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Detection of methicillin-resistant coagulase-negative staphylococci and PVL/mecA genes in cefoxitin-susceptible Staphylococcus aureus (t044/ST80) from unpasteurized milk sold in stores in Djelfa, Algeria

Open ArchivePublished:January 14, 2021DOI:https://doi.org/10.3168/jds.2020-19270

      ABSTRACT

      This study was designed to determine antimicrobial resistance phenotypes and genotypes and virulence factors in Staphylococcus aureus and coagulase-negative staphylococci (CNS) in unpasteurized milk sold in Djelfa, Algeria. Eighty-two unpasteurized cow milk samples were randomly obtained from 82 retail stores in Djelfa and tested to detect staphylococci. Species were identified by biochemical tests and MALDI-TOF. Antimicrobial resistance phenotypes and genotypes were determined by disk diffusion test, PCR, and sequencing. The Staph. aureus isolates were subjected to spa typing, multilocus sequence typing, and detection of virulence genes and the scn gene by PCR and sequencing. Forty-five (54.9%) milk samples were contaminated by staphylococci and 45 isolates were recovered: 10 Staph. aureus (12.2% of total samples) and 35 CNS (42.7%). Resistance to penicillin (blaZ), tetracycline (tetL/tetK), and erythromycin (ermB/msrA/ermC) were the most common phenotypes (genotypes). Three CNS were methicillin-resistant and all were mecA-positive. The Staph. aureus isolates were ascribed to the following lineages [spa type/sequence type/associated clonal complex (number of isolates)]: t267/ST479/CC479 (n = 6), t1510/ST5651/CC45 (n = 1), t359/ST97/CC97/ (n = 1), t346/ST15/CC15 (n = 1), and t044/ST80 (n = 1). The mecA gene was detected in the cefoxitin-susceptible t044/ST80 isolate and co-harbored the lukF/lukS-PV and scn genes. The detection of mecA-PVL-positive Staph. aureus, methicillin-resistant CNS, and multidrug-resistant staphylococcal species indicates a potentially serious health issue and reveals that unpasteurized milk sold in Djelfa city could be a potential vehicle for pathogenic and antimicrobial-resistant staphylococci.

      Key words

      INTRODUCTION

      Staphylococci species are usually considered part of the normal microbiota of humans and animals but they can also act as opportunistic pathogens involved in food-borne diseases and other clinical manifestations. Based on their ability to coagulate rabbit plasma, species of this genus are divided into 2 subgroups: coagulase-positive staphylococci (CPS) and coagulase-negative staphylococci (CNS). Animal-derived foods, such as dairy products, are often found to harbor CPS, mainly Staphylococcus aureus (
      • Mama O.M.
      • Gomez-Sanz E.
      • Ruiz-Ripa L.
      • Gomez P.
      • Torres C.
      Diversity of staphylococcal species in food producing animals in Spain, with detection of PVL-positive MRSA ST8 (USA300).
      ;
      • Titouche Y.
      • Hakem A.
      • Houali K.
      • Meheut T.
      • Vingadassalon N.
      • Ruiz-Ripa L.
      • Salmi D.
      • Chergui A.
      • Chenouf N.
      • Hennekinne J.A.
      • Torres C.
      • Auvray F.
      Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.
      ). The presence of Staph. aureus in milk and milk products is a public health concern, because this pathogen can produce several virulence factors that play an important role in cellular invasion, bacterial growth, and reduction of immune system cells (
      • Jarraud S.
      • Mougel C.
      • Thioulouse J.
      • Lina G.
      • Meugnier H.
      • Forey F.
      • Nesme X.
      • Etienne J.
      • Vandenesch F.
      Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease.
      ;
      • Lozano C.
      • Gomez-Sanz E.
      • Benito D.
      • Aspiroz C.
      • Zarazaga M.
      • Torres C.
      Staphylococcus aureus nasal carriage, virulence traits, antibiotic resistance mechanisms, and genetic lineages in healthy humans in Spain, with detection of CC398 and CC97 strains.
      ). These virulence factors are represented by cell surface components and exoproteins such as enterotoxins, exfoliatins, toxic shock syndrome toxin (encoded by the tst gene), and Panton-Valentine leukocidin (PVL, encoded by the lukF/lukS-PV genes) (
      • Holmes A.
      • Ganner M.
      • McGuane S.
      • Pitt T.L.
      • Cookson B.D.
      • Kearns A.M.
      Staphylococcus aureus isolates carrying Panton-Valentine leucocidin genes in England and Wales: frequency, characterization, and association with clinical disease.
      ). Panton-Valentine leukocidin is a pore-forming toxin that destroys polymorphonuclear cells. It is composed of 2 separate protein units, LukS-PV and LukF-PV, which behave synergistically to produce the toxic effect (
      • Shrestha B.
      Review on Panton Valentine leukocidin toxin carriage among Staphylococcus aureus.
      ). It is also associated with necrotizing pneumonia (
      • Haider S.
      • Wright D.
      Panton-Valentine leukocidin Staphylococcus causing fatal necrotising pneumonia in a young boy.
      ). In addition, Staph. aureus can cause food poisoning due to consumption of food containing one or more preformed enterotoxins. Six staphylococcal enterotoxins (SEA, SEB, SEP, SEC, SED, and SEE) are known to be prevalent (
      • Argudín M.A.
      • Mendoza M.C.
      • Rodicio M.R.
      Food poisoning and Staphylococcus aureus enterotoxins.
      ;
      • Titouche Y.
      • Houali K.
      • Ruiz-Ripa L.
      • Vingadassalon N.
      • Nia Y.
      • Fatihi A.
      • Cauquil A.
      • Bouchez P.
      • Bouhier L.
      • Torres C.
      • Hennekinne J.A.
      Enterotoxin genes and antimicrobial resistance in Staphylococcus aureus isolated from food products in Algeria.
      ). Staphylococcal enterotoxins are heat stable and resistant to inactivation by gastrointestinal proteases (
      • Balaban N.
      • Rasooly A.
      Staphylococcal enterotoxins.
      ).
      The CNS species have long been reported as contaminants. However, the role of this group of bacteria has been revised and they are currently recognized as important causative agents of bacteremia in animals and nosocomial catheter-related bloodstream infections in humans (
      • Schulin T.
      • Voss A.
      Coagulase-negative staphylococci as a cause of infections related to intravascular prosthetic devices: Limitations of present therapy.
      ;
      • Piessens V.
      • Van Coillie E.
      • Verbist B.
      • Supre K.
      • Braem G.
      • Van Nuffel A.
      • De Vuyst L.
      • Heyndrickx M.
      • De Vliegher S.
      Distribution of coagulase-negative staphylococcus species from milk and environment of dairy cows differs between herds.
      ). In dairy cows, they constitute the most prevalent pathogens causing subclinical mastitis, a disease affecting milk production (
      • Piessens V.
      • Van Coillie E.
      • Verbist B.
      • Supre K.
      • Braem G.
      • Van Nuffel A.
      • De Vuyst L.
      • Heyndrickx M.
      • De Vliegher S.
      Distribution of coagulase-negative staphylococcus species from milk and environment of dairy cows differs between herds.
      ). Nonetheless, the risk associated with staphylococci is not limited to their pathogenic properties; it includes their capacity to acquire mobile genetic elements encoding antimicrobial resistance genes via horizontal gene transfer (
      • Gómez P.
      • Casado C.
      • Saenz Y.
      • Ruiz-Ripa L.
      • Estepa V.
      • Zarazaga M.
      • Torres C.
      Diversity of species and antimicrobial resistance determinants of staphylococci in superficial waters in Spain.
      ). It has been suggested that CNS isolated from cattle tend to be more resistant to antimicrobial agents than Staph. aureus, and they often develop multidrug resistance (
      • Klibi A.
      • Jouini A.
      • Gomez P.
      • Slimene K.
      • Ceballos S.
      • Torres C.
      • Maaroufi A.
      Molecular characterization and clonal diversity of methicillin-resistant and -susceptible Staphylococcus aureus isolates of milk of cows with clinical mastitis in Tunisia.
      ). Methicillin resistance, which was initially described in Staph. aureus (MRSA) and later in CNS (MR-CNS), has become an emerging concern in both veterinary and human medicine and in clinical and nonclinical settings. It is conferred primarily by expression of the mecA gene, located within the staphylococcal cassette chromosome (SCCmec;
      • Martínez-Meléndez A.
      • Morfín-Otero R.
      • Villarreal-Treviño L.
      • González-González G.
      • Llaca-Díaz J.
      • Rodríguez-Noriega E.
      • Camacho-Ortíz A.
      • Garza-González E.
      Staphylococcal cassette chromosome mec (SCCmec) in coagulase negative staphylococci.
      ). Methicillin-resistant Staph. aureus are defined as Staph. aureus carrying the mecA gene (or unusually mecB or mecC) or phenotypically showing resistance to oxacillin or cefoxitin. However, oxacillin-susceptible mecA-positive Staph. aureus, also known as cefoxitin-susceptible MRSA (CS-MRSA), have been increasingly reported not only among clinical isolates but also in animals and food of animal origin (
      • Andrade-Figueiredo M.
      • Leal-Balbino T.C.
      Clonal diversity and epidemiological characteristics of Staphylococcus aureus: high prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with clinical isolates in Brazil.
      ).
      In Algeria, 3.5 billion liters of raw milk are produced per year. However, only 18% of this milk is integrated in the industry sector; the rest is sold traditionally to consumers in stores with poor hygienic conditions, either directly (unpasteurized milk) or transformed (skim milk, traditional cheeses, and other products), according to traditional, manual processes (
      • Chenouf N.S.
      • Yabrir B.
      • Hakem A.
      • Messai C.R.
      • Tobbiche F.
      • Titouche Y.
      • Chenouf A.
      • Zitouni A.
      The bacteriological quality of unpasteurized milk and traditional dairy products sold via informal circuit in Djelfa City (Algeria).
      ;
      • ONIL
      Statistics of the National Interprofessional Milk Office. Office National Interprofessionnel du Lait et des Produits Laitiers (ONIL), Algeria.
      ). These products are highly appreciated by both rural and urban populations, despite the risk of carrying food-borne pathogens (
      • Chenouf N.S.
      • Yabrir B.
      • Hakem A.
      • Messai C.R.
      • Tobbiche F.
      • Titouche Y.
      • Chenouf A.
      • Zitouni A.
      The bacteriological quality of unpasteurized milk and traditional dairy products sold via informal circuit in Djelfa City (Algeria).
      ). In this context, few reports have dealt with the genetic characterization of Staph. aureus isolates in Algerian raw milk (
      • Chaalal W.
      • Chaalal N.
      • Bourafa N.
      • Kihal M.
      • Diene S.M.
      • Rolain J.M.
      Characterization of Staphylococcus aureus isolated from food products in Western Algeria.
      ;
      • Matallah A.M.
      • Bouayad L.
      • Boudjellaba S.
      • Mebkhout F.
      • Hamdi T.M.
      • Ramdani-Bouguessa N.
      Staphylococcus aureus isolated from selected dairies of Algeria: Prevalence and susceptibility to antibiotics.
      ;
      • Titouche Y.
      • Hakem A.
      • Houali K.
      • Meheut T.
      • Vingadassalon N.
      • Ruiz-Ripa L.
      • Salmi D.
      • Chergui A.
      • Chenouf N.
      • Hennekinne J.A.
      • Torres C.
      • Auvray F.
      Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.
      ), with no attention given to CNS strains. Accordingly, the present study was designed to estimate the prevalence and the diversity of staphylococci species (CPS and CNS) recovered from unpasteurized milk sold in retail stores of Djelfa city, Algeria, and to investigate their antibiotic resistance mechanisms and virulence traits.

      MATERIALS AND METHODS

      Milk Sampling and Isolation of Staphylococcus Species

      Eighty-two unpasteurized cow milk samples were obtained from small milk stores in Djelfa city during the period from 2016 to 2019. Djelfa is a steppic zone located 300 km south of Algiers and it covers 1.36% of Algeria's total surface area. Cattle breeding is less practiced in the region compared with that of small ruminants (sheep and goats). In terms of milk production, the region is classified among the fourth group (out of 5), producing between 50 and 100 million liters of milk annually (
      • ONIL
      Statistics of the National Interprofessional Milk Office. Office National Interprofessionnel du Lait et des Produits Laitiers (ONIL), Algeria.
      ). In each store, cow milk is collected from several farms (at least 2 farms) and sold on the same day. Upon arrival at the store, milk is kept in plastic containers at room temperature for sale (one large container per store), and other milk products, such as raw cheeses, are kept in refrigerated counters. From each store and container, 100 mL of raw milk was collected once in a small sterile container and immediately transported in a 4°C cooler to the laboratory (one sample per store). Sample processing was carried out according to ISO 6888-3 (
      • ISO (International Organization for Standardization)
      ISO 6888-3: Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 3: Detection and MPN technique for low numbers.
      ), by enrichment of 1 mL of milk in Giolitti-Cantoni broth (Microbiotech, Sétif, Algeria), supplemented with 1% potassium tellurite and incubated at 37°C for 24 h. Afterward, each positive enriched culture (recognized by black coloration due to the reduction of tellurite to metallic tellurium) was inoculated on mannitol salt agar plates (Microbiotech) and incubated at 37°C for 24 to 48 h. Colonies with presumptive Staphylococcus morphology were selected (one isolate/positive sample) and identified using Gram staining, catalase test, coagulase test, and the MALDI-TOF MS technique (Biotyper, Bruker Corp., Billerica, MA).

      Genomic DNA Extraction

      One colony of Staphylococcus grown on brain heart infusion agar (Scharlau, Barcelona, Spain) was suspended in 45 μL of Milli-Q water (Millipore, Burlington, MA) with 5 μL of lysostaphin (1 mg/mL), and incubated in a water bath at 37°C for 10 min. Then, the following reagents were added: 45 μL of Milli-Q water, 5 μL of proteinase K solution (2 mg/mL), and 150 μL of Tris HCl (0.1 M, pH 8). After 2 incubation steps, at 60°C for 10 min and 100°C for 5 min, the final suspension was centrifuged at 17,000 × g for 3 min at room temperature and the supernatant was collected for molecular analysis (
      • Mama O.M.
      • Gomez P.
      • Ruiz-Ripa L.
      • Gomez-Sanz E.
      • Zarazaga M.
      • Torres C.
      Antimicrobial resistance, virulence, and genetic lineages of staphylococci from horses destined for human consumption: High detection of S. aureus isolates of lineage ST1640 and those carrying the lukPQ gene.
      ).

      Antimicrobial Susceptibility and Resistance Genes

      Susceptibility against 11 antimicrobial agents was assessed by the disk diffusion method on Mueller-Hinton agar (Microbiotech) for all isolates (
      • CLSI (Clinical and Laboratory Standards Institute)
      Performance Standards for Antimicrobial Susceptibility Testing.
      ). The antimicrobials under study were as follows (μg/disk, unless otherwise noted): penicillin (10 IU), cefoxitin (30), erythromycin (15), clindamycin (2), enrofloxacin (5), kanamycin (30), gentamicin (10), trimethoprim-sulfamethoxazole (1.25/23.75), chloramphenicol (30), and tetracycline (30) (Oxoid, Basingstoke, UK). In addition, streptomycin activity (10 μg/disk) was tested (
      • CASFM (Comité De l'antibiogramme De La Société Française De Microbiologie)
      ). In parallel, the D-test, involving the placement of an erythromycin disk in proximity to a clindamycin disk, was applied to detect eventual inducible clindamycin resistance (
      • Gómez P.
      • Casado C.
      • Saenz Y.
      • Ruiz-Ripa L.
      • Estepa V.
      • Zarazaga M.
      • Torres C.
      Diversity of species and antimicrobial resistance determinants of staphylococci in superficial waters in Spain.
      ).
      Based on the obtained antimicrobial resistance phenotypes, resistance genes were studied using conventional PCR methods (
      • Gómez P.
      • Casado C.
      • Saenz Y.
      • Ruiz-Ripa L.
      • Estepa V.
      • Zarazaga M.
      • Torres C.
      Diversity of species and antimicrobial resistance determinants of staphylococci in superficial waters in Spain.
      ): penicillin (blaZ), tetracycline (tetK, tetL, and tetM), aminoglycosides [ant(6)-Ia, ant(4′)-Ia, aac(6′)-Ie–aph(2″)-Ia,], macrolides-lincosamides (ermA, ermB, ermC, ermT, msrA, msrB, lnuA), and trimethoprim (dfrG). Regardless of the methicillin-resistant phenotype, PCR and sequencing were performed in all staphylococci isolates to detect the mecA gene.

      Virulence Genes and Molecular Typing in Staph. aureus Isolates

      Staphylococcus aureus isolates were subjected to PCR and sequencing for the detection of genes encoding PVL (lukF/lukS-PV), enterotoxins (sea, seb, and sep), exfoliative toxins (eta and etb), and toxic shock syndrome toxin (tst). Additionally, the scn gene, a marker of the human immune evasion cluster (IEC), was assessed following previously described procedures (
      • Argudín M.A.
      • Mendoza M.C.
      • Rodicio M.R.
      Food poisoning and Staphylococcus aureus enterotoxins.
      ;
      • Mama O.M.
      • Gomez P.
      • Ruiz-Ripa L.
      • Gomez-Sanz E.
      • Zarazaga M.
      • Torres C.
      Antimicrobial resistance, virulence, and genetic lineages of staphylococci from horses destined for human consumption: High detection of S. aureus isolates of lineage ST1640 and those carrying the lukPQ gene.
      ;
      • Titouche Y.
      • Hakem A.
      • Houali K.
      • Meheut T.
      • Vingadassalon N.
      • Ruiz-Ripa L.
      • Salmi D.
      • Chergui A.
      • Chenouf N.
      • Hennekinne J.A.
      • Torres C.
      • Auvray F.
      Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.
      ).
      All recovered Staph. aureus isolates were typed by sequencing the repeat region of the Staphylococcus protein A gene (spa). The spa types were then determined with the Ridom Staph Type software, by detection and assignment of spa repeats (http://spaserver.ridom.de/). Targeted PCR for the detection of the clonal complex (CC)398 clone (
      • Stegger M.
      • Lindsay J.A.
      • Moodley A.
      • Skov R.
      • Broens E.M.
      • Guardabassi L.
      Rapid PCR detection of Staphylococcus aureus clonal complex 398 by targeting the restriction-modification system carrying sau1-hsdS1.
      ), and multilocus sequence typing (MLST) of the 7 housekeeping loci (arcC, aroE, glpF, gmK, pta, tpi, and yqiL) were performed in all Staph. aureus isolates (
      • Mama O.M.
      • Gomez P.
      • Ruiz-Ripa L.
      • Gomez-Sanz E.
      • Zarazaga M.
      • Torres C.
      Antimicrobial resistance, virulence, and genetic lineages of staphylococci from horses destined for human consumption: High detection of S. aureus isolates of lineage ST1640 and those carrying the lukPQ gene.
      ).

      RESULTS

      Milk Contamination and Recovery of Staphylococci Isolates

      Of the 82 unpasteurized milk samples analyzed, 45 (54.9%) were contaminated by either CPS or CNS species and consequently, 45 staphylococci isolates were recovered (one isolate per positive sample). Ten (12.2%) milk samples contained CPS isolates, all of which were identified as Staph. aureus. Regarding CNS, 35 isolates were identified with the following distribution: Staph. sciuri (n = 18), Staph. lentus (n = 14), Staph. hominis (n = 1), Staph. chromogenes (n = 1), and Staph. cohnii (n = 1).
      Among the 45 contaminated milk samples, 27 (60%) came from mixed milk of 3 farms and 18 (40%) from mixed milk of 2 farms. Moreover, in all contamination cases, milk was transported from the farm to the store in mini trucks without refrigeration over 2 to 3 h (data not shown).

      Molecular Typing, Antimicrobial Resistance, and Virulence in Staph. aureus Isolates

      As shown in Table 1, 5 spa types were identified among Staph. aureus isolates: t267, t1510, t359, t346, and t044. They were ascribed to the following lineages (no. of isolates): ST479 (6), ST5651/CC45 (1), ST97/CC97/ (1), ST15/CC15 (1), as well as the pandemic clone ST80 (1).
      Table 1Molecular typing, antimicrobial resistance, and virulence genes in the 10 Staphylococcus aureus isolates recovered from unpasteurized milk samples
      Isolate codeMolecular typing
      spatype = Staph. aureus protein A type; ST/CC = sequence type/clonal complex.
      Antimicrobial resistance phenotype
      PEN = penicillin, ERY = erythromycin, CLI = clindamycin, ENR = enrofloxacin, KAN = kanamycin, TET = tetracycline, *inducible resistance.
      Antimicrobial resistance genotypeVirulence genes
      spa typeST/CC
      X1320t267ST479PEN-ERY-CLI*-TETblaZ, ermB, ermC, msrA, tetLND
      None of the tested genes were detected.
      X1321t267ST479ENRNT
      Not tested.
      ND
      X1322t1510ST5651/CC45PENblaZND
      X1323t267ST479SusceptibleNTND
      X1324t267ST479PEN-KAN-TETblaZ, tetL, tetKND
      X1325t359ST97/CC97PEN-ERY-CLI-TETermB, msrA, tetL, tetKND
      X1326t267ST479PEN-ERY-CLI-TETblaZ, ermB, msrA, tetLND
      X1327t267ST479PEN-KANNDND
      X1922t044ST80SusceptiblemecAlukF/lukS-PV, scn
      X2026t346ST15/CC15PEN-ERY-CLI*-TETmsrA, tetKND
      1 spatype = Staph. aureus protein A type; ST/CC = sequence type/clonal complex.
      2 PEN = penicillin, ERY = erythromycin, CLI = clindamycin, ENR = enrofloxacin, KAN = kanamycin, TET = tetracycline, *inducible resistance.
      3 None of the tested genes were detected.
      4 Not tested.
      Focusing on antimicrobial susceptibility testing, the highest resistance in Staph. aureus isolates was found toward penicillin (7 isolates, 70%), whereas all isolates showed susceptibility to cefoxitin, gentamicin, chloramphenicol, and trimethoprim-sulfamethoxazole. Three Staph. aureus isolates were resistant to at least one antimicrobial agent (enrofloxacin, penicillin, or kanamycin) and 2 were susceptible to all antimicrobials tested. A multidrug-resistant phenotype, defined as resistance to ≥3 drug classes, was found in 5 Staph. aureus isolates (Table 1).
      Genotypically, resistance to penicillin occurred predominantly via production of a β-lactamase encoded by the blaZ gene, in 4 out of 7 resistant Staph. aureus isolates, whereas tetracycline resistance was mediated by tetL or tetK genes. The 4 erythromycin–clindamycin-resistant isolates carried the msrA or ermB/C genes. Interestingly, mecA was detected in the cefoxitin-susceptible t044/ST80 isolate (CS-MRSA) and co-harbored the gene encoding PVL (lukF/lukS-PV). All Staph. aureus isolates were negative for the sea, seb, sep, eta, etb, tst, and scn genes, except for the t044/ST80 isolate, which carried scn (Table 1).

      Antimicrobial Resistance Phenotypes and Genotypes in CNS Isolates

      The antimicrobial resistance phenotypes and genotypes detected among the CNS isolates are summarized in Table 2. Of the 35 CNS isolates, 23 (65.7%) were susceptible to all antimicrobials studied, whereas the remaining 12 were resistant to at least one antimicrobial (34.3%). Among the resistant isolates, 4 were multidrug-resistant (2 Staph. sciuri, 1 Staph. hominis, and 1 Staph. cohnii). High rates of antibiotic resistance were demonstrated against tetracycline (9 isolates, 25.7%) and erythromycin (8 isolates, 22.8%), with tetL/tetK and ermB/msrA/ermC being the most frequent genotypes, respectively. Resistance to penicillin (blaZ) and enrofloxacin were also observed in 3 CNS isolates (8.6%), whereas only one Staph. lentus isolate expressed resistance to trimethoprim-sulfamethoxazole mediated by the dfrG gene. Importantly, 3 CNS isolates (Staph. hominis, Staph. sciuri, and Staph. cohnii) showed methicillin resistance (MR-CNS), and all harbored mecA (3.6% of total samples). The Staph. hominis isolate displayed a multidrug resistance genotype with the following genes: blaZ, mecA, tetK, msrA, lnuA, ant(6)-Ia, ant(4′)-Ia, and aac(6′)-Ie-aph(2″)-Ia.
      Table 2Antimicrobial resistance phenotype/genotype in the 35 CNS isolates
      Species and no. of isolatesAntimicrobial resistance phenotype
      PEN = penicillin, FOX = cefoxitin, ERY = erythromycin, CLI = clindamycin, ENR = enrofloxacin, KAN = kanamycin, GEN = gentamycin, STR = streptomycin, SXT = trimethoprim-sulfamethoxazole, TET = tetracycline.
      Antimicrobial resistance genotype
      Staphylococcus scuiri
       12SusceptibleNT
      None of the antimicrobial resistance genes tested in susceptible isolates.
       1ERY-TETtetK, tetL, ermB
       1ERY-ENR-TETtetK, tetL, ermC, msrA, ermB
       1ERYermB
       1PEN-FOXmecA
       1PEN-TETtetK
       1ERY-ENR-TETtetK, tetL, msrA, ermB
      Staphylococcus lentus
       10SusceptibleNT
       1ERY-SXTermB, dfrG
       1TETtetK
       2ERY-TETtetK, tetL, ermB, ermC
      Staphylococcus hominis
       1PEN-FOX-ERY-CLI-KAN-GEN-STR-SXT-TETblaZ, mecA, tetK, msrA, lnuA, ant(6)-Ia, ant(4′)-Ia, aac(6′)-Ie-aph(2″)-Ia
      Staphylococcus cohnii
       1FOX-ENR-TETmecA, tetK
      Staphylococcus chromogenes
       1SusceptibleNT
      1 PEN = penicillin, FOX = cefoxitin, ERY = erythromycin, CLI = clindamycin, ENR = enrofloxacin, KAN = kanamycin, GEN = gentamycin, STR = streptomycin, SXT = trimethoprim-sulfamethoxazole, TET = tetracycline.
      2 None of the antimicrobial resistance genes tested in susceptible isolates.

      DISCUSSION

      In Algerian society, the consumption of unpasteurized milk and its derivatives constitutes an ancient habit associated with livestock farming. These traditions may constitute a potential health hazard. Generally, the prevalence of Staph. aureus strains from animal-derived food is well documented in Algeria; however, there is a lack of data on CNS. Overall, more than half of milk samples were contaminated by staphylococcal species, with a relatively high incidence of CNS compared with Staph. aureus (42.7% and 12.2% in analyzed samples, respectively), with Staph. sciuri being the most frequently isolated species. It is important to note that only one colony was selected per sample because morphologically only one type of colony was found in each mannitol salt agar plate inoculated; however, the possibility of missing cases of mixed contamination cannot be excluded. Moreover, both the presence of Staph. aureus and the diversity of CNS (and their antimicrobial resistance profiles) might have been underrepresented with the methods used here. Similar observations were reported in Belgium and Austria, where 90 and 94% of bulk milk samples, respectively, contained CNS (
      • Kümmel J.
      • Stessl B.
      • Gonano M.
      • Walcher G.
      • Bereuter O.
      • Fricker M.
      • Grunert T.
      • Wagner M.
      • Ehling-Schulz M.
      Staphylococcus aureus entrance into the dairy chain: Tracking S. aureus from dairy cow to cheese.
      ;
      • De Visscher A.
      • Piepers S.
      • Haesebrouck F.
      • Supre K.
      • De Vliegher S.
      Coagulase-negative Staphylococcus species in bulk milk: Prevalence, distribution, and associated subgroup- and species-specific risk factors.
      ). Carriage rates of Staph. aureus isolates in Algerian raw milk ranged from 19.9 to 34.8% (
      • Chaalal W.
      • Chaalal N.
      • Bourafa N.
      • Kihal M.
      • Diene S.M.
      • Rolain J.M.
      Characterization of Staphylococcus aureus isolated from food products in Western Algeria.
      ;
      • Matallah A.M.
      • Bouayad L.
      • Boudjellaba S.
      • Mebkhout F.
      • Hamdi T.M.
      • Ramdani-Bouguessa N.
      Staphylococcus aureus isolated from selected dairies of Algeria: Prevalence and susceptibility to antibiotics.
      ;
      • Titouche Y.
      • Hakem A.
      • Houali K.
      • Meheut T.
      • Vingadassalon N.
      • Ruiz-Ripa L.
      • Salmi D.
      • Chergui A.
      • Chenouf N.
      • Hennekinne J.A.
      • Torres C.
      • Auvray F.
      Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.
      ), whereas much higher frequencies of 49.4 and 50% were reported in neighboring North African countries (
      • Bendahou A.
      • Abid M.
      • Bouteldoun N.
      • Catelejine D.
      • Lebbadi M.
      Enterotoxigenic coagulase positive Staphylococcus in milk and milk products, lben and jben, in northern Morocco.
      ;
      • Ben Said M.
      • Abbassi M.S.
      • Bianchini V.
      • Sghaier S.
      • Cremonesi P.
      • Romano A.
      • Gualdi V.
      • Hassen A.
      • Luini M.V.
      Genetic characterization and antimicrobial resistance of Staphylococcus aureus isolated from bovine milk in Tunisia.
      ).
      Contamination of raw milk with staphylococcal species may have numerous origins, but is mostly related to poor hygiene practices, from upstream (management of the herd) to downstream (storage of the final product), through milking, handling, and transportation processes (
      • Chenouf N.S.
      • Yabrir B.
      • Hakem A.
      • Messai C.R.
      • Tobbiche F.
      • Titouche Y.
      • Chenouf A.
      • Zitouni A.
      The bacteriological quality of unpasteurized milk and traditional dairy products sold via informal circuit in Djelfa City (Algeria).
      ). Nasal carriage by cows and nasal or hand carriage by farm workers are also important sources of Staph. aureus and CNS (
      • Akkou M.
      • Bouchiat C.
      • Antri K.
      • Bes M.
      • Tristan A.
      • Dauwalder O.
      • Martins-Simoes P.
      • Rasigade J.P.
      • Etienne J.
      • Vandenesch F.
      • Ramdani-Bouguessa N.
      • Laurent F.
      New host shift from human to cows within Staphylococcus aureus involved in bovine mastitis and nasal carriage of animal's caretakers.
      ;
      • Roberts M.C.
      • Garland-Lewis G.
      • Trufan S.
      • Meschke S.J.
      • Fowler H.
      • Shean R.C.
      • Greninger A.L.
      • Rabinowitz P.M.
      Distribution of Staphylococcus species in dairy cows, workers and shared farm environments.
      ). Moreover, the presence of staphylococci species in milk can originate from the udder itself, in the case of intramammary infection, or from the teat canal and udder skin of healthy cows. Emergence of CNS as a major cause of mastitis in dairy cows has been reported not only in Algeria (
      • Saidi R.
      • Mimoune N.
      • Baazizi R.
      • Benaissa M.H.
      • Khelef D.
      • Kaidi R.
      Antibiotic susceptibility of staphylococci isolated from bovine mastitis in Algeria.
      ;
      • Zaatout N.
      • Ayachi A.
      • Kecha M.
      • Kadlec K.
      Identification of staphylococci causing mastitis in dairy cattle from Algeria and characterization of Staphylococcus aureus.
      ), but also in other countries (
      • Klibi A.
      • Maaroufi A.
      • Torres C.
      • Jouini A.
      Detection and characterization of methicillin-resistant and susceptible coagulase-negative staphylococci in milk from cows with clinical mastitis in Tunisia.
      ).
      Genetic diversity was observed among the Staph. aureus isolates. Most of our Staph. aureus isolates belonged to spa type t267, which was previously reported in Algeria in raw milk from healthy cows (
      • Titouche Y.
      • Hakem A.
      • Houali K.
      • Meheut T.
      • Vingadassalon N.
      • Ruiz-Ripa L.
      • Salmi D.
      • Chergui A.
      • Chenouf N.
      • Hennekinne J.A.
      • Torres C.
      • Auvray F.
      Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.
      ), milk from cows with mastitis (
      • Zaatout N.
      • Ayachi A.
      • Kecha M.
      • Kadlec K.
      Identification of staphylococci causing mastitis in dairy cattle from Algeria and characterization of Staphylococcus aureus.
      ), and in nasal swabs of farmers (
      • Akkou M.
      • Bouchiat C.
      • Antri K.
      • Bes M.
      • Tristan A.
      • Dauwalder O.
      • Martins-Simoes P.
      • Rasigade J.P.
      • Etienne J.
      • Vandenesch F.
      • Ramdani-Bouguessa N.
      • Laurent F.
      New host shift from human to cows within Staphylococcus aureus involved in bovine mastitis and nasal carriage of animal's caretakers.
      ). Our findings suggest that this spa type might be circulating within Algerian dairy environments. Similarly, t267 was predominant in Staph. aureus from cattle herds in Tunisia (
      • Klibi A.
      • Jouini A.
      • Gomez P.
      • Slimene K.
      • Ceballos S.
      • Torres C.
      • Maaroufi A.
      Molecular characterization and clonal diversity of methicillin-resistant and -susceptible Staphylococcus aureus isolates of milk of cows with clinical mastitis in Tunisia.
      ). In our study, all CC479 isolates were concomitant with spa type t267. In previous surveys, CC479 was considered a ruminant-associated clone, mainly found in severe bovine mastitis cases (
      • Boss R.
      • Cosandey A.
      • Luini M.
      • Artursson K.
      • Bardiau M.
      • Breitenwieser F.
      • Hehenberger E.
      • Lam T.
      • Mansfeld M.
      • Michel A.
      • Mosslacher G.
      • Naskova J.
      • Nelson S.
      • Podpecan O.
      • Raemy A.
      • Ryan E.
      • Salat O.
      • Zangerl P.
      • Steiner A.
      • Graber H.U.
      Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.
      ;
      • Hoekstra J.
      • Rutten V.P.M.G.
      • Lam T.J.G.M.
      • Van Kessel K.P.M.
      • Spaninks M.P.
      • Stegeman J.A.
      • Benedictus L.
      • Koop G.
      Activation of a bovine mammary epithelial cell line by ruminant-associated Staphylococcus aureus is lineage dependent.
      ). Furthermore, one Staph. aureus isolate was t359/CC97; this lineage has been detected in dairy cattle from South Africa (
      • Schmidt T.
      • Kock M.M.
      • Ehlers M.M.
      Molecular characterization of Staphylococcus aureus isolated from bovine mastitis and close human contacts in South African dairy herds: Genetic diversity and inter-species host transmission.
      ), Italy (
      • Feltrin F.
      • Alba P.
      • Kraushaar B.
      • Ianzano A.
      • Argudin M.A.
      • Di Matteo P.
      • Porrero M.C.
      • Aarestrup F.M.
      • Butaye P.
      • Franco A.
      • Battisti A.
      A livestock-associated, multidrug-resistant, methicillin-resistant Staphylococcus aureus clonal complex 97 lineage spreading in dairy cattle and pigs in Italy.
      ), and China (
      • Zhang L.
      • Gao J.
      • Barkema H.W.
      • Ali T.
      • Liu G.
      • Deng Y.
      • Naushad S.
      • Kastelic J.P.
      • Han B.
      Virulence gene profiles: Alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China.
      ). It has also been reported as an uncommon cause of infections in small ruminants, pigs, and humans (
      • Spoor L.E.
      • McAdam P.R.
      • Weinert L.A.
      • Rambaut A.
      • Hasman H.
      • Aarestrup F.M.
      • Kearns A.M.
      • Larsen A.R.
      • Skov R.L.
      • Fitzgerald J.R.
      Livestock origin for a human pandemic clone of community-associated methicillin-resistant Staphylococcus aureus.
      ).
      A study conducted on 220 Staph. aureus isolates of the CC97 clone collected from different sources (bovine, human, porcine, and caprine origins) in 18 countries indicated that emergent clones of human epidemic community-associated MRSA resulted from livestock-to-human host jumps, as is the case for bovine Staph. aureus CC97 (
      • Spoor L.E.
      • McAdam P.R.
      • Weinert L.A.
      • Rambaut A.
      • Hasman H.
      • Aarestrup F.M.
      • Kearns A.M.
      • Larsen A.R.
      • Skov R.L.
      • Fitzgerald J.R.
      Livestock origin for a human pandemic clone of community-associated methicillin-resistant Staphylococcus aureus.
      ). These results suggest the importance of dairy cattle as a source of zoonotic bacterial pathogens, with eventual spread within human populations. Three other lineages were also detected in the current findings: CC45, CC15, and ST80. They are most often found to be associated with human MRSA isolates and have been reported from patients with atopic dermatitis in Canada (
      • Yeung M.
      • Balma-Mena A.
      • Shear N.
      • Simor A.
      • Pope E.
      • Walsh S.
      • McGavin M.J.
      Identification of major clonal complexes and toxin producing strains among Staphylococcus aureus associated with atopic dermatitis.
      ), and with endovascular infections in Spain (
      • Pérez-Montarelo D.
      • Viedma E.
      • Murcia M.
      • Munoz-Gallego I.
      • Larrosa N.
      • Branas P.
      • Fernandez-Hidalgo N.
      • Gavalda J.
      • Almirante B.
      • Chaves F.
      Pathogenic characteristics of Staphylococcus aureus endovascular infection isolates from different clonal complexes.
      ).
      Recently, the emergence of MRSA-CC15 in Kuwait hospitals has been reported (
      • Udo E.E.
      • Boswihi S.S.
      • Mathew B.
      • Noronha B.
      • Verghese T.
      • Al-Jemaz A.
      • Al Saqer F.
      Emergence of methicillin-resistant Staphylococcus aureus belonging to clonal complex 15 (CC15-MRSA) in Kuwait hospitals.
      ). The European pandemic clone ST80 has already been detected among MRSA in Algerian hospitals, and was responsible for more than one-third of community and nosocomial infections in Algiers in the 2000s decade (35.7 and 35.8%, respectively;
      • Ramdani-Bouguessa N.
      • Bes M.
      • Meugnier H.
      • Forey F.
      • Reverdy M.E.
      • Lina G.
      • Vandenesch F.
      • Tazir M.
      • Etienne J.
      Detection of methicillin-resistant Staphylococcus aureus strains resistant to multiple antibiotics and carrying the Panton-Valentine leukocidin genes in an Algiers hospital.
      ;
      • Antri K.
      • Rouzic N.
      • Dauwalder O.
      • Boubekri I.
      • Bes M.
      • Lina G.
      • Vandenesch F.
      • Tazir M.
      • Ramdani-Bouguessa N.
      • Etienne J.
      High prevalence of methicillin-resistant Staphylococcus aureus clone ST80-IV in hospital and community settings in Algiers.
      ;
      • Alioua M.A.
      • Labid A.
      • Amoura K.
      • Bertine M.
      • Gacemi-Kirane D.
      • Dekhil M.
      Emergence of the European ST80 clone of community-associated methicillin-resistant Staphylococcus aureus as a cause of healthcare-associated infections in Eastern Algeria.
      ). The first detection of the community-associated MRSA CC80 clone in Europe goes back to 1997 in Denmark, as mentioned by
      • Stegger M.
      • Wirth T.
      • Andersen P.S.
      • Skov R.L.
      • De Grassi A.
      • Simões P.M.
      • Tristan A.
      • Petersen A.
      • Aziz M.
      • Kiil K.
      • Cirković I.
      • Udo E.E.
      • del Campo R.
      • Vuopio-Varkila J.
      • Ahmad N.
      • Tokajian S.
      • Peters G.
      • Schaumburg F.
      • Olsson-Liljequist B.
      • Givskov M.
      • Driebe E.E.
      • Vigh H.E.
      • Shittu A.
      • Ramdani-Bougessa N.
      • Rasigade J.-P.
      • Price L.B.
      • Vandenesch F.
      • Larsen A.R.
      • Laurent F.
      Origin and evolution of european methicillin-resistant Staphylococcus aureus..
      , who believed that this lineage evolved from a methicillin-susceptible Staph. aureus ancestor that might have originated from sub-Saharan Africa.
      In the present work, only the ST80 isolate harbored the scn gene, as well as the lukF/lukS-PV genes, which is considered a frequent virulence factor in this lineage. Interestingly, the presence of scn in this isolate suggests its potential human origin. In Algeria, few studies have described detection of PVL-containing Staph. aureus isolates in the cattle environment associated with mastitis cases or in food samples (
      • Benhamed N.
      • Kihal M.
      Phenotypic and genotypic characterization of Staphylococcus aureus agents of dairy cows' mastitis in Algeria.
      ;
      • Chaalal W.
      • Chaalal N.
      • Bourafa N.
      • Kihal M.
      • Diene S.M.
      • Rolain J.M.
      Characterization of Staphylococcus aureus isolated from food products in Western Algeria.
      ).
      The present study demonstrated the absence of the eta, etb, tst, sea, seb, and sep genes among the Staph. aureus isolates. In fact, toxic shock syndrome and exfoliative toxins are more frequently detected among clinical isolates. In the case of food samples, staphylococcal enterotoxins are especially relevant because of their potential implication in food poisoning. In this sense, staphylococcal enterotoxins were frequently detected in raw milk collected in Northern Algeria (
      • Titouche Y.
      • Hakem A.
      • Houali K.
      • Meheut T.
      • Vingadassalon N.
      • Ruiz-Ripa L.
      • Salmi D.
      • Chergui A.
      • Chenouf N.
      • Hennekinne J.A.
      • Torres C.
      • Auvray F.
      Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.
      ).
      The clonal lineage CC398 was not detected among our Staph. aureus isolates. This result could be explained by the fact that this lineage is mainly isolated from pigs (
      • Lozano C.
      • Lopez M.
      • Gomez-Sanz E.
      • Ruiz-Larrea F.
      • Torres C.
      • Zarazaga M.
      Detection of methicillin-resistant Staphylococcus aureus ST398 in food samples of animal origin in Spain.
      ).
      A multidrug-resistant phenotype was detected in 9 of 45 staphylococci (20%), and a variety of resistance genes were also detected. These results indicate a serious public health issue, considering that these microorganisms may be transferred to humans via milk consumption, as well as the danger of therapeutic failures. In agreement with several previous reports (
      • Matallah A.M.
      • Bouayad L.
      • Boudjellaba S.
      • Mebkhout F.
      • Hamdi T.M.
      • Ramdani-Bouguessa N.
      Staphylococcus aureus isolated from selected dairies of Algeria: Prevalence and susceptibility to antibiotics.
      ;
      • Titouche Y.
      • Hakem A.
      • Houali K.
      • Meheut T.
      • Vingadassalon N.
      • Ruiz-Ripa L.
      • Salmi D.
      • Chergui A.
      • Chenouf N.
      • Hennekinne J.A.
      • Torres C.
      • Auvray F.
      Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria.
      ), the highest level of resistance was noted toward penicillin, tetracycline, and erythromycin, with blaZ, tetK, and ermB being the most common corresponding genotypes, respectively. The β-lactam class is known to be extensively used in Algerian dairy cattle against staphylococcal mastitis, which remains the major reason for use of antibiotics in bovines (
      • Saidi R.
      • Mimoune N.
      • Baazizi R.
      • Benaissa M.H.
      • Khelef D.
      • Kaidi R.
      Antibiotic susceptibility of staphylococci isolated from bovine mastitis in Algeria.
      ). Tetracycline has a bacteriostatic effect and is commonly used and recommended in cattle for the treatment of septicemia, respiratory, digestive, genitourinary, and interdigital infections (
      • Barour D.
      • Berghiche A.
      • Boulebda N.
      Antimicrobial resistance of Escherichia coli isolates from cattle in Eastern Algeria.
      ). Likewise, erythromycin, being the second macrolide intended for use in animals, has long been prescribed for acute mastitis caused by gram-positive bacteria in lactating dairy cows because of its good distribution in the mammary gland (
      • Bajwa N.S.
      • Bansal B.K.
      • Srivastava A.K.
      • Ranjan R.
      Pharmacokinetic profile of erythromycin after intramammary administration in lactating dairy cows with specific mastitis.
      ). The long-term use of these antibiotics, considering that they belong to older drug families prescribed in bovines, either for therapeutic or prophylactic reasons, can largely explain the high resistance rates because of selection pressure. From the animal and public health perspectives, Staph. aureus and CNS are of major concern, especially where animal-derived foods and humans intersect, mainly through the food chain. These results provide insight into antibio-resistance in Algerian cattle farms that may be contributing to therapeutic failures in cattle farming, the emergence of resistance by gene transfer between species, and the presence of antibiotic residues in milk. This study highlights the need to reduce and optimize the use of antibiotics in livestock animals.
      The screening for methicillin resistance, performed via cefoxitin disk diffusion test, revealed 3 MR-CNS, all of which carried mecA. These results highlight the role that CNS may play as a potential donor of mecA gene to other staphylococcal species, mainly those with greater pathogenic properties, such as Staph. aureus. Currently, MR-CNS are receiving significant attention in human and animal health and have been described in cattle, animal handlers, and their environment (
      • Venugopal N.
      • Mitra S.
      • Tewari R.
      • Ganaie F.
      • Shome R.
      • Rahman H.
      • Shome B.R.
      Molecular detection and typing of methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci isolated from cattle, animal handlers, and their environment from Karnataka, Southern Province of India.
      ), in companion animals and their owners (
      • Gómez-Sanz E.
      • Ceballos S.
      • Ruiz-Ripa L.
      • Zarazaga M.
      • Torres C.
      Clonally diverse methicillin and multidrug resistant coagulase negative staphylococci are ubiquitous and pose transfer ability between pets and their owners.
      ), and also in clinical isolates from hospitals (
      • Kitti T.
      • Seng R.
      • Saiprom N.
      • Thummeepak R.
      • Chantratita N.
      • Boonlao C.
      • Sitthisak S.
      Molecular characteristics of methicillin-resistant staphylococci clinical isolates from a tertiary hospital in Northern Thailand.
      ). In Algeria, data on MR-CNS are lacking. Therefore, complementary studies with larger sample sizes (from cattle and cattle workers) are required to better investigate the dissemination and epidemiology of MR-CNS in cattle sector.
      Phenotypically, no methicillin resistance was detected among our Staph. aureus isolates by antibiogram. However, the t044/ST80 isolate was found to carry the mecA gene (CS-MRSA). Detection of mecA is not performed routinely in Algerian microbiology laboratories; therefore, misclassification may occur. Detection of emerging MRSA variants, such as CS-MRSA, is increasingly described. This discrepancy between phenotype and genotype could be explained by an eventual default in mecA expression. Methicillin-susceptible mecA-containing Staph. aureus isolates were also reported in university and general hospitals in Brazil (
      • Andrade-Figueiredo M.
      • Leal-Balbino T.C.
      Clonal diversity and epidemiological characteristics of Staphylococcus aureus: high prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with clinical isolates in Brazil.
      ) and Japan (
      • Hososaka Y.
      • Hanaki H.
      • Endo H.
      • Suzuki Y.
      • Nakae T.
      • Nagasawa Z.
      • Otsuka Y.
      • Sunakawa K.
      Characterization of oxacillin-susceptible mecA-positive Staphylococcus aureus: A new type of MRSA.
      ), and in processed food from Europe (
      • Quijada N.M.
      • Hernandez M.
      • Oniciuc E.A.
      • Eiros J.M.
      • Fernandez-Natal I.
      • Wagner M.
      • Rodriguez-Lazaro D.
      Oxacillin-susceptible mecA-positive Staphylococcus aureus associated with processed food in Europe.
      ). To the best of our knowledge, the current paper is the first report of CS-MRSA in Algeria. Further in-depth characterization of the CS-MRSA isolate detected in this study is warranted, to characterize its methicillin resistance expression. The significance of this study lies in its extensive character, describing the prevalence and genetic characterization of both CPS and CNS from raw milk sold in Djelfa city. This study could also be regarded as a food safety survey given that its findings are intended to be communicated and applied across the Algerian dairy chain.

      CONCLUSIONS

      Detection of mecA-PVL-positive Staph. aureus, methicillin-resistant CNS, and multidrug-resistant staphylococcal species indicates a potentially serious health issue and reveals that unpasteurized milk destined for human consumption in Djelfa city could be a vector of pathogenic and antimicrobial-resistant staphylococci. These outcomes require particular attention from all actors in the Algerian dairy industry, but primarily food safety scientists and veterinarians prescribing antibiotics. The use of heat treatment and the application of good hygiene and manufacturing practices can minimize the risk of milk contamination. Greater controls in production and consumption of unpasteurized milk should be implemented.

      ACKNOWLEDGMENTS

      Author NSC received a grant for the year 2018 from the Algerian Ministry of Higher Education and Scientific Research (The PNE Program), under the direction of Carmen Torres. Molecular characterization of strains at the University of La Rioja (Spain) has been supported by project SAF2016-76571-R from the Agencia Estatal de Investigación (AEI) of Spain and the Fondo Europeo de Desarrollo Regional (FEDER) of the European Union. Author OMM received a predoctoral fellowship of Mujeres por África-Universidad de La Rioja (Spain). Author LRR has a predoctoral FPI fellowship from the Universidad de La Rioja (Spain). Author RFF received a predoctoral fellowship from the Ministerio de Ciencia, Innovación y Universidades of Spain (FPU18/05438). Author IC has a doctoral financial support fellowship from Fundação para a Ciência e Tecnologia (FCT; Portugal), through the reference SFRH/BD/133266/2017 (Medicina Clínica e Ciências da Saúde). The authors thank students from Life and Natural Sciences Institute (University of Djelfa, Algeria) for their contribution in sampling and strain isolation. The authors declare no conflicts of interest.

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