Ovine NAS: general findings
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 Table 2 reports the primers used for PCR amplifications.
Table 3 summarizes biofilm formation results, evaluated by the phenotypic characteristics of bacterial colonies grown on Congo Red Agar (CRA) and by PCR detection of icaA/D, bhp, aap, embp, atlE, aae, sdrF, sdrG and agr. Out of 125 isolates examined, only 4 (3.2%) were classified as strong biofilm producers because their colonies exhibited a very black color; the remaining 121 (96.8%) as non-biofilm producers due to bordeaux or red color. One isolate harbored both icaA and icaD genes while two had only the icaA gene: one of them was considered as non-biofilm-producing phenotype. On the other hand, 37 (29.6%), 22 (17.6%) and 63 (50.4%) isolates possessed the bhp, aap and embp genes, respectively (Table 3). Concerning autolysin genes, 50 (40%) isolates were PCR positive for altE while 49 (39.2%) were positive for aae. Concerning adhesion (MSCRAMMS) factors, 59 (47.2%) and 66 (52.8%) isolates harbored sdrF and sdrG genes, respectively. Concerning the agr type, 21 (16.8%) isolates belonged to agr-1 whilst 33 (26.4%) to agr-3. None of the isolates belonged to type 2. The toxin genes analyzed did not produce amplification (data not shown).
Ovine S. epidermidis
S. epidermidis was the most represented ovine NAS. Out of 57 isolates, 2 (3.5%) were strong biofilm producers but only one harbored both icaA/D genes. On the other hand, 54 (94.7%), 30 (52.6%) and 20 (16%) isolates possessed embp, bhp and aap genes, respectively. Concerning autolysin genes, 48 (84.2%) were PCR-positive for atlE and 46 (80.7%) for aae. High percentages were found for MSCRAMMS genes: 87.7% for sdfG and 78.9% for sdrF. 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 3). 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 3 shows that almost all isolates were non-biofilm producers, except one S. caprae and one S. muscae.
Human NAS: general findings
Table 3 summarizes the distribution of virulence 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.
Human S. haemolyticus
Out of 28 S. haemolyticus isolates examined, 16 (57.1%) were classified as strong biofilm producers by CRA because their colonies exhibited a very black color; the remaining 12 as non-biofilm producers due to bordeaux or red color. None of S. haemolyticus isolates harbored icaA/D, bhp and embp. On the contrary, three black (2 from blood and 1 from nasal swab) and one bordeaux (from glans swab) isolates possessed the aap gene. No amplification was observed for atlE, aae, sdrF, sdrG genes and agr locus (Table 3) and for all toxin genes (data not shown).
Human S. epidermidis
Among the 26 S. epidermidis isolates, 22 (84.6%) showed a very black color; ten (38.5%) of them (3 from catheter, 3 from blood, 2 from nasal swab, 1 from skin swab and 1 from pus) harbored both icaA and icaD. The remaining isolates were icaA/D negative. However, 16 (61.5%), 25 (96.1%) and 23 (88.5%) isolates possessed the bhp, aap and embp genes, respectively (Table 3). Out of 10 icaA/D positive isolates, 5 were also positive for the other biofilm genes analyzed. Among the 4 non-biofilm-producing phenotypes, all were icaA/D negative but 3 (2 from pus and 1 from oral swab) harbored aap and embp while 1 (from skin swab) possessed all bhp, aap and embp genes. Data on the prevalence of autolysins and MSCRAMMs 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 and 21/26 (80.8%) harbored 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. 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 and sdrG genes associated with biofilm formation. Among the 9 agr-2se isolates, only 1 (from blood) possessed these genes. Regarding the pyrogenic toxin genes, amplification was not observed in any of the S. epidermidis isolates or in the remaining staphylococci (data not shown).
Minor human NAS
Out of the 4 S. lugdunensis isolates examined, 3 (2 from peritoneal fluid and 1 from vaginal swab) displayed a very black colour on CRA. However, none of them harbored neither icaA/D nor any other gene examined, except the sdrG gene.
Negative results were also obtained for all S. hominis isolates, except for a PCR positivity for aap and sdrG genes. Among the 3 S. capitis isolates, 1 (from skin swab) exhibited black color whereas 2 were phenotypically red but, despite this, all isolates were PCR-positive for icaA and sdrG genes. Table 4 reports the PCR results for S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis. Of note, one bordeaux S. warneri isolate (from fluid drainage) was PCR negative for icaA/D and bhp genes but positive for aap, embp, atlE, aae, sdrG/F and agr genes. However, we were not able to type the agr locus.