Differences in resistance profiles and virulence genes among methicillin-resistant and methicillin-susceptible Staphylococcus aureus of different lineages at a public tertiary hospital

Introduction: Staphylococcus aureus is a major nosocomial pathogen that is associated with high virulence and the rapid development of drug resistance. Methods: We analyzed and compared the antimicrobial resistance, virulence profiles, and molecular epidemiology of 67 S. aureus strains, including 36 methicillin-sensitive (MSSA) and 31 methicillin-resistant (MRSA) strains recovered from a public hospital located in south-eastern Brazil. Results: The clones circulating in this hospital presented a great diversity, and the majority of the strains were related to clones responsible for causing worldwide epidemics: these included USA100 (New York/Japan clone), USA300, and USA600. The 31 MRSA (22 SCCmecII and 9 SCCmecIV) and 36 MSSA strains exhibited low resistance against gentamicin and trimethoprim/sulfamethoxazole. No MRSA strain showed resistance to tetracycline. Virulence gene carriage was more diverse and abundant in MSSA than in MRSA. Of the evaluated adhesion-related genes, ebpS was the most prevalent in both MSSA and MRSA strains. The genes bbp and cna showed a strong association with MSSA strains. Conclusions: Our findings reinforce the idea that MSSA and MRSA strains should be carefully monitored, owing to their high pathogenic potential.


INTRODUCTION
Staphylococcus aureus is a major nosocomial pathogen associated with high virulence and rapid drug resistance development worldwide 1 . The prevalence of methicillinresistant S. aureus (MRSA) has necessitated the implementation of specific therapeutic and expensive prevention measures in hospital settings 2 . However, a recent study revealed the presence of a broad spectrum of virulence genes in the genomes of methicillin-susceptible S. aureus (MSSA) strains that could act as a potential source of infection. Thus, MSSA should be given the same attention as MRSA strains 3 .
A study performed by Jiménez et al. 4 using S. aureus strains isolated from a pediatric population showed that MSSA lineages harbor a lot more virulence genes as compared to MRSA, and this difference was thought to be related to the fitness cost associated with methicillin resistance. The relationship between virulence and resistance was also noted by Seidl et al. 5 , wherein the authors showed that the intrinsic virulence of MRSA strains is similar, or even less than that of MSSA, and that the increase in virulence is associated with the decrease in methicillin resistance levels.
The dynamics of the prevalence of S. aureus clones, including MRSA and MSSA strains, have been recently investigated 6 . As a consequence, the changes in the epidemiological overview have been observed worldwide, revealing the emergence of new clones replacing the previously established ones 7,8 . As monitoring resistance and virulence profiles is important to establish control strategies, here we aimed to analyze and compare the antimicrobial resistance patterns, virulence profiles, and molecular epidemiology of S. aureus strains isolated from a public hospital located in south-eastern Brazil.

Bacterial strains, settings, and ethic statement
We evaluated 67 S. aureus strains isolated from healthcareassociated infections ( The strains were identified as S. aureus using the MicroScan® system (Siemens Healthcare Diagnostics Inc., USA). Bacteria were stored in brain heart infusion medium (Merck, Germany) with 20% glycerol at -20°C. The present research was approved by the Human Research Ethics Committee of the Federal University of Espírito Santo under number 247/2011.

DNA isolation and SCCmec typing
Genomic DNA from S. aureus was extracted following the method described by Schuenck et al. 8 , and used as a template for polymerase chain reaction (PCR). The expression of the gene mecA was evaluated in all the 67 strains included in the study, and SCCmec typing was performed for the samples deemed positive for mecA 10 .

Molecular typing
Pulsed-field gel electrophoresis (PFGE) was performed for all strains after the analysis of genomic DNA macrorestriction with SmaI enzyme in a CHEF-DRIII system (Bio-Rad, USA), as previously described 11 . Band patterns were analyzed with BioNumerics v6.5 (Applied Maths, Belgium) using the Unweighted Pair Group Method (UPGMA) with the arithmetic mean based on Dice coefficients. Strains were considered to belong to the same pulsotype upon sharing at least 80% similarity in the banding patterns or same subtype upon showing identical banding patterns. The clonality of the strains was obtained by comparison with a previously published research 12 .
One strain of each pulsotype of MRSA and two strains of two main pulsotypes of MSSA were further characterized using multi-locus sequence typing (MLST) with internal fragments of seven housekeeping genes (arcC, aroE, glpF, gmK, pta, tpi, and yqiL) amplified using specific primers as per the recommendations described in S. aureus MLST database (http://saureus.mlst.net/). All fragments amplified were purified using the Wizard SV gel and PCR Clean-up System (Promega, EUA) and sequenced using an ABI PRISM® 3130XL Genetic Analyzer (Applied Biosystems, EUA). An allelic number corresponding to a sequence that was already present in the database was assigned to each sequenced housekeeping gene. Sequence types (ST) and clonal complexes (CC) were assigned according to their allelic profiles.

Detection of virulence genes
The presence of five adhesin genes, namely, cna (collagenbinding protein), bbp (bone sialo-binding protein), ebpS (elastinbinding protein), fnbA (fibronectin-binding protein A), and fnbB (fibronectin-binding protein B) was evaluated with PCR. The detection of cna, bbp, ebpS, and fnbB was performed according to the methods described by Tristan et al. 13 , while fnbA was detected as per the method described by Peacock et al. 14 lukS/F genes encoding Panton-Valentine leukocidin (PVL) were also investigated 15 .

Statistical analysis
All statistical analyses were performed with the chi-square and Fisher's test using the BioEstat ® software 5.3 version (Mamiraua, Brazil). The significance level was set at 0.05.

Antimicrobial susceptibility and SCCmec typing
Thirty-one (46%) S. aureus strains were found to be resistant (MIC 50 : 128 µg/mL; MIC 90 : 256 µg/mL) to oxacillin and 36 (54%) were found to be susceptible to oxacillin (MIC 90 : 0.5 µg/ mL). All strains showed susceptibility to vancomycin with an MIC 90 value of 1 µg/mL. The MRSA group included 22 SCCmec type II and nine SCCmec type IV strains. Most of the strains were susceptible to gentamicin (97% of strains from both groups) and trimethoprim/ sulfamethoxazole (92% of MSSA strains and 100% of MRSA strains) ( Table 1). The percentage of MSSA strains resistant to tetracycline was significantly higher than the percentage of MRSA strains resistant to tetracycline (28.0% versus 0%; P = 0.0001). All 22 MRSA SCCmec type II strains were resistant to ciprofloxacin and norfloxacin, while MRSA SCCmec type IV strains showed a significantly reduced resistance to these antibiotics (P = 0.0007). Furthermore, 17 (77.3%) MRSA SCCmec type II strains showed resistance to rifampicin, and significantly differed from the other groups, which were sensitive to this antibiotic (P = 0.00001).

Molecular typing of S. aureus strains
Based on the results of PFGE, we grouped the 67 strains into 16 pulsotypes (A to Q), and 31 MRSA strains were classified into five pulsotypes (A to E) and 12 subtypes ( Table 2). Pulsotype A (n = 22) comprised all MRSA SCCmec type II strains and was distributed into five different subtypes belonging to ST5 (similar to the USA100/New York-Japan clone). The other MRSA strains (n = 9) were presented as type SCCmec type IV with four different pulsotypes (B-D). ST8 was described in pulsotype B (n = 4), which presented a PFGE pattern similar to that of the USA300 clone. Pulsotypes C and D were categorized into ST5 and pulsotype E, similar to the USA600 clone, and were presented as ST45.
The 36 MSSA strains showed high genetic variability and were distributed in 11 pulsotypes (F-Q). One strain of the two predominant pulsotypes (F and G) was selected for MLST analysis. The strain ST1635 (ST5-related) in the pulsotype F and ST30 in the pulsotype G corresponded to the main MSSA genotypes identified in the present study ( Table 3).

Virulence genes and S. aureus lineages
The most prevalent virulence genes identified in the 67 strains were ebpS (82%) and fnbA (51%). We failed to observe differences in the distribution of both genes between MRSA and MSSA strains (P = 0.5 and P = 0.1, respectively) (Figure 1). However, MSSA strains harboring the adhesin genes cna (47% versus 3%) and bbp (31% versus 0%) (P = 0.0002 and P = 0.0024, respectively) were detected. The gene fnbB encoding fibronectin-binding protein was not detected in SCCmec type II strains but was highly prevalent in SCCmec type IV strains (44%, Table 2). Moreover, PVL-encoding genes were detected in 11 strains, including one MRSA SCCmec type II, four MRSA SCCmec type IV, and six MSSA.
The most prevalent gene detected in MSSA strains was epbS (78%), followed by fnbA (61%). Some virulence genes were associated with specific molecular types ( Table 3), i.e., cna and bbp were predominantly detected in the strains of pulsotypes G and H, respectively.

DISCUSSION
The present study provides information about the molecular epidemiology of S. aureus clinical strains from a hospital located in south-eastern Brazil and reveals important findings on the distribution of virulence genes and antimicrobial resistance among MSSA and MRSA strains.   The results of molecular typing of S. aureus strains demonstrate a great diversity in clones circulating in this hospital environment, as all sequence types identified were associated with the important clonal complexes circulating in the American continent (i.e., CC5, CC8, CC45, and CC30). Furthermore, the majority of the strains isolated were related to worldwide epidemic clones such as USA100 (NY/J), USA300, and USA600 16,17 .
The predominant MSSA strains characterized in the present study (ST1635-CC5 and ST30) were frequently detected in epidemiologic studies in Brazil 17,18 . Among MRSA strains, those with SCCmec type II related to worldwide epidemic clones such as USA100 (NY/J) were prevalent. This observation is consistent with the results of the study published by Caiaffafilho et al. 19 , wherein MRSA strains isolated from blood samples from Brazilian patients were studied.
The predominance of SCCmec type II (34/52, 65.4%) was also observed among the MRSA strains isolated from patients with bloodstream and respiratory tract infections during 2015-2016 in the University Hospital of Londrina in the Parana State, Brazil 20 . Interestingly, the predominance of MRSA strains harboring SCCmec II elements was also observed in a study during 2010 to 2013 in the same hospital 21 . These data suggest the shift in the MRSA population and emphasizes on the substitution of the strains harboring SCCmec III with those carrying SCCmec II that is becoming prevalent in some areas 19,20 .
MRSA SCCmec type III strains related to the Brazilian Epidemic Clone (BEC)/ST239 were surprisingly not observed in the present study. This was the main lineage found in Brazilian hospitals in the past several decades. Thus, our findings may 5/7 reflect the changes in the prevalence of MRSA clones involved with nosocomial infections in Brazil. This epidemiologic change has been observed in other national studies, wherein the prevalence of new clones has become increasingly common [18][19][20] .
The largest cassettes (I, II, and III) enhance the survival of MRSA in a hospital environment. However, smaller cassettes such as cassette IV are thought to promote evolutionary advantages through the horizontal transfer of this element 17 .
The high degree of diversity in the genotype of MRSA SCCmec type IV distributed in four different genotypes indicates the polyclonal origin of these strains in the hospital investigated. The same high genotypic diversity observed for MRSA SCCmec type IV was also reported in other similar national studies 8,22 . In addition, many of these strains were similar to the USA300 (ST8) clone, which is a non-multidrug-resistant clone that predominates in community-onset infections 23 .
The MRSA and MSSA strains exhibited low resistance to gentamicin and trimethoprim/sulfamethoxazole. Low resistance to these antibiotics was also observed in S. aureus strains in southern Brazil by Silveira et al. 24 , supporting the potential applicability of gentamicin and trimethoprim/ sulfamethoxazole as empiric agents against S. aureus infections in Brazil. MSSA strains showed a significant resistance to tetracycline, while MRSA strains were deemed sensitive to this antibiotic. Cavalcante et al. 25 observed similar results for tetracycline resistance in MRSA SCCmec type IV, and these authors proposed that such low resistance can be a possible marker of SCCmec type IV. In the present study, rifampicin resistance was high in the USA100 (NY/J) SCCmec type II (77%), contradicting the profile observed for SCCmec type IV (0%) and MSSA strains (0%).
The present study has drawn attention to the occurrence of MSSA strains harboring a broader spectrum of virulence genes as compared to MRSA strains. The occurrence of PVL-encoding genes was similar between MRSA and MSSA strains. Although this exoprotein is traditionally seen in community-acquired MRSA such as the USA300 strain, it has also been identified in MSSA and hospital-acquired MRSA strains 18 . The spread of this gene is a matter of concern, as the clones that produce PVL are generally associated with high mortality rates all over the world 26 .
Among the five adhesion molecules evaluated herein, ebpS was the most prevalent in both MSSA and MRSA strains. The high incidence of ebpS in multiple strains was observed in previous studies, consistent with the ubiquitous distribution of these genes in different S. aureus lineages 27 . Among the microbial surface components that recognize adhesive matrix molecules, FnbA and FnbB play important roles in S. aureus pathogenicity. These proteins promote bacterial attachment to fibrinogen, elastin, and fibronectin and participate in the initiation of the integrin-mediated intracellular uptake of bacteria via epithelial and endothelial cells 28 . In the present study, the distribution of fnbA gene was heterogeneous for both MRSA and MSSA. On the other hand, fnbB gene had heterogeneous distribution only among the MSSA strains; the only positive MRSA strains for fnbB expression were the SCCmec type IV, which were related to the USA300 lineage.
The cna gene was widespread among MSSA strains. However, only one strain, an MRSA-related USA600 clone (Berlin) CC45, presented cna gene. The low prevalence or absence of this gene in MRSA strains is well documented 1 . The role of cna gene product in the pathogenesis of bone infections and in the development of endovascular complications is well documented; however, it may play a less defined role in other infections 29 . In addition, consistent with our observation, the presence of cna was previously related to CC30 and CC45 lineages 29 .
The gene encoding bone sialoprotein binding protein (Bbp) was specifically expressed in MSSA strains, all of which belong to the CC30 lineage. However, in accordance with the findings of a previous study, no MRSA strain was found to be positive for bbp positive 30 . The presence of bbp gene has been associated with osteomyelitis and arthritis in humans 13 . S. aureus ST30 lineages containing the bbp gene have been detected in an orthopedic hospital in Brazil 16 . The relationship of CC30 lineages with the bbp gene has been noted in previous studies, emphasizing the potential role of this gene as a molecular marker for CC30 identification 30,31 .
The acquisition of antibiotic resistance in S. aureus is thought to involve changes in the virulence profile owing to the fitness costs associated with resistance genes 32 . In MRSA strains, this balance between virulence and resistance genes was closely associated with the size of SCCmec element possessed by the bacterium; strains with larger cassettes, such as SCCmec type II, have reduced numbers of virulence factors, while the presence of smaller cassettes, such as that observed in SCCmec type IV, is associated with a greater number of virulence genes, consistent with the observation reported in MSSA strains 32 .
Although this study evaluated a limited number of strains from a single center between April 2011 and February 2012, it showed that the virulence gene carriage was more diverse and abundant in MSSA than in MRSA strains and that the distribution of some of these genes correlated with the specific S. aureus lineages. In addition, tetracycline resistance was related to MSSA strains. Our findings support the hypothesis that MSSA may be potentially more pathogenic, although further studies are warranted to identify the clinical relevance of this phenomenon. As the clinical outcome of S. aureus infections is influenced by both antimicrobial resistance and virulence factors, both these factors should be considered for a better understanding of the development and dynamics of the pathogen.