In this work, 85 strains of Staphylococcus aureus were isolated from samples taken from slaughter poultry in Poland. Attempts were made to determine the prophage profile of the strains and to investigate the presence in their genome of genes responsible for the production of five classical enterotoxins (A–E), toxic shock syndrome toxin (TSST-1), exfoliative toxins (ETA and ETB) and staphylokinase (SAK). For this purpose, multiplex PCR was performed using primer-specific pairs for targeted genes. The presence of the mecA gene was found in 26 strains (30.6%). The genomes of one of the methicillin- resistant S. aureus (MRSA) strains and two methicillin-sensitive S. aureus (MSSA) strains contained the gene responsible for the production of enterotoxin A. Only one MRSA strain and two MSSA strains showed the presence of the toxic shock syndrome toxin (tst) gene. Only one of the MSSA strains had the gene (eta) responsible for the production of exfoliative toxins A. The presence of the staphylokinase gene (sak) was confirmed in 13 MRSA strains and in 5 MSSA strains. The study results indicated a high prevalence of prophages among the test isolates of Staphylococcus aureus. In all, 15 prophage patterns were observed among the isolates. The presence of 77-like prophages incorporated into bacterial genome was especially often demonstrated. Various authors emphasize the special role of these prophages in the spread of virulence factors (staphyloki- nase, enterotoxin A) not only within strains of the same species but also between species and even types of bacteria. Introduction superantigen family [6, 7]. The mechanism of action of supe- rantigens involves the ability to induce non-specific stimula - Staphylococcus aureus has the ability to induce severe tion of T lymphocyte proliferation and cytokine secretion. diseases of animals and humans, that are associated with The accumulation of cytokines in the mammalian body can numerous virulence factors produced by these bacteria, such lead to toxic shock. Another consequence of the presence of as toxins, cell adhesins and secreted exoproteins (coagulase, enterotoxins in the body is their enterotoxicity, resulting in toxic shock syndrome toxin-1, staphylokinase and others) food poisoning. A single bacterial strain may produce any [1–5]. Staphylococcal enterotoxins (SEs) and toxic shock of these toxins separately or in various combinations [2, 8]. syndrome toxin-1 (TSST-1) are exotoxins that belong to the Staphylokinase (SAK) a protein produced by certain S. aureus strains is a fibrin-specific activator of human plasmi- nogen. During the infection process, staphylokinase disrupts Electronic supplementary material The online version of this phagocytosis of bacterial cells, and fibrinolysin, a compo- article (https ://doi.org/10.1007/s0028 4-018-1518-9) contains supplementary material, which is available to authorized users. nent of staphylokinase, facilitates the penetration of staphy- lococci into tissues and their proteolytic degradation . * Agnieszka Marek The ability of S. aureus strains to produce staphylokinase firstname.lastname@example.org can be exploited to trace the ecological origin of a strain. It Sub-Department of Preventive Veterinary and Avian has been observed that S. aureus strains of human biotype Diseases, Institute of Biological Bases of Animal Diseases, can produce enterotoxins much more frequently than strains Faculty of Veterinary Medicine, University of Life Sciences of animal biotypes. However, some researchers claim that in Lublin, Akademicka 13, 20-950 Lublin, Poland strains derived from poultry are also capable of producing Department of Epizootiology and Clinic of Infectious this protein . Diseases, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Głęboka 30, 20-612 Lublin, Poland Vol:.(1234567890) 1 3 Association Between the Methicillin Resistance of Staphylococcus aureus Isolated from… 1257 Mobile genetic elements (MGEs), such as bacteriophages, S. aureus contain prophages integrated into their chro- plasmids, pathogenicity islands, transposons or insertion mosome, their prophage patterns were identified as well. sequences, encode putative virulence factors and molecules Gene polymorphism of twenty-six MRSA isolates by that confer the ability to produce enterotoxins, TSST-1, exfo- Pulsed-field gel electrophoresis (PFGE) with restriction liative toxins or staphylokinase [10–13]. The vast majority of enzymes SmaI and ApaI was performed as well. bacteria contain prophages integrated into their chromosome or as extra-chromosomal elements. Detection of staphylo- coccal prophages by multiplex PCR is comparable in sensi- tivity to detection by hybridization of restriction fragments Methods . A key role in the pathogenesis and virulence of the S. aureus is attributed to temperate bacteriophages of the Sample Collection Siphoviridae family belonging to the order Caudovirales. Based on their lytic activity, morphology and serological The study was conducted on material derived from broiler properties, phages of the Siphoviridae family are classified chickens and turkeys farms located in the area of central- into six phage types: 3A-like virus, 11-like virus, 77-like western Poland between December 2013 and November virus and 187-like virus. Phages of type 77-like fall into two 2015. During this time, samples from 153 flocks were serological subgroups, Fa and Fb, which can be present at collected. Randomly selected birds showing clinical signs the same time as prophages in the genome of bacteria . of disease from each flock were examined. Three to five Phages of the Twort-like type are related to lytic phages and specimens from the affected organs were taken from each belong to the family Myoviridae. Bacteriophages, via lyso- bird. A total of 569 samples from broilers and 446 sam- genic conversion and participation in the spread of patho- ples from turkeys were collected. The samples were taken genic islands, are believed to contribute to S. aureus variabil- from internal organs (heart, liver, tarsal joints and bone ity and the formation of highly virulent strains . They marrow) of birds aged 1 day to 6 weeks (chickens) or 20 can also influence the adaptation of S. aureus to its hosts, weeks (turkeys). The samples were taken from birds show- both human and domesticated animals, by providing new ing the following clinical symptoms: increased mortality, genetic information or facilitating the loss of unnecessary dermatitis and cellulitis, lameness and arthritis, decreased DNA in the new ecological niche. It has been demonstrated weight gain and omphalitis and yolk sac infections. The that S. aureus strains of animal origin may be susceptible size of the flocks from which the samples were collected to the same bacteriophages as human strains, and thus via ranged from 8000 to 44000 birds. phage they can acquire the virulence factors characteristic The material collected (samples of internal organs) was for human strains . plated on a blood agar medium (Blood LAB-AGAR, Bio- Despite efforts to reduce the use of antibiotics in poultry corp, Poland) and Chapman selective medium (Mannitol production in the past few years, there has been a steady Salt LAB-AGAR, Biocorp, Poland) and incubated under aer- increase in the incidence of bacterial infections with mul- obic conditions at 37 °C for 24–48 h, depending on the rate tiple-resistant strains of Staphylococcus species in poultry of growth of the bacteria. Single colonies were transferred flocks. There has also been an enormous increase in the to blood agar in order to isolate pure bacterial cultures and number of methicillin-resistant (MRSA) strains [17, 18]. a preliminary bacteriological characterization was made of Resistance to beta-lactam antibiotics is usually dependent the isolated flora, involving Gram’s staining, cell morphol- on the presence of the mecA gene, which encodes the low- ogy and motility using microscope and type of haemolysis. affinity penicillin-binding protein (PBP)-designated PBP2a In this study, quantitative measurement of the colony was and makes the staphylococci resistant to almost all antibiot- not performed. ics of this group (penicillins, almost all cephalosporins and Isolated bacteria were stored for further testing at − 85 °C carbapenems), of which many are still widely used in both in 50% (v/v) glycerol in brain heart infusion broth (BHIB; human and veterinary medicine [19, 20]. The presence of Sigma). virulence factors and antibiotic resistance genes in isolates of S. aureus indicates the risk carried by a particular strain. The objective of this study was to determine the preva- Statement of Human and Animal Rights lence of methicillin-resistant (MRSA) and methicillin- sensitive S. aureus (MSSA) in samples taken from chicken All procedures performed in studies involving human par- and turkey broilers in Poland, and the associations between ticipants were in accordance with the ethical standards of the methicillin resistance of S. aureus strains isolated the institutional and/or national research committee and with from poultry, their toxin gene profiles and their ability the 1964 Helsinki Declaration and its later amendments or to produce staphylokinase. Because the vast majority of comparable ethical standards. 1 3 1258 A. Marek et al. toxins A and B (eta and etb)—set C, was developed using Characterization of Bacterial Strains eight pairs of primers (Table 1). The conditions of the mul- tiplex PCR reaction were taken from the study by Mehrota The identification of all Staphylococcus strains was carried out using mass spectrometry MALDI-TOF MS using the et al. .The PCR primers used to amplify the sak gene are listed in Table 1. The conditions of the PCR reaction were IVD MALDI Biotyper (Bruker Daltonik, Bremen, Germany) as described by Marek et al. . taken from the study by Sung et al. . The susceptibility of 11 antibiotics was tested using standard disc diffusion method on Mueller–Hinton agar Multiplex PCR for Detection of Prophages in S. aureus Strains plates (CM0337B, Oxoid, UK) using a bacterial suspension with the turbidity adjusted to a 0.5 McFarland standard. The A multiplex PCR assay (set D) for detection of DNA susceptibility of bacteria was determined for the follow- ing agents (Oxoid, England): amoxicillin 25 µg (AML25); sequences specific for 3A-like, 11-like, 77-like, 187-like and Twort-like phages was developed using eight pairs of prim- amoxicillin + clavulanic acid 20 + 10 µg (AMC30); ampicil- lin 10 µg (AMP10); penicillin G 10 units (P10); cefoxitin ers (Table 2). The conditions of the multiplex PCR reaction were taken from the study by Pantůček et al. . 30 µg (FOX30); clindamycin 2 µg (DA2); chloramphenicol 30 µg (C30); erythromycin 15 µg (E15); gentamicin 10 µg Pulsed‑field Gel Electrophoresis (PFGE) (CN10); tetracycline 30 µg (TE30); trimethoprim–sul- phamethoxazole 1:19, 25 µg (SXT25). The categories sus- The genetic relatedness of MRSA isolates was investigated ceptible, intermediate resistant or resistant were assigned on the basis of the Guidelines for Susceptibility Testing . by PFGE of total DNA digested with SmaI or ApaI restric- tion endonucleases . S. aureus non-typeable with SmaI The Minimum Inhibitory Concentrations (MIC) for oxacil- lin were additionally evaluated by the broth microdilution were subjected to ApaI-PFGE and run for 20 h at 6V/cm using pulsed time ranging from 2 to 5 s. The SmaI or ApaI method . For oxacillin, S. aureus strains showing MIC of ≥ 4 µg/ml were determined to be MRSA. For quality con- fragments were electrophoretically separated in a 1% (w/v) agarose (Sigma-Aldrich, Poland) gel using the CHEF Map- trol, S. aureus ATCC 25923, Escherichia coli ATCC 25922 and E. faecalis ATCC 29212 were used in the microdilution per System (BIO-RAD, Poland). The macrorestriction pat- terns were examined by cluster analysis using NTSYSpc ver. tests. 2.02 software (Exeter Software Ltd, USA). The similarity distances between pulsotypes (PFGE patterns) were calcu- Bacterial DNA Extraction lated using the Dice coefficient, and the dendrogram was based on the unweighted pair group method with arithme- Total DNA was extracted from the collected strains inocu- lated individually on blood agar and incubated at 37 °C/24 h. tic average (UPGMA). According to the criteria proposed by Tenover et al., isolates whose PFGE pattern differed in The Novabeads Bacterial DNA kit (Novazym Poland) was used for DNA extraction according to the manufacturer’s more than six restriction fragments (bands) were genetically unrelated and were assigned to different pulsotypes, named protocol. as Arabic letters. Isolates were considered to be related if their pulsotype differed in no more than six restriction bands Detection of the mecA Gene (subtype) and were indicated with the major lettering type followed by a number . SmaI- and ApaI-generated pul- The identification of all MRSA isolates was confirmed by multiplex PCR (set A) targeting the mecA gene encoding sotypes were distinguished by capital- and lower-case Arabic letters, respectively. methicillin resistance. To verify the efficiency of the amplifi- cation, an internal control primer pair targeting an S. aureus- specific nuc region was amplified. PCR for mecA and nuc gene was carried out by the methodology described by Brak- Results stad et al. and Murakami et al. [18, 22]. Isolates of S. aureus that were mecA and nuc positive were considered as MRSA. Sample Collection Prevalence of Toxins and sak Genes in S. aureus A total of 567 bacterial strains belonging to the genus Staph- ylococcus were isolated from the material tested. The Staph- and MRSA Isolates ylococcus strains isolated from the samples belonged to 24 species. Among 24 Staphylococcus species, 85 strains of S. A multiplex PCR assay for detection of genes for staphylo- coccal enterotoxins A to E (sea, seb, sec, sed and see)—set aureus were identified. The remaining strains belonged to the species S. cohnii (27.7%), S. lentus (17%), S. chromogenes B, and for toxic shock syndrome toxin 1 (tst) and exfoliative 1 3 Association Between the Methicillin Resistance of Staphylococcus aureus Isolated from… 1259 Table 1 Nucleotide sequences Primer* Oligonucleotide sequence (5′–3′)** Gene Size of ampli- Control strain References and sizes of PCR products fied product of methicillin resistance, (bp) thermonuclease, enterotoxins (A-E), TSST-1, exfoliative NUC-1GCG ATT GAT GGT GAT ACG GTT nuc 270 ATCC43300  toxins and staphylokinase NUC-AGC CAA GCC TTG ACG AAC TAA AGC MEC-1AAA ATC GAT GGT AAA GGT TGGC mecA 533  MEC-2AGT TCT GGC ACT ACC GGA TTTGC ESA1ACG ATC AAT TTT TAC AGC sea 544 FRI913  ESA2TGC ATG TTT TCA GAG TTA ATC ESB1GAA TGA TAT TAA TTC GCA TC seb 416 ATCC13566  ESB2TCT TTG TCG TAA GAT AAA CTTC ESC1GAC ATA AAA GCT AGG AAT TT sec 257 FRI913  ESC2AAA TCG GAT TAA CAT TAT CCA ESD1TTA CTA GTT TGG TAA TAT CTC CTT sed 334 FRI151m  ESD2CCA CCA TAA CAA TTA ATG C ESE1ATA GAT AAA GTT AAA ACA AGCAA see 170 FRI913  ESE2TAA CTT ACC GTG GACCC GTSSTR-1ACC CCT GTT CCC TTA TCA TC tst 326 FRI1169  GTSSTR-2TTT TCA GTA TTT GTA ACG CC GETAR-1GCA GGT GTT GAT TTA GCA TT eta 93 CCM7056  GETAR-2AGA TGT CCC TAT TTT TGC TG GETBR-1ACA AGC AAA AGA ATA CAG CG etb 226 CCM7056  GETBR-2GTT TTT GGC TGC TTC TCT TG SAK-1TGA GGT AAG TGC ATC AAG TTCA sak 403 ATCC25923  SAK-2CCT TTG TAA TTA AGT TGA ATC CAG G *The sets of primers were synthesized by Genomed S.A, Poland **The concentration of primers was 0.04 µmol Table 2 Multiplex PCR . Primer sequence of staphylococcal phage type, PCR product length and type of protein Phage type Primer*** Primer sequence (5′–3′)** PCR product Sequence coding for length (bp) 3A-like phage SGA1TAT CAG GCG AGA ATT AAG GG 744 Tail fibres SGA2CTT TGA CAT GAC ATC CGC TTGAC 11-like phage SGB1ACT TAT CCA GGT GGY GTT ATTG 405 Hypothetical tail protein SGB2TGT ATT TAA TTT CGC CGT TAGTG 77-like phage SGF1CGA TGG ACG GCT ACA CAG A 155 Hypothetical tail protein SGF2TTG TTC AGA AAC TTC CCA ACCTG SGFa1TAC GGG AAA ATA TTC GGA AG 548 Packaging protein SGFa2ATA ATC CGC ACC TCA TTC CT SGFb1AGA CAC ATT AAG TCG CAC GATAG 147 Packaging protein SGFb2TCT TCT CTG GCA CGG TCT CTT 187-like phage SGL1GCT TAA AAC AGT AAC GGT GAC AGT G 648 Hypothetical capsid protein SGL2TGC TAC ATC ATC AAG AAC ACC TGG Twort-like phage SGD1TGG GCT TCA TTC TAC GGT GA 331 Major capsid protein SGD2GTA ATT TAA TGA ATC CAC GAGAT *S. aureus strain NCTC 8325 was used as positive control **Nucleotide sequences were derived from the published sequences by Pantůček et al  ***The concentration of primers was 0.04 µmol 1 3 1260 A. Marek et al. (8.4%), S. equorum (6.5%), S. saprophyticus (4.4%), S. sci- and amoxicillin (89.8%). Considerably more MSSA isolates uri (3.9%), S. hominis (3.2%), S. xylosus (3.2%), S. arlettae exhibited resistance to gentamicin (18.6%) and tetracycline (2.4%), S. simulans (1%), S. felis (0.9%), S. vitulinus (0.9%), (28.8%). Over half of the MSSA strains were resistant to the S. delphini (0.7%), S. epidermidis (0.7%), S. haemolyticus other three antimicrobial agents, with the highest percentage (0.7%), S. condimenti (0.5%), S. warneri (0.5%), S. alac- of strains, 68.3% resistant to penicillin G. Among MRSA tolyticus (0.4%), S. carnosus (0.4%), S. capitis (0.4%), S. isolates, 100% in vitro susceptibility was not observed in hyicus (0.4%), S. lugdunensis (0.4%), S. schleiferi subsp. any of the eleven antimicrobial agent applied. All MRSA coagulans (0.4%). strains were resistant to penicillin G and ampicillin. A high PCR confirmed the presence of the nuc gene in all 85 percentage of strains were resistant to amoxicillin (96.2%), (100%) S. aureus strains. cefoxitin (85%), amoxicillin + clavulanic acid (84.7%), tet- racycline (84.7%), erythromycin (80.8%) and clindamycin Prevalence of MRSA Strains (73%). Detailed data are presented in Table 3. PCR confirmed the presence of a 533 bp product character - Prevalence of Toxin Genes in S. aureus and MRSA istic of the presence of the mecA gene in 26 (30.6%) strains. Isolates Phenotypic Susceptibility of the Isolated Bacteria The results of the multiplex PCR for five classical entero- to Selected Antimicrobial Agents toxins (A–E) showed that the genome of one of the MRSA strains contained the gene responsible for the production of The minimum inhibitory concentration (MIC) against oxa- enterotoxin A. Four strains carried the gene responsible for cillin showed that 24 isolates of S. aureus (28.2%) were the production of enterotoxin B and one strain contained the resistant to this antibiotic. All strains showing a MIC value gene responsible for the production of enterotoxin D. None indicating resistance to oxacillin also possessed the mecA of the MRSA strains had genes responsible for the produc- gene. tion of enterotoxins C and E (Table 4). As a result of the susceptibility testing of the isolated S. In the case of MSSA strains (n = 59), the presence of aureus strains to 11 selected antimicrobial agents, 100% sus- two strains capable of producing enterotoxin A was con- ceptibility in in vitro conditions for cefoxitin was observed firmed. Five strains showed the presence of the gene respon- among of 59 MSSA strains. A relatively high percentage of sible for the production of enterotoxin B, two strains had MSSA strains were found to be susceptible to clindamycin genes responsible for the production of enterotoxin C and (96.6%), trimethoprim–sulphamethoxazole (96.6%), chlo- one strain had the gene responsible for the production of ramphenicol (93.3%), amoxicillin + clavulanic acid (93.2%) enterotoxin D. None of the MSSA strains carried the gene Table 3 Phenotypic , Antibiotic MRSA strains n = 26 MSSA strains n = 59 antimicrobial resistance of S. aureus strains isolated from R I Resistance rate R I Resistance broiler chickens and turkeys (%) rate (%) Amoxicillin 23 2 96.2 5 1 10.2 Amoxicillin + clavulanic acid 12 10 84.7 4 – 6.8 Ampicillin 24 2 100 32 – 54.2 Penicillin G 26 – 100 38 2 68.3 Cefoxitin 22 – 85 – – – Clindamycin 14 5 73 – 2 3.4 Chloramphenicol 6 1 26.9 1 3 6.7 Erythromycin 17 4 80.8 31 – 52.5 Gentamicin 5 – 19.2 10 1 18.6 Tetracycline 20 2 84.7 11 6 28.8 Trimethoprim–sulphamethoxazole 3 1 15.3 2 – 3.4 The resistance rate was calculated as the number of intermediate and resistant isolates divided by the total number of isolates R resistant, I intermediate The susceptibility o11 antibiotics was tested using standard disc diffusion method on Mueller–Hinton agar plates 1 3 Association Between the Methicillin Resistance of Staphylococcus aureus Isolated from… 1261 Table 4 Prophage content and prevalence of staphylokinase and toxins genes of the 26 MRSA S. aureus isolates estimated by multiplex PCR Lysogenic type Number of MRSA (%) Presence of the gene PFGE pulsotype strains (n = 26) sea tst eta sak seb etb sec sed see Single lysogenic strains 11-like (SGB) 2 7.7 – – – – A, a2 77-like (SGFa) 4 15.4 – – – 4 a1, a4, a5, a8 77-like (SGFb) 4 15.4 seb (n = 1) 2 a1, a2 Double lysogenic strains 3A (SGA)—11 (SGB) 1 3.8 – – – – d 3A(SGA)—77a (SGFa) 4 15.4 sea (n = 1) 1 – 2 a3, a11, a12, c seb (n = 1) 11 (SGB)—77b (SGFb) 2 7.7 – – – 1 a1, a9 77a (SGFa)—77b (SGFb) 3 11.5 sed(n = 1) 1 a1, a6, a7 77b (SGFb)—187 (SGL) 1 3.8 seb(n = 1) – – 1 B Triple lysogenic strains 3A (SGA)—11 (SGB)—77a (SGFa) 1 3.8 – – – – C 3A (SGA—77a(SGFa)- 77b (SGFb) 2 7.7 seb (n = 1) – – 1 a1, b 11 (SGB)–77a(SGFa)—77b (SGFb) 2 7.7 – – – 1 a10, a13 MRSA methicillin-resistant S. aureus, sea, seb, sec, cec, see enterotoxin A,B,C,D,E, tst toxic shock syndrome toxin-1, eta, etb exfoliative toxins A, B, sak staphylokinase responsible for the production of enterotoxin E (Table 5; or lysogenic types among MRSA isolates were single lyso- Fig. 1 in supplementary materials). genic 77-like (Fa subtype), 77-like (Fb subtype) and dou- Regarding TSST-1 and exfoliative toxins A and B, only ble lysogenic 3A-like-77-like (Fa subtype). Among MSSA one MRSA strain and two MSSA strains showed the pres- isolates, the most prevalent prophage pattern was 11-like, ence of the tst gene. None of the MRSA strains had genes followed by single lysogenic 77-like (Fb subgroup), double responsible for the production of exfoliative toxins A and B, lysogenic 3A-like-11-like and 11-like-77-like (Fb subgroup); and only one of the MSSA strains had the eta gene (Tables 4, detailed data are presented in Tables 4 and 5 and Fig. 2 in 5 and Fig. 3 in supplementary materials). supplementary materials. Prophages of the Twort type were not identified in any of the 85 S. aureus strains tested, while Detection of the Staphylokinase (sak) Gene prophage 187-like was identified in the genome of one of the MRSA strains. The presence of the sak gene was demonstrated in 13 strains of MRSA and in 5 strains of MSSA. Detailed data are shown in Tables 4 and 5. Pulsed‑Field Gel Electrophoresis (PFGE) Prophage Pattern in S. aureus Strains Only three of the MRSA isolates were digested by SmaI enzyme and belonged to three different pulsotypes named as Multiplex PCR successfully distinguished the prophage A, B and C (Table 4 and Figs. 4, 5 in supplementary materi- 3A-like (SGA), 11-like (SGB) and 77-like (SGF) sero- als). However, all twenty-six MRSA isolates were typeable groups, the 77-like a (SGFa) and b (SGFb) subgroups and using the restriction enzyme ApaI. The macrorestriction pro- 187-like (SGL). files obtained, following enzyme ApaI digestion, are pre- In our study, four serotypes and two subtypes of sented in Figs. 6, 7 and 8 in supplementary materials. Analy- prophages were detected. Furthermore, all MRSA and sis of the phylogenetic relationship between twenty-three MSSA isolates contained at least one prophage incorpo- of MRSA strains distinguished 16 macrorestriction profiles rated in their genome. A total of 11 prophage patterns were following digestion with ApaI endonuclease. Six isolates identified among MRSA strains and 13 prophage patterns belonging to the pulsotype a1 and three isolates belonging among MSSA strains. The most prevalent prophage patterns to the pulsotype a2 showed the same ApaI-PFGE patterns 1 3 1262 A. Marek et al. Table 5 Prophage content and prevalence of staphylokinase and toxins genes of the 59 MSSA S. aureus isolates estimated by multiplex PCR Lysogenic type Number of MSSA strains (n = 59) (%) Presence of the gene with respective PCR pattern sea tst eta sak seb etb sec sed see Single lysogenic strains 3A-like (SGA) 5 8.5 sea (n = 1) 11-like (SGB) 11 18.6 1 77-like (SGFa) 3 5.1 77-like (SGFb) 7 11.9 seb (n = 1) Double lysogenic strains 3A (SGA)—11 (SGB) 7 11.9 seb (n = 1) 1 sec (n = 1) 3A(SGA)—77a (SGFa) 2 3.4 sea (n = 1) 1 11 (SGB)—77b (SGFb) 7 11.9 seb (n = 1) A (n = 1) 11 (SGB)—77a (SGFa) 2 3.4 77a (SGFa)—77b (SGFb) 4 6.8 sed (n = 1) Triple lysogenic strains 3A (SGA)- 11 (SGB)—77a (SGFa) 3 5 2 3A (SGA)- 11 (SGB)—77b (SGFb) 4 6.8 1 11 (SGB)—77a (SGFa)—77b (SGFb) 3 5 seb (n = 1) Quadruple lysogenic strains 3A-like (SGA)—11-like (SGB)—77a 1 1.7 seb (n = 1) 1 (SGFa)—77b (SGFb) sec (n = 1) MSSA methicillin-sensitive S. aureus, sea, seb, sec, cec, see enterotoxin A,B,C,D,E, tst toxic shock syndrome toxin-1, eta, etb exfoliative toxins A, B, sak staphylokinase (Fig. 1). The genetic similarity of the remaining 14 MRSA for two and four isolates, respectively. The overall MRSA strains ranged from 65 to 98% (Fig. 1). prevalence in this study was 30.6%. Oxacillin-resistant S. aureus are considered resistant to other β-lactam agents, i.e. penicillins, β-lactam/β-lactamase inhibitor combinations, Discussion cephems and carbapenems. This is because most cases of documented of MRSA infections have responded poorly to The threat of Staphylococcus aureus is due to its ease of β-lactam therapy, or because convincing clinical data that transmission between animals and humans and its patho- document clinical efficacy for those agents have not been genicity. It has been demonstrated that livestock-associated presented . While all the MSSA strains tested in the S. aureus strains (LA-MRSA) originated in humans and present study were susceptible to cefoxitin and oxacillin, vice versa [34, 35]. Here, we characterized the presence over half of them were found to be resistant to three anti- of a group of genes responsible for toxin production and biotics applied. Among the strains of MSSA, the most fre- methicillin resistance in S. aureus isolated from slaughter quently observed resistance included lack of susceptibility poultry. Determination of the oxacillin MIC is a method to penicillin G, ampicillin and erythromycin. The present recommended by the CLSI for the detection of meticillin study confirmed the presence of the mecA gene responsible resistance in routine testing; however, some recent studies for resistance to methicillin in twenty-six of the S. aureus have reported low sensitivity and low specificity of oxacillin strains which were all resistant to penicillin G and ampi- compared with cefoxitin . Therefore, cefoxitin is con- cillin (Table 3). Resistance to methicillin is heterogene- sidered to be a better predictor than oxacillin for the detec- ous, which can cause some difficulty in determining these tion of heteroresistance because it is a stronger inducer of characteristics under in vitro conditions, and may lead to penicillin-binding protein 2a (PBP2a) [36, 37]. In our study, complications in the preparation of the test and the interpre- discrepancies between phenotypic oxacillin and cefoxitin tation of results . The literature data indicate that most susceptibility and the presence of mecA gene were observed multi-resistant strains of Staphylococcus aureus exhibit a 1 3 Association Between the Methicillin Resistance of Staphylococcus aureus Isolated from… 1263 in the Netherlands, where MRSA was isolated from 16.0% of chicken meat samples and 35.3% of retail turkey meat samples . Most MRSA isolates have been shown to be resistant to several classes of antibiotics and more than 80% of them produce penicillinases . The research conducted on poultry farms indicate that the farm staff is exposed to an increased risk of MRSA colonization compared to the general population . The results of the study published by Richter et al. indicate that the prevalence of MRSA in the investigated turkey meat production farms in the southwest of Germany reached 90%. In addition, among these isolates, the majority were livestock-associated MRSA . Bacteriophages play an important role in the biology of S. aureus. Through horizontal gene transfer and lysogenic phage conversion associated with virulence factors, they can convert a non-virulent strain of staphylococcus into a viru- lent one [1, 19, 41]. Interestingly, the toxins of the superan- tigens are detected more often among methicillin-resistant staphylococci. For example, enterotoxin A is more com- monly produced by MRSA strains . The use of β-lactam antibiotics and fluoroquinolones in sub-inhibitory concentra- tions has been shown to induce the emergence of prophages from latent states. This results in the replication of the viral genome and the amplification of the genes encoded therein, including lysogenic conversion genes [12, 43]. The results of our study indicated a high prevalence of prophages among the test isolates of S. aureus. In all, Fig. 1 Dendrogram showing genetic similarity among twenty-three 15 prophage patterns were observed among the isolates. MRSA isolates digested with ApaI restriction endonuclease The most prevalent prophage patterns or lysogenic types among MRSA isolates were single lysogenic 77-like, while among MSSA isolates the most prevalent prophage pattern lack of susceptibility to erythromycin and tetracycline, and was 11-like. The prophages detected least frequently were less often to gentamycin and chloramphenicol . Moreo- 3A-like (SGA) (5.8%) and 187-like (SGL) (3.8%) (Tables 4, ver, MRSA strains are also considered to be resistant to all 5). Similar results have been obtained by other authors, who cephalosporins and other β-lactam antibiotics regardless of studied the types of bacteriophages presented in human the results of tests conducted under in vitro conditions . methicillin-resistant S. aureus strains . However, the In our study, the most frequently observed resistance among most prevalent prophage type detected in the genome of MRSA strains included lack of susceptibility to penicillin human MRSA strains by Pantůček et al. was 3A-like . G, ampicillin, amoxicillin, cefoxitin, amoxicillin + clavu- The difference may be due to the fact that the microorgan - lanic acid, oxacillin and tetracycline. Over half of the tested isms were isolated in different geographic locations and strains also showed a lack of sensitivity to erythromycin and from different animal species. clindamycin. The rates of resistance of MRSA to chloram- The results of our study indicate that as many as 18 (13 phenicol, gentamycin and trimethoprim–sulphamethoxazole MRSA and 5 MSSA) of the strains tested had the sak gene were much lower (< 30%) than those to other antibiotics. responsible for the production of staphylokinase. Some These data are disturbing, given the fact that S. aureus authors believe that the activity of phage-encoded virulence strains were isolated from poultry flocks located only in the factors such as staphylokinase is specific for human target area of central-western Poland. For comparison, in research molecules, indicating tight host/pathogen coevolution . conducted by Persoons et al. (2009) in Belgium, MRSA Matthews and Novick suggest that strains of bovine origin was isolated from 8 broiler chickens from two of the 14 carry sak-containing phages less frequently than human farms sampled. In the MRSA-positive flocks, the number of isolates . Our knowledge about prophage patterns in S positive samples varied between 1/5 (20%) and 5/5 (100%) aureus of poultry origin is poorly known. There have been . However, data published in 2009 by de Boer et al. indi- reports suggesting that serogroup F (77-likevirus) and B cate that the highest prevalence of MRSA in poultry was (11-likevirus) phages of Staphylococcus aureus are capable 1 3 1264 A. Marek et al. of expressing staphylokinase [31, 47, 48,]. The results of prophage induction from the cells of S. aureus into Listeria our study indicate that most of the S. aureus strains that pos- monocytogenes has been observed as well, having spontane- sessed the sak gene also had 77-like prophages incorporated ously occurred in cow’s milk . into their genome. Pulsed-field gel electrophoresis (PFGE) is the most As well as its importance as a livestock and community- applied and effective genetic typing method for epidemiolog- associated pathogen, S. aureus is also a well-known cause ical studies and investigation of foodborne outbreaks caused of food intoxication [2, 3, 8, 49]. It is estimated that the by different pathogens, including Staphylococcus aureus. actual number of foodborne illnesses caused by S. aureus Traditionally, human MRSA isolates have been typed by is much higher than those reported . The relationship pulsed-field gel electrophoresis (PFGE), using SmaI as the between the production of enterotoxin A and the presence restriction enzyme . The advantages of using PFGE are of bacteriophages and the enterotoxin A (sea) gene in the good discriminatory power and good reproducibility at the DNA of temperate bacteriophages was first described by interlaboratory level when standardized protocols are used. Betley et al. . Coleman et al. showed that, depending on It is possible that livestock-associated MRSA (LA-MRSA) the phenotype observed in lysogenic cells, bacteriophages is not typeable by this method, as the activity of SmaI is converting enterotoxin A can simultaneously convert staphy- blocked due to methylation of the restriction site . In our lokinase [16, 25]. Among the S. aureus strains tested, the study, only three of the MRSA isolate was typeable by SmaI presence of the sea gene was found in three strains (one enzyme digestion. The remaining twenty-three isolates were MRSA and two MSSA), two of which also had the sak typeable using the restriction enzyme ApaI, an alternative gene. Interestingly, strains with both the sea and sak genes to SmaI as mentioned above. It is likely that the twenty- had the same prophage pattern (3A-77a) (Tables 4, 5). A three MRSA isolates that have not been digested by the SmaI relationship between the production of other toxins (toxic enzyme and have been digested with the ApaI enzyme are shock syndrome and exfoliative toxins) and the presence of of animal origin. Similar observations were made by Bens prophages in the genome of S. aureus bacteria has also been et al. (2006) when examining their isolates collected at a demonstrated . In research conducted by El-Adawy et al. pig farm . (2016), the genes encoding the toxic shock syndrome toxin DNA restriction analysis using ApaI as the restriction (tst), sea, seb, sec and see, and genes for exfoliative toxins enzyme revealed sixteen different patterns, which means (eta/etb) were not found in any turkey and broiler chicken that in the case of MRSA there were six and three identical isolates . In our study, the presence of genes (tst-1) was strains with the same macrorestriction profiles (Fig. 1). This detected in three of the 85 strains tested, while only one may suggest a cross infection by these bacteria in different strain had the gene responsible for producing exfoliatin A individuals within the poultry flock. (eta). The MRSA strain that possessed the tst-1 gene had the 3A-77a prophage pattern, while the other two MSSA strains had prophage patterns 11-like and 3A-11-77b. In contrast, Conclusions the MSSA strain in which the exfoliatin A gene was detected had prophage pattern 11-77b (Tables 4, 5). We confirmed the presence of 30,6% positive strains of The results are particularly disturbing given that exfolia- MRSA in food production animals (chickens and turkeys), tive toxins (ETs) produced by S. aureus strains are the major which as livestock are in close contact with humans (farm- causative agents of blistering skin disorders in humans. Lit- ers, farm co-workers, veterinarians). The results strongly erature data indicate that staphylococcal scaled-skin syn- suggest that people working with livestock are at a potential drome (SSSS) caused by antibiotic-resistant strains of S. risk of becoming MRSA carriers and hence at an increased aureus has recently emerged as a serious problem . risk of infections caused by MRSA. This might compli- The toxic shock syndrome toxin 1 (tst-1) gene is located cate MRSA control measures in human healthcare, urging within mobile genetic element such as staphylococcal SaP11 research into risk factors and transmission routes. Also, the pathogenicity island, which by horizontal transfer can occur relative high frequency of some virulence genes in strains of through bacteriophage transduction. Studies conducted on S. aureus originated from slaughtered poultry in this study strains of S. aureus isolated from healthy humans showed may reflect the potential hazard to consumers. that a significant percentage of isolates (24.3%) possessed The S. aureus strains we studied harboured at least one or the gene for TSST-1 . Therefore, it is believed that up to three prophages. In consequence, high diversity among many healthy individuals carry toxin-producing strains of prophages results in the high potential of the isolate to pro- S. aureus. Some pathogenicity islands of S. aureus can be duce a wide range of virulence factors. In our studies, the transduced to other staphylococcal species: S. chromoge- presence of 77-like prophages incorporated into bacterial nes, S. intermedius, S. xylosus and S. epidermidis . The genome was especially often demonstrated. Various authors transfer of the SaPI1 pathogenicity island associated with emphasize the special role of these prophages in the spread 1 3 Association Between the Methicillin Resistance of Staphylococcus aureus Isolated from… 1265 13. Malachowa N, DeLeo FR (2010) Mobile genetic elements of of virulence factors (staphylokinase, enterotoxin A) between Staphylococcus aureus. Cell Mol Life Sci 67:3057–3071 Staphylococcus strains [31, 48]. From the point of view of 14. Doskar J, Pallová P, Pantůcek R, Rosypal S, Růzicková V, human medicine as well as veterinary medicine, it is also Pantůcková P, Kailerová J, Klepárník K, Malá Z, Bocek P (2000) particularly disturbing that S. aureus virulence factors can Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F be transferred via mobile genetic elements not only within prophages in lysogenic strains. Can J Microbiol 46:1066–1076 strains of the same species but also between species and even 15. Ackermann HW, DuBow MS (1987) Viruses of prokaryotes, vol 2. types of bacteria. Natural groups of bacteriophages. CRC Press, Boca Raton 16. Coleman DC, Sullivan D, Russell RJ (1989) Staphylococcus Acknowledgements This research did not receive any specific grant aureus bacteriophages mediating the simultaneous lysogenic con- from finding agencies in the public, commercial or not for profit version of β-lysin, staphylokinase and enterotoxin A: molecular sectors. mechanism of triple conversion. J Gen Microbiol 135:1679–1697 17. de Boer E, Zwartkruis-Nahuis JT, Wit B, Huijsdens XW, de Neel- ing AJ, Bosch T, van Oosterom RA, Vila A, Heuvelink AE (2009) Compliance with Ethical Standards Prevalence of methicillin-resistant Staphylococcus aureus in meat. Int J Food Microbiol 134:52–56 Conflict of interest The authors have no conflicts of interest to declare. 18. Murakami K, Minamide W, Wada K, Nakamura E, Teraoka H, Watanabe S (1991) Identification of methicillin-resistant strains Open Access This article is distributed under the terms of the Crea- of staphylococci by polymerase chain reaction. J Clin Microbiol tive Commons Attribution 4.0 International License (http://creat iveco 29:2240–2244 mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- 19. Chambers HF (1997) Methicillin resistance in Staphylococci: tion, and reproduction in any medium, provided you give appropriate Molecular and biochemical basis and clinical implications. Clin credit to the original author(s) and the source, provide a link to the Microbiol Rev 10:781–791 Creative Commons license, and indicate if changes were made. 20. Clinical and Laboratory Standards Institute (2015) Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. CLSI document M100-S25. Wayne PA 35: 64–72 21. 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