Dissemination of Methicillin-Susceptible CC398 Staphylococcus aureus Strains in a Rural Greek Area

A large collection of Staphylococcus aureus including a. 745 clinically significant isolates that were consecutively recovered from human infections during 2012–2013, b. 19 methicillin-susceptible (MSSA), randomly selected between 2006–2011 from our Staphylococcal Collection, c. 16 human colonizing isolates, and d. 10 strains from colonized animals was investigated for the presence and the molecular characteristics of CC398. The study was conducted in Thessaly, a rural region in Greece. The differentiation of livestock-associated clade from the human clade was based on canSNPs combined with the presence of the φ3 bacteriophage and the tetM, scn, sak, and chp genes. Among the 745 isolates, two MRSA (0.8% of total MRSA) and thirteen MSSA (2.65% of total MSSA) were found to belong to CC398, while, between MSSA of our Staphylococcal Collection, one CC398, isolated in 2010, was detected. One human individual, without prior contact with animals, was found to be colonized by a MSSA CC398. No CC398 was identified among the 10 S. aureus isolated from animals. Based on the molecular markers, the 17 CC398 strains were equally placed in the livestock-associated and in the human clades. This is the first report for the dissemination of S. aureus CC398 among humans in Greece.


Introduction
Since 2005, the livestock-associated clonal complex (CC) 398 Staphylococcus aureus have been detected mainly in pigs or other food-producing animals (cattle or poultry), as well in horses, dogs, black rats or food of animal origin [1]. These strains, that can cause mainly superficial skin and soft tissue infections (SSTIs), affect primarily individuals in direct contact with animals, e.g., farm personnel, veterinarians, veterinary students or slaughterhouse workers [1][2][3][4][5]. The majority of these strains are methicillin-resistant [6,7]. In 2009, a new sub-population of ST398 methicillin-sensitive S. aureus (MSSA), was observed in community households in northern Manhattan, while, soon thereafter, this clone was reported as the causal agent of necrotizing pneumonia and invasive bloodstream infections in individuals not in direct animal contact in European countries or China [8][9][10][11][12][13]. Nowadays, according to whole-genome analytical and epidemiological studies, S. aureus CC398 strains cluster into two distinct phylogenetic clades, a livestock clade and a basal human clade, that predates the livestock clade [14][15][16]. Main characteristics of the livestock-associated S. aureus CC398 clade is the loss of φ3 bacteriophage (FSa3) and the acquisition of a Tn916-like transposon carrying the tetM gene; human S. aureus CC398 clade harbours FSa3, a beta-haemolysin (hlb) converting phage, that carries the human-specific immune evasion cluster (IEC) of genes. IEC genes encode proteins that interact specifically with the human immune response; chp (chemotaxis inhibitor protein) encodes an inhibitor of chemokine responses and blocks neutrophil recruitment, sak (staphylokinase) encodes an inhibitor of defensins, and scn (staphylococcal complement inhibitor) encodes an inhibitor of complement formation and neutrophil phagocytosis [14][15][16]. Recently, Stegger et al showed that these two phylogenetic clades, the livestock-associated and the human one, can be rapidly differentiate based on single-nucleotide polymorphisms (canSNP_748, canSNP_1002 and canSNP_3737) combined with the presence of scn and tetM genes [16].
Thessaly is a rural area of Central Greece with a population of 1.200.000. Predominant activity in the region is livestock production (approx. 2.000.000 sheep, 220.000 poultry, 140.000 pigs, 132.000 cows, 3.000 horses). Objective of this study was to investigate the presence and the molecular characteristics of CC398 S. aureus (MRSA or MSSA) in a population of 745 clinical staphylococcal isolates, recovered from human infections in a rural Greek area, during 2012-2013. In addition, the existence of CC398 was also examined in a small size of nasal samples obtained from healthy individuals and from food-producing animals (mainly from sheep) the same period.

Ethics Statement
The research protocol concerning the human specimens was approved by the Ethics Committee of the General University Hospital of Larissa (Permit Number: 358). A written informed consent was obtained from the patients and the human individuals. The data were analyzed anonymously. In addition, samples from 512 animals (332 sheep, 100 pigs and 80 cows), randomly collected, were used in the study. The 38 farms from sheep of which samples were collected, were commercial flocks, managed semi-intensively. On the other hand, the four pig farms, included in the study, were representative of the majority of pig farms of our area, while, the median number of sows was 180 (min: 150, max: 320). In all farms, the prevailing production system catered for fattening pigs; piglets were born at those farms and weaned at the age of 3 to 4 weeks, for subsequent slaughtering at 90-120 kg live bodyweight, when 20-to 24-weekold. The flocks were under veterinary care, which was provided by private practicing veterinarians. The data of the animals were obtained from veterinarians who collected samples during standard animal care, while, the owners of the farms were asked to sign a consent form and fill in a questionnaire, before sampling. No animals were euthanised during the study and efforts taken to ameliorate animal suffering. The study did not involve any experimentation, but was based in nasal swabs, that had been collected from the sheep for routine diagnostic purposes in the participating flocks. Diagnostic veterinary procedures are not within the context of relevant EU legislation for animal experimentations (Directive 86/609/EC) and may be performed in order to diagnose animal diseases and improve animal welfare.

Bacterial strains
The strain collection included four groups (A, B, C, D) with different origin. The main group, the group A, consisted of 745 S. aureus isolates (253 MRSA and 492 MSSA), consecutively collected from clinically significant samples (e.g., pus, blood) from 412 out-patients and 333 inpatients, all admitted to the five hospitals of Thessaly during the period 2012-13. In addition, in order to elucidate a. if CC398 S. aureus circulated in our area prior the study period, and b. if this clone colonizes nasal cavity of human individuals and livestock-associated animals, three other smaller groups (B, C, D) were also included. The group B consisted from 19 randomly selected MSSA from the Staphylococcal Collection of Thessaly, recovered between 2006-11 from significant specimens (abscess, blood); group C included 16 S. aureus (2 MRSA and 14 MSSA) isolated from nasal samples collected from 92 healthy individuals (with no staphylococcal infection during the six months previous to sampling), of which 42 people were occupational contact with animals (32 sheep farmers and 10 pig farmers); group D included 10 S. aureus (MSSA) recovered from 512 nasal swabs of livestock animals (332 sheep, 100 pigs and 80 cows) with no clinical staphylococcal infection. One staphylococcal isolate was tested from each person or animals. Limitation of this study is that the size of tested samples of group B, C, and D were small and were not estimated according to statistical rules.
During the study, demographic (name, age, gender, residence, occupation, travels, previous hospitalizations) and clinical information (underlying disease, antibiotic therapy) were collected from humans from whom samples were collected. Similar data were collected regarding animals from which samples were collected.
All nasal samples, obtained either from humans or animals, were enriched in tryptic soy broth (OXOID, Basingstoke, England) with 7% NaCl at 37°C for 24 h and then plated onto manitol salt agar (OXOID, Basingstoke, England) and cultured at 37°C for 24 h. Hospital-and community-associated strains were defined according to published criteria [17].

Molecular methods
The initial screening test for CC398 strains was based on the presence of aroE allele 35, the most characteristic for the detection of this clonal complex. According to multilocus sequence typing data, this allele is present only in 1.46% of all sequence types. Complete MLST was performed to all isolates bearing aroE allele 35. In all CC398 strains, agr groups, SCCmec types, spa types and genes encoding PVL (lukS/lukF-PVL) and toxic shock syndrome toxin-1 (tst) were also defined by using PCRs [19]. Moreover, antimicrobial resistance genes (mecA, mecC, ermA, ermB, ermC, ermT, lnuA, lnuB, lnuC, lnuD, vgaA, tetM, tetL, tetO, tetK, dfrD, dfrK, dfrG, and dfrS1) were detected by PCR, as described before [19,20]. The differentiation between livestock-associated and human CC398 strains was based a. on the determination of canSNP_748, canSNP_1002 and canSNP_3737 as described by Stegger et al, b. on the presence of FSa3 confirmed by integrase3 gene (φ3int) PCR amplification combined with the absence of functional beta-hemolysin gene (hlb), and c. on the presence of scn, chp and sak genes by PCR [16,21,22]. To obtain information on the location of IEC genes, PCRs were performed using relevant combinations of specific primers for scn, sak and chp, as previously reported by van Wamel et al [22]. PCR products were sequenced and results obtained were compared with the Staphylococcus aureus phage phiNM3 complete genome (DQ530361).

Results
According to screening, aro35 allele was detected in 17 isolates: 15 isolates of group A (15/745), one of group B (1/19) and one of group C (1/16). No isolate was found to be aro35-positive from group D (0/10).
The complete MLST scheme revealed that all these 17 isolates belonged to CC398. The majority of them (13 isolates) was ST398, whereas, four strains belonged to new variants, namely ST2684 (two isolates), ST2759 (one isolate) or ST2978 (one isolate) ( Table 1). Eleven of these isolates were recovered from SSTIs or abscesses, five from bloodstream infections and one from nares (Table 1). All CC398 strains belonged to agr1 and distributed in seven spa types (Table 1). According to patients' demographic data, only four strains could be classified as hospital-associated (Table 1). All people were adults; none had reported prior contact and occupation with animals or travel in countries with an increased prevalence of ST398.
Susceptibility testing results are in Table 1. Two strains were oxacillin-resistant and carried mecA gene; resistance to erythromycin was associated with presence of ermA, ermC and ermT, resistance to lincomycin with presence of lunA, lnuB and vgaA, resistance to tetracycline with presence of tetM gene and resistance to sulfamethoxazole-trimethoprim with presence of dfrG. No strain carried either lukS/lukF-PVL or tst genes.
According to canSNP assays, nine strains were placed in the livestock clade (see Table 1). Eight of them were tetracycline-resistant and carried the tetM gene, while, only two were MRSA. As anticipated, all were negative for the presence of FSa3 and belonged to spa t011 or t034, types strongly related to the livestock-associated clade as previously reported [16]. On the other hand, according to canSNPs, eight strains were placed in the human clade; their spa types were t468, t1255, t1451, t13982 and t6606 (see Table 1). All of them were tetracycline susceptible and carried the FSa3 with various combinations of the scn, chp and sak genes; five strains were scn+/ chp+, while the remaining three strains were sak+/ chp+(see Table 1). This latter combination is described for the first time, while both genes were located to FSa3 [22,23]. All erythromycin-resistant strains of the human clade carried the ermT gene.

Discussion
Although the molecular epidemiology of MRSA in Greece has been investigated, there was a limited surveillance of MSSA [17,24]. Until now, one MRSA ST398 has been detected in 2009 in Athens, as well as one MSSA, details of which have been reported before. Both isolates were recovered from blood samples [13]. To the best of our knowledge, this is the first report from Greece indicating that, during 2012-2013, among a large collection of 745 clinical staphylococcal isolates from infected humans, 0.8% of MRSA (2/253 isolates in group A) and 2.65% of MSSA (13/492 isolates in group A) belonged to CC398. In addition, one human individual, without prior contact with animals, was found to be colonized by a CC398 MSSA.  Based on the canSNPs combined with the presence of FSa3 nine and eight isolates were placed in the livestock and in the human clade, respectively. Our results were in concordance with previous reports, that have demonstrated that, the detection of some resistant genes, such as the tetM and the ermT genes, could be used as rapid tools for differentiate livestock-associated clade from the human CC398 clade [25,26].
It was surprising to find that in samples collected from animals (group D) no CC398 S. aureus was found. This could be explained by firstly the majority of the animals tested were sheeps and secondary the size of tested samples was very small. So, no concrete conclusions about the existence of CC398 in animal populations (D) could be obtained. However, the detection of one livestock-associated CC398 MSSA, spa t034 in 2010, indicates that the livestock clade already existed in the area and indicates the need for a wider surveillance in collaboration with veterinary researchers, which is in progress.
In conclusion, the present study underscores the presence of the two major host-associated S. aureus CC398 clades in a rural Greek area. The majority of isolates was MSSA and caused a large spectrum of human infections. Although, the prevalence rate of CC398 MSSA was not found to be high (2.65%) as compared to those reported from other countries [25,26], the presence of this clone in patients and healthy individuals emphasizes the need of epidemiological surveillance for MSSA.