Comparative characterisation of human and ovine non-aureus staphylococci isolated in Sardinia (Italy) for antimicrobial susceptibility profiles and resistance genes

We present the comparative characterisation of 195 non-aureus staphylococci (NAS) isolates obtained from sheep (n = 125) and humans (n = 70) in Sardinia, Italy, identified at the species level by gap gene polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis with AluI. Isolates were tested phenotypically with a disc diffusion method and genotypically by PCR, for resistance to 11 antimicrobial agents including cationic antiseptic agents. Among the ovine isolates, Staphylococcus epidermidis (n = 57), S. chromogenes (n = 29), S. haemolyticus (n = 17), S. simulans (n = 8) and S. caprae (n = 6) were the most prevalent species, while among human isolates, S. haemolyticus (n = 28) and S. epidermidis (n = 26) were predominant, followed by S. lugdunensis and S. hominis (n = 4). Of the 125 ovine isolates, 79 (63.2%) did not carry any of the resistance genes tested, while the remainder carried resistance genes for at least one antibiotic. The highest resistance rates among ovine isolates were recorded against tetracycline (20.8%), and penicillin (15.2%); none was resistant to methicillin and two exhibited multidrug resistance (MDR); one of which was positive for the antiseptic resistance smr gene. By contrast, most human isolates (59/70, 84.3%) were resistant to ⩾1 antimicrobials, and 41 (58.6%) were MDR. All 52 (74.3%) penicillin-resistant isolates possessed the blaZ gene, and 33 of 70 (47.1%) harboured the mec gene; of these, seven were characterised by the Staphylococcal Chromosomal Cassette (SCCmec) type IV, 6 the type V, 5 of type III and one representative each of type I and type II. The majority (57.1%) was erythromycin-resistant and 17 isolates carried only the efflux msrA gene, 11 the methylase ermC gene and an equal number harboured both of the latter genes. Moreover, 23 (32.8%) were tetracycline-resistant and all but one possessed only the efflux tetK gene. qacA/B and smr genes were detected in 27 (38.6%) and 18 (25.7%) human NAS, respectively. These results underline a marked difference in species distribution and antimicrobial resistance between ovine and human-derived NAS.


Introduction
Sardinia, an island located in the middle of the Mediterranean Sea, with a population is around 1.6 million inhabitants, has approximately 3.5 million milking Sarda sheep, corresponding to half of the total Italian national stock. A considerable part of the regional economy relies on dairy sheep farming, mainly for pecorino cheese production; as a consequence, the control of intra-mammary infections is of the greatest importance for dairy farmers. Several reports indicate that non-aureus staphylococci (NAS) are the most prevalent bacteria recovered from subclinical mastitis of sheep and goats [1][2][3][4], thus creating opportunities for cross colonisation and infection among sheep and farmers, due to their antimicrobial resistance and pathogenicity gene pools. Of note, NAS have emerged as relatively frequent nosocomial agents capable of causing infection in debilitated or compromised patients as well as their association with catheter-related and other indwelling medical device-related infections [5].
In the last decade, a significant increase of antibiotic-resistant infections has been recorded among ovine NAS, especially for beta-lactams and tetracyclines, which are commonly used in veterinary practice for mastitis treatment [6,7]. Two mechanisms confer penicillin resistance in staphylococci, the most common being production of β-lactamase, encoded by the blaZ gene. The other mechanism is due to a penicillin-binding protein transpeptidase (PBP2a), encoded by the mecA gene [8], which is carried on a mobile chromosomal element, the Staphylococcal Chromosomal Cassette mec (SCCmec) [9]. SCCmec types are defined by the recombinase (ccr) gene complex and the class of the mec gene complex [10]. Recently, a novel PBP2a homologue has been described as encoded by mecC [11].
Many cationic antiseptic agents such as quaternary ammonium compounds (QACs) are widely used as surface germicides within healthcare facilities [12,13]. Although issues of antibiotic resistance have been widely investigated, knowledge on the occurrence of antiseptic resistance genes (qacA/B, smr, qacG, qacH, qacJ) in staphylococci from dairy animals is limited [7,14].
In most veterinary and clinical laboratories, differentiation of NAS species is based on phenotypic reactions which may be unreliable, particularly for animal isolates. Consequently, several genotypic methods are increasingly being applied for species-level identification [15]. This approach combined with genotyping by multilocus sequence typing (MLST) for the differentiation of strain populations, allows an informative analysis and insight into the evolving epidemiology of bacterial species groups in relation to their pathogenicity and antimicrobial resistance [16].
The objective of this study was to compare the molecular characteristics of NAS isolated from ovine mastitis with those from human clinical specimens, and specifically to: (1) identify NAS isolates using genotypic techniques; (2) determine their antimicrobial susceptibility profiles, and distribution of antimicrobial and antiseptic resistance genes and (3) determine the genetic relatedness of isolates within species by MLST.

Ovine
In total, 125 NAS isolates were collected from sheep milk samples in different provinces of Sardinia (Italy) over a period of 9 months (April-December 2017). The isolates belonged to a bank of NAS used for the preparation of inactivated autogenous vaccines, according to the Italian Ministerial Decree no. 287/1994. Basic identification of staphylococci was determined by colony morphology, Gram-stain, catalase and coagulase tests, clumping factor production (Staphylase Test, Oxoid, UK), and growth on mannitol salt agar (Becton Dickinson, Quebec, CDN).

Human
During the same period, 70 NAS isolates were collected from clinical specimens from 70 different patients attending clinical departments at three major hospitals in Sardinia. Isolates were anonymised without patient identifiers and thus individual consent was not required: 90% of the human NAS were recovered from hospitalised patients in intensive care unit, haematology and orthopaedics.

Species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)
Species identification was based on PCR amplification and PCR-RFLP of the glyceraldehyde-3-phosphate dehydrogenase gene (gap) [17]. Sequencing of the gap gene was used to identify non-speciated isolates. Briefly, 15 μl of amplicons were digested in a 30 μl volume containing 10× buffer, 0.5 μl of 10 mg/ml acetylated BSA (Promega, Madison, USA) and 1 μl of 10 U/μl FastDigest AluI endonuclease (Thermo Fisher Scientific, City, Country). Samples were incubated at 37°C for 15

Ovine NAS
S. chromogenes, S. haemolyticus and minor ovine NAS S. chromogenes (n = 29) and S. haemolyticus (n = 17) were the next most prevalent species isolated from ovine milk samples. Only one of the 17 S. haemolyticus isolates was resistant to Te (tetK gene) ( Table 4). Among S. chromogenes, 11 isolates were resistant to a single antibiotic and four to two agents. The highest resistance was found against Pn (n = 7), followed by Te (n = 6) and Er (n = 3). All Pn-resistant isolates had the blaZ gene while, among the six S. chromogenes isolates resistant to Te, five were positive for tetK and one for tetM. Of the three Er-resistant isolates, one had both ermB and ermC, one only ermC, and the other, ermB (Table 5).
Regarding the remaining ovine isolates, two of four S. simulans were resistant to Te (tetK or tetM gene), one to Er (ermC gene) and one to St. Two of the three S. caprae were resistant to both Pn and Te (blaZ and tetK genes) and one to Pn alone (blaZ). The single Pn-resistant isolate of S. warneri contained the blaZ gene (Table 5).

S. haemolyticus
All, but two, of 28 isolates of this species were resistant to one or more antimicrobials, with high rates to Pn (n = 25, 89.3%), Km (n = 23, 82.1%), Er (n = 22, 78.6%), Ox/Kx (n = 18, 64.3%) and Te (n = 15, 53.6%; the great majority being MDR) ( Table 4). All 18 Ox-resistant S. haemolyticus isolates were mecA positive with SCCmec type V represented by six and SSCmec type IV in three. A single isolate of SCCmec type VII with recombinase genes ccrA5 and ccrB5 was also PCR-positive for the ccrAB SHP allotype. Eight isolates were non-typeable for SCCmec (Table 4). All Pn-resistant isolates harboured the blaZ gene. Among the 22 Er-resistant isolates, five possessed only the ermC gene, eight only the msrA gene, eight carried simultaneously ermC and msrA genes, and one was positive for both ermA and msrA genes ( Table 4). All 15 Te-resistant isolates had the tetK gene. Antiseptic-resistance genes: qacA/B alone, or smr alone, were detected in 42.8% (n = 12) and in 21.4% (n = 6) of the S. haemolyticus isolates, respectively. An additional three isolates possessed both qacA/B and smr genes.

S. epidermidis
All, but one, of the 26 S. epidermidis isolates were resistant to ⩾1 antibiotic ( . Four isolates were non-typeable for SCCmec and were also PCR-negative for ccrAB5 and ccrAB SHP . All Pn-resistant isolates were positive for the blaZ gene; six of 15 Er-resistant isolates were positive only for the msrA gene, five the ermC gene and four both ermC and msrA genes. The four Te-resistant isolates carried the tetK gene, one of them also had the tetL gene. Nine (34.6%) isolates possessed the qacA/B gene alone and the same number only the smr gene. Three isolates from blood, skin swab and seminal fluid carried both genes. Interestingly, the smr gene was detected in the antimicrobial-susceptible S. epidermidis isolated from pus. MLST analysis showed that ST59 and ST5 were each represented by six isolates and ST80 by five. ST981 proved to be a novel type.

Minor human NAS
Three of the four S. hominis isolates were MDR and one of them, isolated from urine, was including Er (ermC gene) and Te (tetK gene); one of them, isolated from urine, was SCCmec type I. Of note, each of the four S. hominis isolates was positive for the blaZ gene and two also harboured the qacA/B gene (Table 5). S. pasteuri and S. capitis isolates were resistant to Er (both ermC and msrA genes) and Pn (blaZ gene), respectively. S. saprophyticus subsp. bovis, resistant to Te, possessed the tetK gene. In the S. warneri isolate, collected from an ear swab and susceptible to all antibiotics tested, the qacA/B gene was found (Table 5).

Discussion
This study is the result of an integrated collaboration between veterinary and human health care professionals to determine whether NAS recovered from human infections share some

Epidemiology and Infection
genetic characteristics with those circulating in sheep. In the current study, 125 NAS from ovine mastitis and 70 from human clinical specimens were assigned to species level by PCR-RFLP of the gap gene. Among the ovine isolates, S. epidermidis was the most common followed by S. chromogenes and S. haemolyticus, while among human isolates S. haemolyticus and S. epidermidis were predominant, followed by S. lugdunensis and S. hominis. S. epidermidis is widely recognised to be the most prevalent NAS recovered from ovine mastitis and human clinical specimens [1,7,29]. This study confirms our previous findings that ovine isolates of this species is the major reservoir of antimicrobial resistance genes, in particular for tetracycline and penicillin [6,7]. However, the frequency of MDR in sheep isolates was considerably lower than that for human isolates (1.6% vs. 58.6%). Among the latter group, MDR was associated more frequently with S. haemolyticus (35.7%) than S. epidermidis (18.6%). This finding is in agreement with previous reports of resistance to a wide range of antimicrobials among S. haemolyticus from  human clinical specimens, and also supports the view that this species may constitute an important reservoir for the transfer of resistance genes to other Staphylococcus species [30,31]. None of the ovine NAS was methicillin resistant in contrast to 53.8% of human isolates. The mecA gene is a constituent of the mobile genetic element SCCmec, which acts as a vehicle for horizontal transfer of antibiotic resistance genes [32]. Among the oxacillin-resistant S. epidermidis, we found type III to be the predominant SCCmec, followed by type IV, the latter being common in human isolates of the species [32,33], while SCCmec type III is more widely distributed among NAS species; the mechanism responsible for this is not understood. The identification of SSCmec type V as the most prevalent in S. haemolyticus is consistent with previous reports of the high frequency of class C2 mec-ccrC complexes (type V) in S. haemolyticus isolated from outpatients living in Algeria, Mali, Moldova, Cambodia and China [32,33]. By contrast, S. haemolyticus collected in South Brazil and India mainly harboured SCCmec type I [34,35]. We identified ccrA5-ccrB5 recombinase genes in one of the S. haemolyticus isolates from a human glans specimen. This gene complex has only been reported in S. pseudintermedius from animals [21], and to the best of our knowledge, this is the first report describing the detection of SCCmec type VII in S. haemolyticus from human clinical specimens. The ccrAB SHP allotype was described by Pi et al. [22] in S. haemolyticus isolates resistant to methicillin and harbouring arginine catabolic mobile element (ACME) cluster genes. This group suggested that ccrAB SHP had a similar function to the known ccr allotype, that is, to catalyse the integration and excision of SCCmec and ACME. Our inability to establish the SSCmec type for eight of the S. haemolyticus studied here could be attributed to the presence of novel ccr allotypes.
The plasmid-located tetK gene was the most prevalent determinant encoding tetracycline resistance, indicating that the resistance mechanism is mainly mediated by the tetracycline efflux pump [36]. In S. aureus, a strong association between tetK gene and SCCmec type V has been reported by Larsen et al. [37] but we did not observe any such correlation in our NAS isolates, although the tetk gene was amplified in oxacillin-negative isolates. The detection of msrA gene encoding an ATP-dependent efflux pump among the erythromycin-resistant NAS is consistent with other reports from France [38] and Tunisia [39]. The inappropriate use of disinfectants can lead to a concentration gradient in the environment, which could select for isolates with reduced susceptibility to these agents. Here, we found a substantial difference between animal and human NAS as QAC resistance was identified in only a single ovine isolate compared to 42 (60%) of 70 human isolates. The ovine S. epidermidis strain harboured the smr gene whereas in human isolates, qacA/B genes were more prevalent (37.1%) than the smr gene (25.7%); both genes were similarly distributed in S. haemolyticus and S. epidermidis of human origin (was 70% and 69%, respectively). The predominance of qacA/B genes compared to the smr gene was also observed in NAS from Hong Kong [40] and Turkey [13]. Furthermore, it is noteworthy that almost all MDR-S. haemolyticus isolates from nasal, skin and oral swabs of patients in intensive care unit were PCR-positive for qacA/B genes.
NAS may therefore play a clinically significant role as reservoirs of resistance genes, especially S. epidermidis in sheep and S. haemolyticus in humans. The carriage of such genes in human NAS and the prevalence of MDR isolates were markedly higher compared with those of ovine origin. Similarly, no methicillin-resistant strains were found in the latter group which may reflect a more appropriate use of antimicrobials in humans as compared with dairy ruminants. Moreover, these findings may suggest that human NAS represent reservoirs of resistance genes which are transmissible to sheep isolates especially if they constitute part of the commensal skin microbiota of animal care or farm workers. On the other hand, the role of sheep NAS as reservoirs of resistance genes for human NAS seems reasonably lower. Nevertheless, this will require further studies, as well as periodic surveillance measures to monitor the spread of antimicrobial genes and maintain control.
Concerning the presence of sequence types in the two species, MLST genotyping of S. epidermidis revealed that ST225, ST6 and ST100 predominated in sheep, compared with T59, ST5 and ST80 in humans. Reports of sequence typing studies on animal-derived S. epidermidis are limited and currently none has analysed ovine milk isolates. Among human S. epidermidis, ST2 has usually been reported to be the most prevalent in humans [16,41], but only one representative isolate of this ST was identified in our collection. Similarly, to our knowledge, only two studies have so far reported ST5 to be more common than ST2 [42,43], which might be indicative of a different distribution in Sardinia from other geographical areas. Finally, it is noteworthy that one S. epidermidis ovine isolate was typed as ST5, which might suggest a possible human to animal transmission. This highlights the risk for strain and gene passage in this direction and underscores the need for periodic surveillance measures.
In conclusion, we believe that this is the first study to investigate and compare the occurrence of antimicrobial/antiseptic resistance and associated genetic determinants in human and ovine NAS in Italy. Our results document the great importance of NAS as reservoirs of resistance genes, in particular S. epidermidis for sheep and S. haemolyticus for humans. As many of the resistance determinants are located on transmissible plasmids, we propose that periodic surveillance might provide important information relevant to the control of animal and human infections, and thus help to limit the spread of MDR bacteria from humans to animals.