ lump cheeses, this study was focused on staphylococci in local Slovak ewes’ milk lump cheeses. Isolated staphylococci were tested for susceptibility to antimicrobial substances-enterocins as well as to two lantibiotic bacteriocins, such as nisin and gallidermin. The aim follows two lines: basic research in connection with staphylococci in ewes’ lump cheese, and further possible utilization of bacteriocins to protect cheese. 2. Materials and Methods 2.1. Sample Collection, Isolation, and Identification of Staphylococci A total of fifty-four fresh ewes’ milk lump cheeses from various local farm producers in central Slovakia were sampled at farms and transported in our laboratory. Samples were treated using the standard microbiological method specified by the International Organization for Standardization (ISO). They were mixed in peptone water (Merck, Darmstadt, Germany) using Stomacher (Masticator, IUL, Madrid, Spain) and decimal dilutions in Ringer solution were prepared (ratio 1:9, Merck, Germany). Diluted samples were spread onto selective media, Baird-Parker agar supplemented with egg yolk and tellurite, and on Mannitol Salt agar (MSA; Becton and Dickinson, Sparks, MA, USA), as recommended with ISO and cultivated at 37 ◦C for 24–48 h. Total bacterial count was expressed in log 10 colony forming unit per gram (log 10 CFU/g). Different colonies (80) were picked up, checked for purity and submitted for identification using the matrix–assisted laser desorption ionization time-of-flight spectrometry identification system (MALDI-TOF MS, Brucker Daltonics, Billerica, MA, USA) based on protein “fingerprints“ [15]. Lysates of bacterial cells were prepared according to the producer’s Foods 2020, 9, 1335 3 of 12 instructions (Bruker Daltonics, Billerica, MA, USA). The results were evaluated using the MALDI Biotyper 3.0 (Bruker Daltonics, Billerica, MA, USA) identification database. Taxonomic allocation was evaluated on the basis of highly probable species identification (value score 2300–3000), secure genus identification and/or probable species identification (2000–2299) and probable genus identification (1700–1999). Positive controls were those included in the Bruker Daltonics database. Colonies with the same MALDI-TOF score value were excluded. For subsequent testing, identified strains were maintained on MSA agar or Baird-Parker agar (Becton and Dickinson, Sparks, Maryland, USA) and stored using the Microbank system (Pro-Lab Diagnostic, Richmond, VA, Canada). 2.2. API Staph Profile-Identity Strip System Identification was also checked with API STAPH strips, which are miniaturized biochemical tests with a specially adapted database. The strips contain dehydrated test substrates in individual microtubes. Each microtube contains a tested medium and isolate inoculated in API STAPH medium added to it. After 18–24 h incubation at 37 ◦C results are read and interpreted with reference to the information contained in the manual. Control strains are those involved in the test manual or Bergey‘s Manual of Systematic Bacteriology (Staphylococcus xylosus ATCC 35663; S. simulans