Nasal and pharyngeal carriage of methicillin-resistant Staphylococcus sciuri among hospitalised patients and healthcare workers in a Serbian university hospital

There has been a paucity of data on methicillin-resistant Staphylococcus sciuri (MRSS) epidemiology in European healthcare settings. The aim of the study was to determine the prevalence of nasal and pharyngeal carriage and diversity of MRSS among inpatients and healthcare workers (HCWs) in the largest healthcare centre in Serbia, and to assess performance of different methods for MRSS screening. Nasal and pharyngeal swabs were obtained from 195 patients and 105 HCWs in different departments. Each swab was inoculated directly onto MRSA-ID, oxacillin-resistance screening agar and mannitol salt agar (MSA) with 2 mg/L of oxacillin. After inoculation, each swab was dipped in Mueller-Hinton broth with 6.5% NaCl and after overnight incubation, subcultured onto oxacillin-MSA. Characterisation of isolated MRSS strains was determined by antimicrobial susceptibility testing, PFGE, SCCmec typing and antimicrobial resistance genes detection. MRSS nasal and pharyngeal carriage rate was high (5%) in our hospital and department-variable. PFGE revealed a possible cross-transmission of MRSS between a patient and an HCW, and dissemination across hospital wards. All analysed isolates were multidrug resistant. Fusidic acid resistance was discovered in 93.7% of isolates, but fusA mutations in EF-G and fusB/C genes were not detected. SCCmec regions of MRSS contained elements of classic methicillin-resistant S. aureus type III. Broth enrichment prior to isolation on oxacillin-MSA was superior to direct cultivation on different media with a sensitivity/specificity of 100% and 88.5%, respectively. MRSS is a significant coloniser of patients and HCWs in the hospital. Further research is needed to investigate the clinical significance of the bacterium in our settings.


Isolation and identification
After the collection, all samples were processed within 2 h. Each swab was inoculated directly onto MRSA-ID (bioMérieux, France), oxacillin resistance screening agar (ORSA; HiMedia, India) and mannitol salt agar (MSA; bioMérieux, France) supplemented with 2 mg/L of oxacillin. All inoculated media were incubated aerobically at 35˚C and observed after 24 h and 48 h of incubation. The order of plating first alternated between the three media for every 100 samples. After inoculation, each swab was dipped in 3 mL of Mueller-Hinton broth (MHB; bioMérieux, France) supplemented with 6.5% NaCl and after incubation for 24 h at 35˚C, subcultured onto MSA supplemented with 2 mg/L of oxacillin, which was thereafter incubated for up to 48 h at 35˚C aerobically.

Antimicrobial susceptibility testing
Susceptibility to cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, fusidic acid, erythromycin, gentamicin, kanamycin, linezolid, mupirocin, quinupristin-dalfopristin, penicillin, rifampin, tetracycline, tobramycin and trimethoprim/sulfamethoxazole (BioRad, USA) was tested by disk diffusion method and to vancomycin and teicoplanic by Etest (bioMérieux, France) in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendation (http://www.eucast.org). In order to identify MLSb phenotypes, a double-disk diffusion test (D test) was performed with erythromycin (15 μg) and clindamycin (2 μg), following the procedure recommended by EUCAST. Determination of minimum inhibitory concentration (MIC) of fusidic acid was performed by broth microdilution method in accordance with EUCAST recommendation. The molecular mechanism(s) of fusidic acid resistance in fusidic acid resistant isolates was investigated by testing isolates for the presence of the fusB and fusC genes using a multiplex PCR and by investigating isolates for the presence of fusA gene mutations by nucleotide sequencing [17].

Molecular typing
PFGE was performed as described previously [6]. According to the criteria proposed by Tenover et al. [22], isolates whose PFGE pattern differed in more than six restriction fragments (bands) were genetically unrelated and were assigned to different pulsotypes (A-I). Isolates were considered to be closely related if their pulsotype differed in no more than three restriction bands and were assigned to pulsotype C1-C3.

Statistical analysis
Demographic characteristics (age and gender) of patients and HCWs and descriptive information regarding department, underlying diagnosis and duration of hospitalisation (Table 1, S1 Table) were described and compared by means of the χ2 test using SPSS 21+ statistical software.

Carriage rate
MRSS carrier prevalence in the two groups was 12/195 (6.1%) in patients and 3/105 (2.9%) in HCWs. Distribution of MRSS carriers and non-carriers stratified by population characteristics is shown in Table 1. Patients with surgical underlying diagnosis (p = 0.01), of female gender (p = 0.05) and hospitalised 7 days (p = 0.05) were more frequently colonised with MRSS.
The presence of MRSS in nasal and throat swab specimens obtained from patients and HCWs is presented in Table 2. The introduction of pharyngeal swabs in screening procedure increased MRSS carriage rate in patients by 16.7%, whereas in HCWs MRSS was recovered only from nasal samples.

Molecular typing and antimicrobial susceptibility
In total, 16 MRSS isolates were further analysed and the results of phenotypic and genotypic characterisation are presented in Table 3.
Based on PFGE typing, genetically unrelated MRSS could be divided into nine different pulsotypes (A-I). The largest group consisted of strains belonging to pulsotype B (25%) only found in patients from different departments (ED and SD). SCCmec typing revealed that most of the MRSS strains (75%) from the study contained elements of classic SCCmec type III (mec class A and ccr type 3). Among 37 additional MRSS strains investigated from our collection, the combination of mec class A and ccr type 3 was detected in 17/37 (45.9%) strains, while only the presence of mec class A gene complex was identified in the remaining 20/37 (54.1%) strains.
All tested isolates were resistant to two or more antibiotics classes besides beta-lactam antibiotics, i.e. they were multidrug resistant. High levels of resistance were observed for aminoglycosides (93.8%) and fusidic acid (93.8%). Two (12.5%) and four (25%) isolates were Of the 15 MRSS isolates that exhibited resistance to aminoglycosides, the aacA-aphD, aphA 3 and aadC genes were detected in 100%, 46.7%, and 20% isolates, respectively. The erm (C) gene was responsible for the constitutive MLS b resistance in 6.3% of isolates, whereas the lnu(A) gene was revealed in 31.3% of MRSS isolates. The tet(M) gene was identified in 6.3% of MRSS isolates that were also phenotypically resistant to tetracycline. The cat221 gene was discovered in a chloramphenicol-resistant isolate which was grouped in pulsotype D. However, in the remaining three isolates resistant to chloramphenicol (pulsotypes F and H), the PCR product with primers specific for cat221 were obtained, but the products had a higher molecular weight than the positive control.
MICs for fusidic acid showed low-to intermediate-resistance (2-16 mg/L), indicating presence of a fusB like resistance mechanisms. However, no amplification was obtained with the applied fusB/fusC PCR. Neither did sequencing of the EFG-G reveal mutations that correlated with the obtained MIC values. However, alterations in EFG-G defined two different clusters (A and B) and two single isolates with deviating amino acid substitutions (Table 4), whereas only substitution of K with N in position 342 (55-32-7 and 55-32-215) is a major alteration.
It was noticed that a pair of primers designed by Kondo et al. [16] to detect the mecA gene, also discover the presence of S. sciuri mecA homologue. The expected product of 286 bp was observed not only in isolated MRSS but also in methicillin-susceptible S. sciuri strains, including reference strains S. sciuri subsp. sciuri CCM 3473, S. sciuri subsp. rodentium CCM 4657 and S. sciuri subsp. carnaticus CCM 4835. Additionally, this 286 bp product was not obtained with S. vitulinus CCM 4511, S. lentus CCM 3472, or S. aureus ATCC 25923, which do not possess mecA homologue.
There was a good concordance between the observed antibiotic resistance profiles and the pulsotypes: pulsotype B and F (Table 3). Two S. sciuri isolates found in one swab taken from the anterior nares of a hospitalised patient were classified into pulsotypes B and G. Evaluation of methods for MRSS isolation Table 5 summarises the results of the comparison of different methods and media used for MRSS isolation. Broth enrichment prior to isolation on MSA with oxacillin was superior to direct cultivation method on different media in detection of MRSS isolates with a sensitivity and specificity of 100% and 88.5% after 48h of incubation.

Discussion
Isolation of S. sciuri, including methicillin-resistant strains, from nose of healthy and hospitalised individuals has previously been reported in low rates among healthy children (0.3%) [23], at hospital admission (0.4%) [24], or among persons in contact with horses (2.4%) [25]. To the best of our knowledge, this work represents the first comparison of MRSS strains isolated from patients and HCWs associated with the same ward/department during the same period. Carriage rate was surprisingly high in the Emergency (6.3%) and Surgical Departments (5.7%), but lower in the Medical Department (1.5%). Furthermore, the introduction of pharyngeal swabs in screening procedure increased MRSS carriage rate in patients by nearly 20%.
To determine the exact pattern of carriage in investigated individuals, non-carriers, intermittent carriers, or persistent carriers, longitudinal studies are required. However, persistent carriers are usually colonised by a higher load of a single strain over a long period, while intermittent carriers may carry different strains over time [26]. Semiquantitative analysis performed in our cross-sectional study, enabled us to conclude that most MRSS carriers were intermittent, because more than half of the strains were recovered from the samples after the broth enrichment step, indicating the presence of S. sciuri in very low numbers, while the remaining isolates produced only one to seven colonies during primary isolation.
As far as the origin of S. sciuri in humans is concerned, its transmission may occur via contact with animals. Another possible source for colonisation of humans with S. sciuri is food. In a previous study, a high rate of colonisation (10.5%) of hospital environment with S. sciuri was shown [6]. Among S. sciuri strains isolated from that hospital environment, 64.3% were resistant to methicillin. These results confirm the previous hypothesis of Couto et al. [9] that S. sciuri strains isolated from nasal passages and pharynx are acquired from the environment rather than from other sources. Yet, human to human transmission should also be considered.
PFGE analysis revealed nine pulsotypes within the population of MRSS strains. A high genetic diversity of S. sciuri strains isolated from hospital environment has also been reported in a previous study [6]. In the present investigation, isolation of MRSS with the same pulsotypes B and E from different individuals in different departments indicates possible dissemination of this bacterium between patients and hospital wards. In addition, MRSS strains with pulsotype E were isolated in the same department from a hospitalised patient and an HCW indicating a possible transmission between patient and HCW. A high resolution typing technique like whole genome sequencing would have been useful to substantiate this hypothesis. Despite the fact that the clinical significance of S. sciuri may remain controversial (partially as the consequence of frequent non-identification of CoNS to the species level), the capacity of this bacterium to carry resistance determinants is well established [3,27]. This feature was confirmed in the present study, since resistance to various classes of antibiotics was detected in all tested MRSS strains. These strains probably acquired resistance to antibiotics in order to survive the high selective pressure in the hospital environment, where the antibiotic selective pressure is high. S. sciuri shows low level of natural resistance to lincosamides, i.e. licomycin and clindamycin [3], and therefore intermediate resistance to clindamycin was expected. However, it is interesting to note that 93.7% of strains were resistant to fusidic acid. This high proportion in S. scuiri isolates has also been confirmed in recent studies in animal species, pointing to a natural resistance of S. scuiri [3,28]. Fusidic acid resistance can either be caused by mutations in the EF-G-encoding fusA gene or by presence of the fusB, fusC and fusD genes which encode EF-G proteins protected from fusidic acid binding [3,27]. Therefore, we analysed fusidic acidresistant MRSS isolates for fusA mutations in EF-G, but could not find any. MICs of analysed isolates were in the low to intermediate range, pointing to a fusB-like mechanism, however fusB/fusC genes were not detected. Additionally, Schoenfelder et al. tested S. sciuri for fusD gene, which was also negative, indicating the presence of different mechanism mediating fusidic acid resistance that is still to be determined [3].
CoNS are believed to constitute a reservoir of SCCmec elements for S. aureus. Therefore, we analysed SCCmec regions of isolated MRSS strains. SCCmec typing revealed mec class A in all tested MRSS strains, but ccr type 3 was discovered in 12 (75%) strains. This mec class/ccr type combination corresponds to SCCmec type III [29]. In remaining four strains ccr type could not be determined. Presence of SCCmec type III or only mec class A in MRSS was also observed in other studies [30][31][32]. The obtained data suggest that all MRSS possess at least mec class complex A. Finally, we tested 37 previously described MRSS strains isolated from humans, dogs, hospital environment and public transportation [1,2,6,8]. SCCmec type III, i.e. combination of mec class A and ccr type 3, was detected in 45.9% of strains, while in the remaining strains only the presence of mec class A gene complex could be demonstrated. The high prevalence of non-typeable SCCmec cassettes in S. sciuri confirms the potential divergence of ccr and mec complexes as previously suggested [33] and indicates the presence of novel SCCmec elements [34].
S. sciuri is CoNS that can be easily misidentified as S. aureus because both species are mannitol-fermenting organisms that may grow as yellow colonies on blood agar and may give positive Slidex Staph Plus agglutination test and Staphaurex test [2,7]. Furthermore, MRSS can grow on chromogenic and selective media for MRSA strains giving the colonies of same colour, which is shown in our study. In accordance with this finding, the identity of isolated MRSA/MRSS strains on screening media has to be confirmed with biochemical tests (coagulase, oxidase and/or novobiocin susceptibility test) or in automated systems. Additionally, the importance of broth enrichment for accurate detection of MRSS in clinical sample is confirmed, as shown for MRSA [12].

Conclusions
MRSS is a significant nasal and pharyngeal coloniser of patients and HCWs in this hospital. Molecular typing revealed the possibility of cross-transmission of MRSS between a patient and an HCW and dissemination through hospital. S. scuiri may serve as a reservoir and an important hub for exchange of the mecA gene and other resistance genes among staphylococci. In order to improve the detection of MRSS, broth enrichment prior to isolation on MSA with oxacillin represented the optimal available choice for isolating this bacterium.
Supporting information S1