Virulence Factors in Staphylococcus Associated with Small Ruminant Mastitis: Biofilm Production and Antimicrobial Resistance Genes

Small ruminant mastitis is a serious problem, mainly caused by Staphylococcus spp. Different virulence factors affect mastitis pathogenesis. The aim of this study was to investigate virulence factors genes for biofilm production and antimicrobial resistance to β-lactams and tetracyclines in 137 staphylococcal isolates from goats (86) and sheep (51). The presence of coa, nuc, bap, icaA, icaD, blaZ, mecA, mecC, tetK, and tetM genes was investigated. The nuc gene was detected in all S. aureus isolates and in some coagulase-negative staphylococci (CNS). None of the S. aureus isolates carried the bap gene, while 8 out of 18 CNS harbored this gene. The icaA gene was detected in S. aureus and S. warneri, while icaD only in S. aureus. None of the isolates carrying the bap gene harbored the ica genes. None of the biofilm-associated genes were detected in 14 isolates (six S. aureus and eight CNS). An association was found between Staphylococcus species and resistance to some antibiotics and between antimicrobial resistance and animal species. Nine penicillin-susceptible isolates exhibited the blaZ gene, questioning the reliability of susceptibility testing. Most S. aureus isolates were susceptible to tetracycline, and no cefazolin or gentamycin resistance was detected. These should replace other currently used antimicrobials.


Introduction
Mastitis is the inflammation of the mammary gland, mainly due to intramammary infection (IMI). In small ruminants, this disease is considered a serious economic issue due to the mortality of lactating females, cost of treatment, reduced milk yield and quality [1,2], as well as a public health concern associated with risk of consumer food poisoning [3,4]. Several pathogens can cause mastitis in small ruminants; however, species of staphylococci are the most frequently isolated microorganisms from goat and sheep milk [2,[5][6][7][8].
Staphylococcus aureus is one of the main pathogens associated with mastitis in small ruminants [9]. Incidence of clinical mastitis in sheep due to this bacterium may reach 20% with a mortality rate between 25% and 50%, and the affected mammary halves in surviving animals are frequently destroyed. Chronic mastitis may cause a 25 to 30% reduction in milk yield from the affected udder [10].
Coagulase negative staphylococci (CNS), although not as virulent as S. aureus, often cause subclinical mastitis in small ruminants [5,[11][12][13]. This type of infection, most times not detected by the farmer, clearly reduces milk production, also changing milk composition, indirectly impairing the milk product's properties [14]. CNS are the most prevalent pathogens of the mammary gland in goats and sheep with subclinical mastitis, affecting

Staphylococci Identification
Excellent (96 to 99% probability) and very good (93 to 95% probability) identification was observed for most Staphylococcus. Unidentified isolates and isolates with low discrimination results were confirmed by 16S rRNA gene sequencing.
In the CNS group, S. caprae was the most found species and was isolated only from goat's milk samples. It is a commensal organism that prevails in the skin of the goat udder [19] This species is most commonly found in cases of goat mastitis [37,[45][46][47], but it was also isolated from sheep [5,48], buffalo [17], and cow's milk [49].
In this study, other Staphylococcus species were only isolated from goats: S. warneri, S. capitis, S. hominis, S hyicus, and S. equorum. This was probably because the sheep sampling was smaller, since all these species have been isolated before from sheep milk by several other authors [44].

Genes Associated to Biofilm
We investigated the presence of coa and nuc genes in all 137 staphylococcal isolates, mainly for identification purposes and due to historical reasons. In fact, the ability of a strain to produce coagulase, encoded by the coa gene, is the basis of the primary classification of staphylococci in coagulase-positive or coagulase-negative [16].
All S. aureus isolates (35) harbored the coa gene, as well as isolate B200E1, not identified to the species level. Based on this result, this isolate was probable also S. aureus. Therefore, the 101 Staphylococcus isolates not carrying the coa gene were confirmed as CNS. Furthermore, in the present study, different amplicons of the coa gene with band sizes ranging from 400 to 900 bp were detected (Figure 1), as already reported by others [52][53][54][55]. In fact, the coa gene also has a discriminatory power between isolates because of the heterogeneity of its 3' variable region containing 81-bp tandem short sequence repeats (SSR) [56][57][58].  (1), and Staphylococcus sp. (1). Furthermore, an association was found between the Staphylococcus species and the presence of the nuc gene (χ2 = 70.968, df = 14, p < 0.001). In fact, all S. aureus harbor the nuc gene, while most CNS (70/101) do not. However, the nuc gene was also detected in more than 50% of the isolates in some CNS species: S. warneri (4/7), S. lentus (3/5), S. auricularis (3/4), and S. hyicus (3/3).
The presence of the nuc gene was used in the past to identify S. aureus [23,25]. The nuc gene is present in most S. aureus isolates; however, some isolates not carrying this gene have been described [59,60]. Moreover, the nuc gene has also been detected in other species of Staphylococcus, both CPS and CNS [61,62].
For the detection of the biofilm production genes, bap, icaA, and icaD, the 44 nucpositive biofilm-producing isolates were selected. nuc-positive biofilm-producing staphylococci and biofilm-associated genes are shown in Table 1.
PT goat S. aureus The presence of the nuc gene was used in the past to identify S. aureus [23,25]. The nuc gene is present in most S. aureus isolates; however, some isolates not carrying this gene have been described [59,60]. Moreover, the nuc gene has also been detected in other species of Staphylococcus, both CPS and CNS [61,62].
For the detection of the biofilm production genes, bap, icaA, and icaD, the 44 nucpositive biofilm-producing isolates were selected. nuc-positive biofilm-producing staphylococci and biofilm-associated genes are shown in Table 1.
The bap gene was amplified in eight isolates: S. chromogenes (5), S. auricularis (1), S. simulans (1), and S. warneri (1). None of the S. aureus nuc-positive biofilm-producing isolates carries the bap gene. In fact, the bap gene has been reported mainly in S. aureus strains isolated from cattle [24,63,64]. However, Martins et al. [65] have detected the bap gene in four sheep milk S. aureus isolates. In our study, 8 out of 18 CNS nuc-positive biofilmproducing isolates harbored the bap gene. The bap gene encodes a cell wall associated protein named Bap (for biofilm associated protein), which enhances biofilm formation as it mediates bacterial primary attachment to abiotic surfaces and intercellular adherence [35]. Other studies have reported the presence of the bap gene in several CNS isolates [66].
The presence of the icaA gene was detected in seven isolates: S. aureus (5) and S. warneri (2). On the other hand, the icaD gene was present in 19 S. aureus isolates. Furthermore, five S. aureus isolates carried both icaA and icaD genes simultaneously. Xu, Tan, Zhang, Xia, and Sun [59] detected the icaD gene in 20 out of 28 S. aureus bovine mastitis isolates, while it was not detected in any of the 76 CNS analyzed. The same authors reported the absence of the icaA gene in all analyzed staphylococcal isolates [59].
No isolate carrying the bap gene harbored the ica operon genes, as reported before by other authors [67]. However, Marques et al. [68] found one single bovine mastitis S. aureus isolate (out of 20) that simultaneously carried bap, icaA, and icaD.
None of the three biofilm-associated genes were detected in 14 of the nuc-positive biofilm-producing isolates: S. aureus (6) and CNS (8). Other authors have also reported the absence of bap, icaA, and icaD genes in biofilm-producing S. aureus [24,69,70]. Despite no association being found between the presence of the nuc gene and biofilm production, most biofilm-producing isolates harbored the nuc gene (53.4%), while it was only detected in about 35% of the non-producers. Nevertheless, Kiedrowski, Kavanaugh, Malone, Mootz, Voyich, Smeltzer, Bayles, and Horswill [28] described an inverse correlation between Nuc thermonuclease activity and biofilm formation and confirmed the important role for eDNA in the S. aureus biofilm matrix.
Apparently, CNS produce biofilm mainly via Bap, as already suggested by Zuniga et al. [71], who found the bap gene to be more frequently present in CNS when compared to CPS.
Meanwhile, most S. aureus seem to form biofilm through PIA since they harbor the icaA and icaD genes. Other authors have reported that a low prevalence of the bap gene in S. aureus indicates that the ica operon-dependent mechanism may be the main responsible for the adhesion and biofilm formation in this species [68]. Notwithstanding, it has been reported that biofilm synthesis in S. aureus can also be encoded by the bap gene [72].
Other biofilm formation mechanisms in staphylococci not harboring the classical biofilm-production genes, bap, icaA, and icaD, need to be explored. Furthermore, some of the isolates not carrying bap, icaA, and icaD also did not harbor the coa gene, which has been reported as associated with biofilm formation [18]. However, the nuc gene might be an important factor to consider since all 44 isolates were biofilm producers and harbored the nuc gene, although Nuc has been referred to as a biofilm inhibitor [27,28].

Antimicrobial Resistance
Out of 137 staphylococcal isolates analyzed for antimicrobial susceptibility, 15 were multidrug resistant, 36 were non-susceptible to two antimicrobial categories, and 61 to one antimicrobial category, according to the classification proposed by Magiorakos et al. [73]. Moreover, no antimicrobial resistances were detected in 24 staphylococcal isolates.
Susceptibility patterns of CPS and CNS isolates are shown in Figure 2. For most antimicrobials tested, a higher percentage of resistant isolates was observed among CNS when compared to CPS. Vasileiou et al. [76] also reported more resistant CNS isolates than S. aureus. However, mastitis caused by CNS responds much better to antimicrobial treatment than S. aureus mastitis [75].
One S. aureus and one CPS Staphylococcus sp. were found to be resistant to oxacillin, while CNS oxacillin resistant isolates belonged to eight species: S. chromogenes (5)  No staphylococci resistant to cefazolin and gentamycin were identified. Moreover, no non-susceptible S. aureus isolates were found to amoxicillin + clavulanic acid. A number of CNS isolates, although resistant to penicillinase-labile penicillins, were susceptible to amoxicillin + clavulanic acid, which was expected due to the inhibitory action of clavulanic acid against β-lactamases [79]. Regarding CNS isolates, none were found to be resistant to neomycin.
Regarding tetracycline, most S. aureus isolates (32/35) were susceptible, while nonsusceptible isolates belonged to the following CNS species: S. caprae (4), S. haemolyticus (3), S. lentus (2), S. capitis (1), S. hominis (2), S. rostri (1), and S. warneri (1). Tetracycline has been widely used in veterinary medicine, and other studies have reported a higher percentage of resistant isolates: 42.8% [82] and 28.9% [45]. On the contrary, our results show a relatively low percentage of non-susceptible isolates (12.4%). In recent years, there has been an abusive use of more recent antimicrobial molecules, such as cephalosporins and quinolones, that may justify the observed reversal in the patterns of resistance to tetracyclines. To avoid the use of critically important antimicrobials for human medicine, tetracyclines, gentamycin, or cefazolin, a first-generation cephalosporin, may be an option for the control of mastitis in small ruminants. However, there should be a tight control over the development of antimicrobial resistances.

Antimicrobial Resistance Genes
The 44 biofilm producing isolates were selected for the detection of antimicrobial resistance genes involved in the resistance to β-lactams and tetracyclines, namely, blaZ, mecA, mecC, tetK, and tetM. Table 2 shows the antimicrobial genes detected in each isolate, along with its antimicrobial resistance profile.
No staphylococcal isolates harboring the mecA or mecC genes were detected, although two isolates were found to be non-susceptible to oxacillin and cloxacillin simultaneously, one only to oxacillin and seven to cloxacillin alone. According to the CLSI (2016), oxacillin disk diffusion testing is not reliable for detecting methicillin resistance, at least in S. aureus, and cefoxitin should be used for disk diffusion testing. However, Barrero-Domínguez, Luque, Galán-Relaño, Vega-Pla, Huerta, Román, and Astorga [45] also did not detect the mecA gene in a cefoxitin-resistant MRSA strain. Thus, other resistance mechanisms cannot be excluded, namely, overproduction of β-lactamase, modified penicillin-binding proteins, distinct SCCmec elements, as well as putative mecA mutations [84,85]. Furthermore, Becker et al. [86] have recently reported the presence of a mecB gene in a MRSA strain, negative for both mecA and mecC genes. However, concerning mecC detection in our study, we cannot conclude that the isolates with a negative PCR result did not harbor the mecC gene, since no positive control strain was available.
An association was found between the resistance to penicillin (χ 2 = 11.650, df = 1, p < 0.05) and ampicillin (χ 2 = 15.828, df = 1, p < 0.001) and the presence of the antimicrobial resistance gene blaZ. The association between resistance to penicillin and ampicillin and the presence of the antimicrobial resistance gene blaZ has been reported before by other authors [87,88]. However, no association was detected between the resistance to oxacillin and cloxacillin and the presence of the antimicrobial resistance gene mecA for this subgroup of 44 isolates.
Only one S. aureus isolate carrying the tetK and another one carrying the tetM gene were identified. Both showed resistance to tetracycline. A S. warneri tetracycline-resistant isolate did not harbor either tetK or tetM (Table 3). El-Razik, Arafa, Hedia, and Ibrahim [82] found a S. intermedius isolate showing intermediate resistance to tetracycline, not harboring tetK, tetL, tetM, and tetO genes.

Milk Samples Collection and Bacteriological Analyses
A total of 328 small ruminants (258 goats and 70 sheep), belonging to 23 both traditional and industrial dairy farms in Portugal and Brazil, were used to collect 646 half-udder milk samples (508 from goats and 138 from sheep).
Milk samples were aseptically collected in a sterile bottle after the teat was carefully disinfected with 70% ethanol and the first flush was rejected. The samples were kept refrigerated and transported to the laboratory. Ten microliters of each milk sample were plated onto MacConkey agar (Oxoid, Hampshire, UK, CM0007) and onto blood agar (BA) (Oxoid, Hampshire, UK; CM0271 with 5% sheep blood) and incubated at 37 • C for 24 h to 48 h.
Colonies from BA were transferred to brain heart infusion agar (BHI) (Oxoid, Hampshire, UK, CM1136) and again incubated at 37 • C for 24h for primary identification of the Staphylococcus genus through morphological and biochemical characteristics, namely, colony morphology, Gram staining, and catalase reaction, according to Markey et al. [89].
Identification of the species level of all isolates was performed by automated compact system VITEK 2 (bioMérieux, Marcy l'Etoile, France) using GP ID cards following the manufacturer's instructions. Biochemical identification was confirmed by 16S rRNA gene sequencing whenever necessary, using the primers described previously [90].

Biofilm Detection
Biofilm production was evaluated following the procedures described by Merino et al. [91] with some modifications. In brief, isolates were grown overnight in trypticase soy broth (TSB) at 37 • C. This overnight culture was diluted 1:40 in TSB supplemented with 0.25% glucose, and 200 mL of this cell suspension was used to inoculate microplates. After 24 h of incubation at 37 • C, the microplates were washed three times with 200 µL H 2 O, dried in an inverted position, and stained with 100 µL of 0.25% crystal violet for 2 to 3 min at room temperature. Afterwards, the microplates were rinsed again three times with H 2 O, dried, the dye dissolved in 200 µL ethanol-acetone (80:20), and the absorbance measured at 620 nm. Each assay was performed in triplicate and repeated three times. Staphylococcus epidermidis ATCC 12,228 and ATCC 35,984 were used as negative and positive controls, respectively. A blank control was also used.
For the interpretation of AST results, the CLSI clinical breakpoints M100-S25 were used [78]. Isolates showing intermediate resistance, now called "susceptible increased exposure" [93], were considered non-susceptible. Moreover, isolates resistant to three or more antimicrobial categories were considered multidrug resistant [73].

Molecular Characterisation of Staphylococcal Isolates
The presence of coa and nuc genes was investigated in all staphylococcal isolates. nucpositive biofilm-producing isolates were selected for the detection of the biofilm production genes, bap, icaA, and icaD, and the antimicrobial resistance genes blaZ, mecA, tetK, and tetM. The presence of the mecC gene was investigated only for nuc-positive biofilm-producing isolates, which were simultaneously resistant to oxacillin and cloxacillin and did not harbor the mecA gene.

Rapid DNA Extraction
Total DNA was extracted as described previously [94]. Bacterial cultures were grown for 24 h in BHI (Oxoid, Hampshire, UK, CM1136). After this period, they were transferred to microtubes with 200 µL of ultrapure water and centrifuged at 12,000× g for two minutes. Two hundred microliters of sterile saline solution (8.5%) were added to the pellet and centrifuged again at 12,000× g for two minutes. Subsequently, 100 µL of 0.05 M NaOH was added to the pellet and boiled for four minutes, then placed immediately on ice. Afterwards, 600 µL of ultrapure water was added to the microtubes and centrifuged at 4000× g for three minutes. Subsequently, 400 µL were transferred to a new microtube and stored at −20 • C until use.
Amplified DNA fragments were stained with 1X Red Gel (Biotium, Fremont, CA, USA) and run on 1.5% (w/v) agarose gels with 0.5X TBE (Tris-borate-EDTA) buffer. Different NZYDNA Ladders (NZYtech, Lisbon, Portugal) were used as molecular weight markers, depending on the size of the PCR products.
Agarose The primers used for amplification of the different genes are listed in Table 3.  For the detection of the coa gene, different primer sequences were used. Staphylococcus aureus ATCC 25,923 was used as positive control. The first pair of primers, coa-F and coa-R, amplified a 676 bp fragment [55]. For the amplification of the nuc gene, primers nuc-F and nuc-R, amplifying a 267 bp DNA fragment, were used [95]. S. aureus ATCC 25,923 was used as positive control and S. epidermidis ATCC 12,228 as negative control. The amplification program was the following: 5 min at 94 • C, followed by 37 cycles, consisting of 94 • C for 1 min, 55 • C for 30 s, and 72 • C for 30 s, ending with a final extension step at 72 • C for 7 min.
For detecting the bap gene, primers bap-F and bap-R were used for the amplification of a 971 bp fragment [35]. No positive control strain was available. The amplification program was as follows: 94 • C for 2 min, followed by 35 cycles of 94 • C for 45 s, 56.5 • C for 45 s, and 72 • C for 50 s, and finally, 72 • C for 5 min.
Primers icaA-F and icaA-R were used for the amplification of a 1315 bp fragment of the icaA gene [96]. S. epidermidis ATCC 35,984 was used as positive control. The following amplification program was used: 92 • C for 5 min, followed by 30 cycles of 92 • C for 45 s, 49 • C for 45 s, and 72 • C for 1 min, and a final extension step of 7 min at 72 • C.
For the icaD gene, primers icaD-F and icaD-R were used to amplify a 381 bp fragment [96]. S. epidermidis ATCC 35,984 was used as positive control. The same amplification program as for icaA was used, except for the extension step within the cycles, which was 72 • C for 30 s.
The presence of the blaZ gene was detected using primers blaZ-F and blaZ-R, which amplified a 517 bp fragment [97]. Staphylococcus aureus ATCC 29,213 was used as positive control and S. aureus ATCC 25,923 as negative control [100]. The amplification program was as follows: 94 • C for 4 min, followed by 37 cycles of 94 • C for 1 min, 50.5 • C for 30 s, and 72 • C for 30 s, and finally, 72 • C for 5 min [97].
To detect the mecA gene, primers mecA-F and mecA-R were used to amplify a fragment of 532 bp [98]. Staphylococcus epidermidis ATCC 35,984 was used as positive control [101] and S. aureus ATCC 25,923 as negative control [102]. The following amplification program was used: 94 • C for 2 min, followed by 29 cycles of 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 30 s, and a final extension of 5 min at 72 • C.
Primers mecC-F and mecC-R were used to amplify a 138 bp fragment [99]. No positive control strain was available. The following amplification program was used: 95 • C for 2 min, followed by 30 cycles of 94 • C for 30 s, 50 • C for 30 s, and 72 • C for 30 s, and a final extension of 10 min at 72 • C.
Primers tetK-F and tetK-R were used to amplify a 360 bp fragment of the tetK gene [59]. No positive control strain was available. For the amplification of the tetM gene, tetM-F and tetM-R were used to amplify a fragment of 158 bp [59]. No positive control strain was available. The amplification program for both tet genes was: 94 • C for 2 min, followed by 29 cycles of 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 30 s, with a final step of 5 min at 72 • C.

Data Analysis
The chi-square test of association was used: to assess the relationship between the presence of the nuc gene with Staphylococcus species; to investigate if the presence of the nuc gene was associated with biofilm production; to check if the resistance to antimicrobials was associated with bacterial species and with the animal species from which these were isolated. For the abovementioned analyses, all 137 isolates were considered.
For the subgroup of 44 nuc-positive biofilm-producing isolates, the chi-square test of association was performed to evaluate the putative relationship between phenotypic resistance to antimicrobials and the presence of four resistance genes.
Multiple correspondence analysis (MCA) was used as an exploratory data analysis technique to detect a structure in the relationships between bacterial species and resistance to selected antimicrobials, divided either into two (susceptible and resistant) or three classes (susceptible, intermediate, and resistant), depending on the antimicrobial.
All statistical analyses were performed using the software STATISTICA Version 12 (StatSoft, Inc., Tulsa, OK, USA).

Conclusions
Mastitis aetiology showed to be diverse in the two small ruminant species studied. The most abundant species was S. caprae, which, however, was only present in goats.
The nuc gene was detected in 67 isolates, of which only 35 were S. aureus. Most CNS did not harbor this gene; however, it was detected in more than 50% of S. warneri, S. lentus, S. auricularis, and S. hyicus. Although many studies still consider the nuc gene as the sole character to identify S. aureus, our results have clearly demonstrated that this gene is insufficient, because it is present in numerous staphylococcal isolates other than S. aureus.
Most staphylococci were biofilm producers. The bap gene was only detected in CNS, while ica operon genes were mainly detected in S. aureus isolates, suggesting that CNS produce biofilm mainly via Bap, and most S. aureus form biofilm through PIA. Furthermore, biofilm-producing staphylococcal isolates not harboring the classical biofilm-production genes bap, icaA, and icaD carry the nuc gene. Therefore, the role of the Nuc thermonuclease in staphylococci biofilm formation needs to be further investigated.
Antimicrobial resistance seems to be a growing concern in the treatment of sheep and goat mastitis, with only a low number of isolates (18%) not showing any antimicrobial resistances. Furthermore, CNS were generally more resistant to antimicrobials than CPS. Additionally, an association between animal species and resistance to some antimicrobials was found, suggesting different managing systems for the two species.
All staphylococcal isolates were susceptible to cefazolin and gentamycin. Furthermore, all S. aureus isolates were shown to be susceptible to amoxicillin + clavulanic acid and most (32/35) to tetracycline. The use of these antimicrobials to control mastitis may be encouraged to avoid the use of others critically important for human medicine that are currently being used, such as third generation cephalosporins and quinolones. Nevertheless, antimicrobial susceptibility tests cannot be neglected, as the development of resistant strains is always a problem.
Regarding antimicrobial resistance genes, nine penicillin-susceptible isolates exhibited the blaZ gene, highlighting the poor reliability of conventional methods for susceptibility testing. Moreover, no staphylococcal isolates harboring the mecA or mecC genes were detected among those found to be non-susceptible to oxacillin. Hence, other methicillin resistance mechanisms need to be explored.