Comparative profiling of agr locus, virulence, and biofilm-production genes of human and ovine non-aureus staphylococci.

BACKGROUND: In a collaboration between animal and human health care professionals, we assessed the genetic characteristics shared by non-aureus staphylococci (NAS) infecting humans and dairy ewes to investigate their relatedness in a region concentrating half of the total National sheep stock. We examined by PCR 125 ovine and 70 human NAS for biofilm production, pyrogenic toxins, adhesins, autolysins genes, and accessory gene regulator (agr) locus. The microtiter plate assay (MPA) was used for the phenotypic screening of biofilm production. Ovine NAS included S. epidermidis, S. chromogenes, S. haemolyticus, S. simulans, S. caprae, S. warneri, S. saprophyticus, S. intermedius, and S. muscae. Human NAS included S. haemolyticus, S. epidermidis, S. hominis, S. lugdunensis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis.
RESULTS: Phenotypically, 41 (32.8%) ovine and 24 (34.3%) human isolates were characterized as biofilm producers. Of the ovine isolates, 12 were classified as biofilm-producing while the remaining 29 as weak biofilm-producing. All 24 human isolates were considered weak biofilm-producing. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA), while the bhp, aap, and embp genes coding biofilm accumulation proteins were present in both non-producing and biofilm-producing isolates. Fifty-nine sheep NAS (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the agr locus: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans. Concerning virulence genes, 40, 39.2, 47.2%, 52.8, 80 and 43.2% of the sheep isolates carried atlE, aae, sdrF, sdrG, eno and epbS respectively, against 37.1, 42.8, 32.8, 60, 100 and 100% of human isolates. Enterotoxins and tsst were not detected.
CONCLUSIONS: Considerable variation in biofilm formation ability was observed among NAS isolates from ovine and human samples. S. epidermidis was the best biofilm producer with the highest prevalence of adhesin-encoding genes.

Background

Half of the total Italian dairy sheep stock is farmed in Sardinia, an island located in the Mediterranean Sea. Sardinia has approximately 3.5 million dairy sheep, with a human population of around 1.6 million inhabitants. Accordingly, a relevant part of the regional economy relies on dairy sheep farming, and controlling intra-mammary infections (IMI) is crucial.

Sheep mastitis prevalence is estimated to range from 5 to 30%, and several reports indicate that non-aureus staphylococci (NAS) are the most prevalent microrganisms causing subclinical disease in small ruminants [1-4]. Therefore, the exchange of colonizing and pathogenic microorganisms, with their antimicrobial-resistance and pathogenicity gene pools, can occur among sheep and farmers. NAS have recently gained attention as nosocomial agents causing frequent infections in debilitated or compromised patients, mainly associated with catheters and other indwelling medical devices [5]. NAS, and in particular S. epidermidis, can produce a multicellular biofilm that decreases the antibiotic concentration within the colony, promotes multiplication, and enhances the survival of invading bacteria [6]. Biofilm formation can be best assessed by the microtiter plate assay (MPA), as it produces a quantitative result by measuring the optical density of the stained biofilm [7, 8]. The main constituent of the NAS biofilm matrix is a linear 1,6-linked glycosaminoglycan, also known as polysaccharide intercellular adhesin (PIA), synthesized by proteins encoded by the intercellular adhesion (ica) operon. Among the ica genes, icaA and icaD have an essential role in biofilm production [9]. The coexistence of both icaA/D genes leads to the full phenotypic expression of the capsular polysaccharide [9]. However, PIA-independent biofilms involving accumulation-associated protein (Aap), biofilm homologue protein (Bhp) and extracellular matrix-binding protein (Embp) have also been reported [10, 11].

Generally, NAS can produce several virulence factors that contribute collectively to colonization and invasion of host cells and tissues, as well as evasion of immune responses [12]. Virulence factors include the autolysins AtlE and Aae [13, 14], and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that mediate initial adhesion to different surfaces and promote colonization and serum protein binding [15]. The best known S. epidermidis MSCRAMMs are the fibrinogen-binding protein SdrG [16], and the collagen/keratin-binding protein SdrF [17, 18].

Furthermore, the production of various toxins can also contribute to NAS virulence [19], including staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1) [20]. Five serological types of SEs are typically known (SEA to SEE), but new types of SEs (SEG to SE1V) have also been identified and characterized [21, 22]. The quorum-sensing system (QS) agr, i.e. accessory gene regulator [23, 24] regulates biofilm formation, intercellular communication, and numerous virulence factors including toxins and autolysins. Three distinct genetic groups (types 1, 2, and 3) based on the agr locus polymorphism have been described in S. epidermidis [25], but data on the genetic polymorphisms of the agr locus in different species of NAS were not available in the scientific literature at the beginning of this investigation.

In this study, we compared the molecular characteristics of NAS isolated from the milk of sheep with mastitis and human clinical specimens with the following aims: 1) assess the biofilm production characteristics by phenotypic and genotypic methods, 2) carry out genotypic screening for a set of MSCRAMMs, autolysins, enterotoxins and tsst-1 genes and 3) investigate the agr locus and its genetic polymorphism.

Results

Ovine NAS

We analyzed a total of 125 isolates, including S. epidermidis (n = 57), S. chromogenes (n = 29), S. haemolyticus (n = 17), S. simulans (n = 8), S. caprae (n = 6), S. warneri (n = 5), S. saprophyticus (n = 1), S. intermedius (n = 1) and S. muscae (n = 1). Table 1 reports the isolates included in the study, while Supplementary Table S1 reports the primers used for PCR amplifications.

Table 1

Distribution of ovine and human NAS isolates according to the specimen origin

Ovine NASa
Source	S. epi	S. chr	S. hae	S. sim	S. cpr	S. war	S. sap	S. int	S. mus
Milk	57	29	17	8	6	5	1	1	1
Human NASa
Source	S. hae	S. epi	S. lug	S. hom	S. cap	S. war	S. xyl	S. pas	S. sap
Nasal swab	6	5	–	–	–	–	–	–	–
Blood	4	6	–	1	–	–	–	–	–
Skin swab	2	3	–	–	2	–	1	–	–
Pus	1	3	–	–	–	–	–	1	–
Peritoneal fluid	2	–	2	–	–	–	–	–	–
Seminal fluid	4	–	–	–	–	–	–	–	–
Injury	–	3	1	–	–	–	–	–	–
Ear swab	2	–	–	–	–	1	–	–	–
Oral swab	1	1	–	–	–	–	–	–	1
Urine	2	–	–	1	–	–	–	–	–
C.V.Cb	–	2	–	–	–	–	–	–	–
Ulcer swab	–	1	–	–	–	–	–	–	–
F.V.C.b	–	1	–	–	–	–	–	–	–
Peritoneal swab	1	–	–	–	–	–	–	–	–
Vaginal swab	–	–	1	–	–	–	–	–	–
Glans swab	1	–	–	–	–	–	–	–	–
Pleural fluid	–	–	–	1	–	–	–	–	–
Fluid drainage	–	–	–	–	–	1	–	–	–
B.L. fluidb	1	–	–	–	–	–	–	–	–
N.P. aspirateb	–	–	–	–	1	–	–	–	–
Bile	1	–	–	–	–	–	–	–	–
Biopsy	–	1	–	–	–	–	–	–	–
Prosthesis	–	–	–	1	–	–	–	–	–
Total	28	26	4	4	3	2	1	1	1

aIsolate abbreviations: S. hae, S. haemolyticus; S. epi, S. epidermidis; S. chr, S. chromogenes; S. sim, S. simulans; S. lug, S. lugdunensis; S. hom, S. hominis; S. cpr, S. caprae; S. S. cap, S. capitis; S. war, S. warneri; S. xyl, S. xylosus; S. pas, S. pasteuri; S.int, S. intermedius; S. sap, S. saprophyticus subsp. Bovis; S. mus, S. muscae

bSpecimen abbreviations: C.V.C central venous catheter, F.V.C femoral venous catheter, B.L.fluid bronchoalveolar lavage fluid, N.P aspirate, nasopharyngeal aspirate

Table 2 summarizes the biofilm formation results. Out of 125 isolates examined, 41 (32.8%) were classified as biofilm producers; of these 29 (23.2%) were classified as weak biofilm-producers (WBP) while 12 (9.6%) as biofilm-producers (BP). Only one isolate harbored both icaA and icaD genes while two had only the icaA gene. On the other hand, 37 (29.6%), 22 (17.6%) and 63 (50.4%) isolates harbored the bhp, aap and embp genes, respectively (Table 2). For autolysin genes, 50 (40%) isolates were PCR positive for altE while 49 (39.2%) were positive for aae. Concerning adhesion factors (MSCRAMMs), 59 (47.2%), 66 (52.8%), 100 (80%) and 54 (43.2%) isolates harbored sdrF, sdrG, eno and epbS, respectively. All isolates were negative for clfA (Table 3). Regarding the agr type, 21 (16.8%) isolates belonged to agr-1 while 33 (26.4%) to agr-3. None of the isolates belonged to type 2 (Table 4). No amplification was obtained for the toxin genes analyzed.

Table 2

Phenotypic characterisation of biofilm production by MPA and genotypic detection by PCR of ica, bhp, aap and embp genes from 125 ovine and 70 human NAS isolates

OVINE Isolates (n)	Biofilm production (MPA)				Biofilm genes
					PIA						Proteinaceous factors
	WBPa		BPb		icaA		icaD		icaA/D		bhp		aap		embp
	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
S. epi (57)	17	30	11	19	1	1.7	1	1.7	1	1.7	30	52.6	20	16	54	94.7
S. chr (29)	0	0	1	3.4	0	0	0	0	0	0	4	13.8	0	0	8	27.5
S. hae (17)	0	0	0	0	0	0	0	0	0	0	0	0	2	11.7	1	5.8
S. sim (8)	7	87.5	0	0	0	0	0	0	0	0	0	0	0	0	0	0
S. cpr (6)	3	50	0	0	1	16.6	0	0	0	0	1	16.6	0	0	0	0
S. war (5)	0	0	0	0	0	0	0	0	0	0	2	40	0	0	0	0
S. sap (1)	1	100	0	0	1	100	0	0	0	0	0	0	0	0	0	0
S. int (1)	1	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0
S. mus (1)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Total (125)	29	23.2	12	9.6	3	2.4	1	0.8	1	0.8	37	29.6	22	17.6	63	50.4
HUMAN Isolates (n)	Biofilm production (MPA)				Biofilm genes
					PIA						Proteinaceous factors
	WBPa		BPb		icaA		icaD		icaA/D		bhp		aap		embp
	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
S. hae (28)	1	3.6	0	0	0	0	0	0	0	0	0	0	4	14.3	0	0
S. epi (26)	9	34.6	0	0	10	38.5	10	38.5	10	38.5	16	61.5	25	96.1	23	88.5
S. lug (4)	3	75	0	0	0	0	0	0	0	0	0	0	0	0	0	0
S. hom (4)	4	100	0	0	0	0	0	0	0	0	0	0	4	100	0	0
S. cap (3)	3	100	0	0	3	100	0	0	0	0	0	0	0	0	0	0
S. war (2)	2	100	0	0	0	0	0	0	0	0	0	0	1	50	1	50
S. xyl (1)	1	100	0	0	1	100	0	0	0	0	0	0	0	0	0	0
S. pas (1)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
S. sap (1)	1	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Total (70)	24	34.3	0	0	14	20	10	14.3	10	14.3	16	22.8	34	48.6	24	34.3

Isolate abbreviations: S. hae, S. haemolyticus; S. epi, S. epidermidis; S. chr, S. chromogenes; S. sim, S. simulans; S. lug, S. lugdunensis; S. hom, S. hominis; S. cpr, S. caprae; S. S. cap, S. capitis; S. war, S. warneri; S. xyl, S. xylosus; S. pas, S. pasteuri; S.int, S. intermedius;S. sap, S. saprophyticus subsp. bovis; S. mus, S. muscae

aWBP weak biofilm-producing isolate

bBP biofilm-producing isolate

Table 3

Results of testing 125 ovine and 70 human NAS isolates for autolysins and MSCRAMMS genes by PCR

OVINE Isolates (n)	Autolysins				MSCRAMMS
	atlE		aae		sdrF		sdrG		clfA		clfB		fnbA		fnbB		bbp		cna		fib		eno		epbS
	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
S. epi (57)	48	84.2	46	80.7	45	78.9	50	87.7	0	0	0	0	0	0	0	0	0	0	0	0	0	0	56	98	52	91
S. chr (29)	1	3.4	0	0	9	31	13	22.8	0	0	2	7	2	7	1	3.4	0	0	2	7	2	7	16	55	2	7
S. hae (17)	1	5.8	1	5.8	1	5.8	1	5.8	0	0	0	0	0	0	0	0	1	6	0	0	0	0	15	88	0	0
S. sim (8)	0	0	1	12.5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
S. cpr (6)	0	0	0	0	1	16.6	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	6	100	0	0
S. war (5)	0	0	1	20	3	60	2	40	0	0	0	0	0	0	0	0	0	0	0	0	0	0	4	80	0	0
S. sap (1)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	100	0	0
S. int (1)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	100	0	0
S. mus (1)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	100	0	0
Total (125)	50	40	49	39.2	59	47.2	66	52.8	0	0	2	1.6	2	1.6	1	0.8	1	0.8	2	1.6	2	1.6	100	80	54	43.2
HUMAN Isolates (n)	Autolysins				MSCRAMMS
	atlE		aae		sdrF		sdrG		clfA		clfB		fnbA		fnbB		bbp		cna		fib		eno		epbS
	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
S. hae (28)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	28	100	0	0
S. epi (26)	25	96.1	26	100	21	80.8	26	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	26	100	26	100
S. lug (4)	0	0	0	0	0	0	4	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	4	100	0	0
S. hom (4)	0	0	0	0	0	0	4	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	4	100	0	0
S. cap (3)	0	0	0	0	0	0	3	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3	100	0	0
S. war (2)	1	50	1	50	1	50	2	100	1	50	0	0	0	0	0	0	0	0	0	0	0	0	2	100	0	0
S. xyl (1)	0	0	1	100	1	100	1	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	100	0	0
S. pas (1)	0	0	1	100	0	0	1	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	100	0	0
S. sap (1)	0	0	1	100	0	0	1	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	100	0	0
Total (70)	26	37.1	30	42.8	23	32.8	42	60	1	50	0	0	0	0	0	0	0	0	0	0	0	0	70	100	26	100

Isolate abbreviations: S. hae, S. haemolyticus; S. epi, S. epidermidis; S. chr, S. chromogenes; S. sim, S. simulans; S. lug, S. lugdunensis; S. hom, S. hominis; S. cpr, S. caprae; S. S. cap, S. capitis; S. war, S. warneri; S. xyl, S. xylosus; S. pas, S. pasteuri; S.int, S. intermedius;S. sap, S. saprophyticus subsp. bovis; S. mus, S. muscae

Table 4

Results of testing 125 ovine and 70 human NAS isolates for agr locus by PCR

OVINE Isolates (n)	agr locus
	agr 200 bp		Type 1		Type 2		Type 3
	n	%	n	%	n	%	n	%
S. epi (57)	57	100	21	36.8	0	0	33	57.8
S. chr (29)	1	3.4	0	0	0	0	0	0
S. hae (17)	1	5.8	0	0	0	0	0	0
S. sim (8)	0	0	0	0	0	0	0	0
S. cpr (6)	0	0	0	0	0	0	0	0
S. war (5)	0	0	0	0	0	0	0	0
S. sap (1)	0	0	0	0	0	0	0	0
S. int (1)	0	0	0	0	0	0	0	0
S. mus (1)	0	0	0	0	0	0	0	0
Total (125)	59	47.2	21	16.8	0	0	33	26.4
HUMAN Isolates (n)	agr locus
	agr 200 bp		Type 1		Type 2		Type 3
	n	%	n	%	n	%	n	%
S. hae (28)	0	0	0	0	0	0	0	0
S. epi (26)	26	100	17	65.4	9	34.6	0	0
S. lug (4)	0	0	0	0	0	0	0	0
S. hom (4)	0	0	0	0	0	0	0	0
S. cap (3)	0	0	0	0	0	0	0	0
S. war (2)	1	50	0	0	0	0	0	0
S. xyl (1)	0	0	0	0	0	0	0	0
S. pas (1)	0	0	0	0	0	0	0	0
S. sap (1)	0	0	0	0	0	0	0	0
Total (70)	27	38.6	17	24.3	9	12.8	0	0

Isolate abbreviations: S. hae, S. haemolyticus; S. epi, S. epidermidis; S. chr, S. chromogenes; S. sim, S. simulans; S. lug, S. lugdunensis; S. hom, S. hominis; S. cpr, S. caprae; S. S. cap, S. capitis; S. war, S. warneri; S. xyl, S. xylosus; S. pas, S. pasteuri; S.int, S. intermedius;S. sap, S. saprophyticus subsp. bovis; S. mus, S. muscae

S. epidermidis

S. epidermidis was the most represented ovine NAS. Out of 57 isolates, 17 (30%) were classified as WBP and 11 (19%) as BP; only one non-BP harbored both icaA/D genes. On the other hand, 54 (94.7%), 30 (52.6%) and 20 (16%) isolates harbored embp, bhp and aap, respectively. Concerning autolysin genes, 48 (84.2%) were PCR-positive for atlE and 46 (80.7%) for aae. MSCRAMM genes were found in high percentages: 98% for eno, 91% for epbS, 87.7% for sdfG and 78.9% for sdrF. No amplification was obtained for clfA/B, fnbA/B, bbp, cna and fib (Table 3). All S. epidermidis isolates were positive for the agr locus: 33 (57.8%) belonged to agr-3se while 21 (36.8%) to agr-1se (Table 4). Three isolates were non-typeable.

S. chromogenes, S. haemolyticus, and minor ovine NAS

S. chromogenes (n = 29) and S. haemolyticus (n = 17) were the most prevalent species in ovine milk samples after S. epidermidis. Table 2 shows that only 1 S. chromogenes was classified as BP while 7 (87.5%) S. simulans and 3 (50%) S. caprae were classified as WBP. The details are reported in Table 2.

Human NAS

The distribution of biofilm, autolysins, and MSCRAMMs genes in the 70 human NAS, including S. haemolyticus, S. epidermidis, S. lugdunensis, S. hominis, S. capitis, S. warneri, S. xylosus, S. pasteuri and S. saprophyticus subsp. bovis is shown in Tables 2 and 3.

S. haemolyticus

Out of 28 S. haemolyticus isolates examined by MPA, only 1 (3.6%) was classified as WBP. None of the S. haemolyticus isolates harbored icaA/D, bhp and embp. On the contrary, the WBP (from blood) and other 3 non-BP (from nasal swab, blood, and glans swab) isolates possessed the aap gene. No amplification was observed for atlE, aae, sdrF/G, clfA/B, fnbA/B, bbp, cna, fib, epbS, agr locus (Table 3), and toxin genes. However, all isolates harbored the eno gene.

Among the 26 S. epidermidis isolates, 9 (34.6%) were classified as WBP; 4 of them (2 from catheter and 2 from blood) harbored both icaA and icaD whereas the remaining 5 isolates were icaA/D negative. The other 5 icaA/D positive isolates, classified as non-BP, were also positive for the other biofilm genes analyzed. Out of 17 non-BP and icaA/D negative isolates, 3 (2 from pus and 1 from oral swab) harbored aap and embp while 1 (from skin swab) harbored all bhp, aap, and embp genes. Overall, 16 (61.5%), 25 (96.1%) and 23 (88.5%) isolates carried the bhp, aap and embp, respectively (Table 2). Data on the prevalence of autolysins and MSCRAMM genes by PCR are shown in Table 3. Concerning autolysin genes, all isolates were PCR positive for aae and almost all (25/26 = 96.1%) were positive for altE. Regarding adhesion factors, all S. epidermidis isolates were positive for sdrG, eno, and epbS, while 21/26 (80.8%) for sdrF. In four of them (2 from pus, 1 from biopsy and 1 from skin swab), a PCR product smaller than the expected size was observed. Sequence analysis of these amplicons showed the absence of an 84 bp fragment. In contrast, no amplification was observed for clfA/B, fnbA/B, bbp, cna, and fib. Determination of the agr type was performed in all S. epidermidis isolates: 17 (65.4%) belonged to agr-1se whilst 9 (34.6%) to agr-2se (Table 4). Among the 17 agr-1se isolates, only 3 (1 from femoral venous catheter, 1 from nasal swab and 1 from pus) carried simultaneously icaA/D, bhp, aap, embp, atlE, aae, sdrF sdrG, eno, and epbS, associated with biofilm formation. Of these, 1 was WBP and two non-BP. Among the 9 agr-2se isolates, only 1 (from blood) possessed these genes and it was a WBP isolate. Regarding the pyrogenic toxin genes, amplification was not observed in the S. epidermidis isolates or the remaining staphylococci.

Minor human NAS

Out of the 4 S. lugdunensis isolates examined, 3 (2 from peritoneal fluid and 1 from vaginal swab) were considered as WBP. However, they harbored only sdrG and eno.

All 4 S. hominis isolates were phenotypically WBP but were PCR-positivity only for aap, sdrG, and eno. All 3 WBP S. capitis isolates were PCR-positive for icaA, sdrG, and eno. Tables 3 and 4 report the PCR results for S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis. Of note, one WPB S. warneri isolate (from fluid drainage) was negative for icaA/D and bhp genes but was positive for the aap, embp, atlE, aae, sdrG/F, clfA, eno and agr genes. However, we were not able to type the agr locus.

Discussion

We established a collaboration between animal and human health care professionals aimed at understanding if non-aureus staphylococci (NAS) responsible for human diseases share genetic similarities with those circulating in sheep, in consideration of the high number of dairy sheep farmed in the island and of the prominent role of these bacteria as mastitis agents.

A total of 195 NAS isolates, 125 from ovine mastitis and 70 from human clinical specimens, were analyzed for biofilm production and presence of autolysins, pyrogenic toxins, and MSCRAMM genes. We also typed agr alleles by PCR because the quorum sensing system regulates many virulence determinants involved in staphylococcal infections, including autolysins, adhesins, and toxins [19]. In sheep, the primary NAS detected were S. epidermidis followed by S. chromogenes and S. haemolyticus. At the same time, in humans we found primarily S. haemolyticus and S. epidermidis, followed by S. lugdunensis, S. hominis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis. S. haemolyticus has been associated with septicemia in neonates and skin infections; S. epidermidis is the main pathogen isolated in catheter-associated bloodstream infections (BSI); S. lugdunensis can cause acute endocarditis; S. hominis and S. capitis may induce BSI in neonates; S. warneri is associated with device-related bone and joint infections, while S. pasteuri, S. xylosus and S. saprophyticus subsp. bovis are not associated with a particular clinical infection, and their appearance as nosocomial pathogens could be related to previous contact with animals, mainly pig, cattle, sheep, and goats [26]; S. epidermidis represents the most frequently isolated species from ovine mastitis and human clinical specimens [1, 26, 27].

Overall, 65 NAS were able to form biofilm in vitro; however, the percentage of biofilm producers in sheep isolates was slightly lower than in human isolates. Moreover, we found a correlation between biofilm production and ica operon presence only in 5 S. epidermidis isolates, 4 human and 1 ovine. Some authors proposed to use this correlation as a pathogenesis marker to distinguish invasive from commensal isolates [28, 29]. However, we and others [11, 30, 31] demonstrated that PCR positivity for icaA/icaD genes can also be found in non-biofilm producers. Since the correlation between biofilm production and positivity for ica, bhp, aap, and embp genes is not clearly defined, we suggest considering all isolates that possess such genes as potentially invasive. In this work, only one S. epidermidis with these characteristics was isolated from ovine mastitis while the other 4 derived from catheters and blood. Noteworthy, a high positivity for the genes encoding the bifunctional adhesins/autolysins AtlE and Aae was found in both animal and human S. epidermidis isolates [3, 5]. In addition to bacteriolytic activity, AtlE and Aae act as adhesins by binding noncovalently to vitronectin and by causing the release of extracellular DNA (eDNA), a critical adherence/aggregation factor in biofilm formation [32]. The presence, of atlE and aae in S. epidermidis was accompanied by a high prevalence of embp, sdrG, sdrF, eno and epbS, all genes that mediate adherence to substrates containing fibronectin, fibrinogen, collagen, laminin and elastin, respectively [4, 33–35]. The ability of S. epidermidis to bind these substrates might represent a relevant mechanism by which it can adhere to and colonize different host sites. The eno gene was the only gene found in all NAS analyzed, except for S. simulans. In human NAS, the prevalence is 100%. Therefore, the ability of NAS to bind laminin, a major component of basal membrane of the vasculature, might play a possible role in to tissue invasion and blood dissemination.

The agr locus is a regulatory system that responds to host and environmental stimuli and controls the production of many virulence factors [24]. In S. epidermidis, three distinct agr groups have been recognized [25]. Li et al. [36] have linked the genetic polymorphism of the agr locus to pathogenicity; group-1se was associated with pathogenicity, while healthy people mainly carried group-2se. In our human S. epidermidis isolates, agr-1se was predominant (n = 17), followed by agr-2se (n = 9). It is interesting to notice that almost all isolates possessing ica genes belonged to agr-1se. This may suggest a correlation of these virulence genes with a specific agr locus. However, other 8 icaA−/D− isolates were present in the group-1se. The feature shared by all 17 isolates belonging to this group was the PCR positivity for the atlE, aae, sdrG, eno and epbS genes. On the other hand, among the 9 isolates grouped in the agr-2se, 1 (from blood) was icaA+/D+, while the remaining ones were ica-negative. The common denominator of these 9 isolates was the PCR positivity for aap, aae, sdrG, embp, eno, and epbS. These findings suggest that the relationship between agr groups and S. epidermidis pathogenicity will require further investigation. As observed in our previous study [30], agr-3se (n = 33) was predominant among ovine S. epidermidis isolates followed by agr-1se (n = 21). These results may suggest a possible transmission of S. epidermidis isolates from the milkers to the ewes.

Unlike S. chromogenes, S. haemolyticus, S. warneri and S. muscae from ovine mastitis and S. pasteuri from human specimens, the other NAS were classified as WBP by the microplate adhesion technique but did not harbor icaA/D. According to Fredheim et al. [37], S. haemolyticus mainly produces a PIA-independent biofilm. However, we detected only the aap (2/17) and embp (1/17) genes in the present study by PCR. Only 4 human S. haemolyticus isolates possessed the aap gene coding a protein that mediates biofilm formation in strains lacking the ica genes [16]. Our data suggest that ica and bhp genes do not contribute significantly to S. haemolyticus biofilms’ protein components.

In S. aureus and in many other bacteria, toxins are critical contributors to aggressive virulence, even though S. epidermidis is not generally accepted as an enterotoxin producer [38, 39]. Based on our findings, the primary enterotoxin genes (sea, seb, sec, sed and see) and the tsst-1 gene were absent in all ovine and human NAS analyzed. On the contrary, Pedroso et al. [16] and Da Cunha et al. [40] detected high percentages of sea and sec genes in coagulase-negative staphylococci from hospitals of Brazil; also, Giormezis et al. [39] found a higher number of isolates positive for tsst among NAS from hospitals in Greece.

Conclusion

In conclusion, we detected intercellular adhesion genes (icaAB) and other genes related to biofilm formation only in S. epidermidis, although we found icaA in ovine S. caprae and S. saprophyticus, and in human S. capitis and S. xylosus. The remaining isolates carried few virulence determinants. The ability to form biofilm observed in NAS isolates, especially S. epidermidis, might constitute a significant virulence factor facilitating colonization, infection, diffusion, and resistance.

Methods

Isolate collection

Ovine isolates: A total of 125 NAS were isolated from sheep milk samples that routinely arrive at the Istituto Zooprofilattico Sperimentale della Sardegna. Milk samples, collected from farms with mastitis problems in different provinces of Sardinia (Italy), were analyzed over 9 months (April-December 2017) (Table 1). The geographic distribution of these isolates is reported in Supplementary Fig. S1. Isolates were identified as: S. epidermidis (n = 57), S. chromogenes (n = 29), S. haemolyticus (n = 17), S. simulans (n = 8), S. caprae (n = 6), S. warneri (n = 5), S. saprophyticus (n = 1), S. intermedius (n = 1), and S. muscae (n = 1), by means of PCR-RFLP [27].

Human isolates: During the same period, 70 NAS isolates were collected from different clinical specimens at the microbiology laboratories of three major Sardinia hospitals. Isolates were anonymized without patient identifiers. Isolates and their origin are summarized in Table 1; 90% of the human NAS were recovered from hospitalized patients in intensive care unit, hematology, and orthopedics. The 70 isolates were identified by PCR-RFLP as S. haemolyticus (n = 28), S. epidermidis (n = 26), S. lugdunensis (n = 4), S. hominis (n = 4), S. capitis (n = 3), S. warneri (n = 2), S. xylosus (n = 1), S. pasteuri (n = 1), and S. saprophyticus subsp. bovis (n = 1) [27].

Statements of owner consent or patient consent were not required in this case since personal or sensitive data never accompanied samples. All Isolates were anonymized regarding the originating animal, flock, or patient, and were processed for phenotypic and molecular analyses without any original information linked to them.

Phenotyping evaluation of biofilm production by the microtiter plate assay (MPA)

All 195 isolates were tested using the MPA technique, described by Vasileiou et al. [8] with some modifications. Briefly, a colony of each isolate was inoculated into a tube containing 1 mL Tryptone Soy Broth (TSB, Oxoid, Basingstoke, UK) for 16 h at 37 °C. Overnight culture was diluted 1:40 with TSB containing 0.25% glucose, and 200 μL per well were seeded in a sterile 96-well flat-bottomed microplate (Thermo Fisher, Rodano, IT) at 37 °C for 24 h. After three washes in PBS pH 7.4, the microplate was dried at 45 °C for 20 min, and wells were then stained with 1% crystal violet for 15 min at room temperature. After three washes with distilled water and subsequent drying at 45 °C for 20 min, 200 μL of 33% acetic acid were added to each well. Biofilm growth was measured at 630 nm in a microplate spectrophotometer (Multiskan GO, Thermo Fisher). Uninoculated wells containing TBS with glucose served as blanks. In each microplate, S. epidermidis ATCC 35984 and S. epidermidis ATCC 12228 were included as the positive and negative controls, respectively. Each isolate and both controls were tested in triplicate, and the assay was repeated two times at different dates. Isolates were classified into three categories based upon the median OD of isolates and positive and negative controls: biofilm-producing (OD isolate ≥ OD of the positive control), weak biofilm-producing (OD negative control < OD isolate < OD positive control) and non biofilm-producing (OD isolate ≤ negative control).

Detection of biofilm, autolysins, MSCRAMMs and pyrogenic toxins genes

Genomic DNA was extracted from all 195 NAS isolates and Reference Strains (RS) according to Onni et al. [41]. Single-tube PCRs were performed for detecting genes related to biofilm production (icaA/D, bhp, aap, embp) [30, 31, 42, 43], autolysins (atlE and aae) [44, 45], MSCRAMMs (encoding clumping factor-clfA/B, fibronectin-binding protein-fnbA/B, encoding bone sialoprotein-binding protein-bbp, collagen-binding protein-cna, fibrinogen-binding protein-fib, laminin-binding protein-eno, elastin-binding protein-ebpS, and serine-aspartate repeat protein-sdrF/G) [19, 30, 46–49] and pyrogenic toxins (sea, seb, sec, sed, see and tsst-1) [50-52]. Primer sets are reported in Supplementary Table S1. PCR tests were carried out in a GeneAmp9700 DNA thermal cycler (Applied Biosystems, now Thermo Fisher Scientific, Waltham, MA, USA). The following RS were used as positive controls: S. epidermidis ATCC 35984 (icaA/D, bhp, aap, embp, atlE, aae, sdrF, sdrG), S. aureus ATCC 25923 (clfA/B, bbp, epbs), S. aureus ATCC 33591 (fnbA/B, cna, fib, eno), S. aureus ATCC 13565 (sea), S. aureus ATCC 14458 (seb), S. aureus ATCC19095 (sec), S. aureus ATCC 23235 (sed), S. aureus ATCC 27664 (see) and S. aureus ATCC 33586 (tsst-1).

Typing of agr alleles

A 200 bp conserved region of the agr operon was amplified as described previously [19]. For isolate typing, we targeted agr-1 to agr-3 sequences [53]. As controls, we used S. epidermidis ATCC 35984 (agr-1), S. epidermidis isolate 1037 (agr-2s) and S. epidermidis isolate 43,027 (agr-3). Supplementary Table S1 reports primer sets and related references.

Additional file 1: Supplementary Table S1. Oligonucleotide primers used for the detection of genes related to biofilm production (icaA/D, bhp, aap and embp), autolysins (atlE, aae), MSCRAMMs (sdrG, sdrF, clfA, clfB, fnbA, fnbB, bbp, cna, fib, eno and epbS), pyrogenic toxins (sea, seb, sec, sed, see and tsst-1) and for agr typing.

Additional file 2: Supplementary Figure S1. Map of Sardinia showing the location of all 125 non-aureus staphylococci isolates in every municipality. No copyright permission was required.