Staphylococcus aureus : pathogenicity , antimicrobial resistance and clinical implications Staphylococcus aureus : patogenicidade , resistência antimicrobiana e implicações clínicas . Staphylococcus aureus : patogenicidad , resistencia a los antimicrobianos e implicaciones clínicas

Objective: To conduct a reflection about the pathogenicity, antimicrobial resistance, and clinical implications of the infections caused by Staphylococcus aureus. Method: Reflexive analysis, supported by theoretical references about the pathogenicity, antimicrobial resistance, and clinical implications of the staphylococcal infections. Results: The pathogenesis of S. aureus infections is complex and depends on the host characteristics, expression of virulence factors and ability to develop resistance to antimicrobials. Methicillin-resistant S. aureus (MRSA) is related to an advancement of healthcareand community-acquired infections, being vancomycin the primary therapeutic option. Infections caused by S. aureus with reduced vancomycin susceptibility (hVISA/VISA) have been associated with treatment failures and increased mortality. Conclusion: With the evolution of S. aureus to MRSA, hVISA and VISA, the treatment of staphylococcal infections has become a major challenge for the medical clinic, and an adequate and early antibiotic therapy is essential for decreasing morbidity and mortality rates related to this microorganism.


INTRODUÇÃO
S. aureus is one of the most frequent causes of healthcare-associated and communityassociated infections, which present high mortality and morbidity rates 1,2 .Due to its pathogenic potential it is responsible for a large range of infections characterized for diverse clinical manifestations, including local conditions as much as high lethality systemic infections 3,4 .
Infections caused by S. aureus are related to its large amount of virulence factors that contribute to the establishment and permanence of infectious processes [5][6][7] .The accessory gene regulator (agr) is the major quorum sensing system related with the control of virulence genes in S. aureus, being responsible for the most part of its virulence factors expression 8,9 .
The increase of staphylococcal infections

STAPHYLOCOCCUS AUREUS
S. aureus are gram-positive cocci, nonmotile, non-spore forming bacteria with diameters of 0.5 -1.5 µm, that microscopically are visualized in clusters 17,18 .These bacteria grows in non-selective culture media in optimal growing conditions at 37 ºC.It has high capacity of adaptation, being able to survive and multiply in hostile environments.They are facultative anaerobes, grow on mannitol salt agar, have a beta-hemolysis pattern in blood agar and produce catalase, coagulase and DNAse enzymes 17,19 .
As an opportunistic microorganism, S.
aureus behaves as both commensal and pathogen.They are normally found colonizing skin microbiota and sites, such as, nasopharynx, armpits, perineum and gastrointestinal tract; but due to its pathogenic potential it can cause a large variety of infections, mostly in immunity reduction cases or compromittment of the skin barrier 20 .

Pathogenicity
S. aureus is one of the most frequent causes of healthcare-associated and communityassociated infections, presenting a high mortality and morbidity rates 1,2 .Its transmission occurs mainly by direct contact with colonized or infected individuals and/or by contact with contaminated surfaces or objects 21,22 .
The majority of infections caused by S.
aureus are noticed to happen in asymptomatic individuals, colonized from short to long-term periods, resulting in disease when the immune system is compromised.Worldwide about 20 to 30 % of the population are persistent carriers and 60 % are periodical carriers of S. aureus 23,24 .
Asymptomatic carriers are troubling due to the fact that even showing no clinical symptoms of an infectious disease they are potential sources of infection and can help to disseminate the pathogen on the environment 25 .
Asymptomatic carrier status is even more worrying when it is a health professional.in four groups, agrI, agrII, agrIII and agrIV based on the specific AIP-AgrC receptor binding [34][35][36][37][38][39][40] .An AIP can only work as an agonist for its own allelic group, so that bacteria with different agr groups interfere in accessory proteins regulation of each other 34,36 .
A relation between agr groups (I-IV) and the infection type has been described for S. Penicillinase is able to degrade penicillin betalactam ring, inactivating its activity to inhibit the bacterial cell wall synthesis 48,50 .
The introduction of methicillin, a semi-

Staphylococcal cassette chromosome mec
The mecA gene is a part of the mec complex present in the mobile genetic element called SCCmec (Figure 2).This region is bracketed by direct repeats, that contain integration site sequence recognized by cassete chromosome recombinases (ccr) and by a pair of inverted repeats.Also, it contains J regions (standing for junkyard), that are useful to classify the SCCmec in different subtypes [59][60] .that encodes resistance to cadmium and is found mostly in SCCmec III 60,61 .
The SCCmec types are defined by the combination of distinct classes from mec complex and diverse types of ccr complex and the subtypes are organized according to the differences in J regions 52,60,62,66 .Until now, there are 12 SCCmec types (I-XII) described, and this classification is extensively used in MRSA molecular typing 57,68   b Class C1: IS431 upstream and downstream of mecA are in the same direction.
c Class C2: IS431 upstream and downstream of mecA are in the opposite direction.
.This antimicrobial acts by inhibiting the cell wall synthesis of gram-positive microorganisms by binding to the carboxyl terminus of D-alanine-D-alanine residues of the peptide precursors, forming a stable noncovalent complex, preventing the elongation of the peptidoglycan in the cell wall [93][94][95] .

CA
With the advancement of MRSA infections associated with the irrational use of antimicrobials, vancomycin has become the main therapeutic option 10 .The constant use of this glycopeptide and, consequently, the increase in selective pressure, resulted in the appearance of vancomycin intermediate S. aureus (VISA) in 1996 in Japan, called Mu50 96 .The next year also in Japan, the first S. aureus with heterogeneous vancomycin resistance (hVISA), known as Mu3, was isolated 97 .
In 2002 in Michigan, USA, the first clinical infection with vancomycin-resistant S. aureus (VRSA) was described.In Brazil, the first isolate with this characteristic was reported in São Paulo in a 35-year-old patient with recurrence of skin and soft tissue infections 98 .The presence of VRSA has also been reported in other countries 99- 101 .It is believed that this resistance is mediated preventing vancomycin binding and inhibiting its action on bacterial cell wall synthesis 99,101,102.The American Clinical and Laboratory Standards Institute (CLSI) 56 manual ranks as susceptible to vancomycin isolates with MICs less than or equal to 2 μg/mL (VSSA), intermediate MIC between 4 and 8 μg/mL (VISA), and resistant MIC higher than or equal to 16 μg/mL (VRSA).

Staphylococcus aureus with heterogeneous vancomycin resistance
Isolates of hVISA are characterized by the presence of a subpopulation with reduced susceptibility to vancomycin.In general, they are isolated from vancomycin-sensitive MRSA when analyzed by conventional methods, ie, with MIC lower or equal to 2 μg/mL, but with a subpopulation of approximately 10 -5 to 10 -6 cells exhibiting intermediate levels of resistance to vancomycin, with MIC higher than or equal to 4 μg/mL [103][104][105][106] .hVISA strains have heterogeneous morphology, presence of small colonies, low growth rate, reduced autolysis and hemolysis, thick cell wall and with reduced susceptibility to vancomycin (Figure 3) [107][108][109] .a Low growth rate and reduced hemolytic activity (Figure 3A).
The mechanism of acquisition of hVISA and VISA phenotypes is not fully elucidated but is mainly related to mutations in two component regulatory systems (TCRS), vraRS and walKR, and in the gene encoding the beta subunit of RNA polymerase, rpoB.It causes the thickening of the bacterial cell wall that entails the trapping of the vancomycin molecules and, consequently, hinders its action at the binding site 95,102,108,110- 113 .
After its first description in 1997 in Japan  The confirmatory method, considered gold standard for the detection of the hVISA phenotype, is the Population Analysis Profile -Area Under the Curve (PAP-AUC).However, this is a time-consuming, laborious and expensive method to be applied in routine clinical microbiology laboratory 104,105,[130][131][132] .
This methodology is based on calculating the area under the curve (AUC) generated after the growth of different cell densities (10 -1 and 10 -7 UFC/mL) in BHI agar containing various concentrations of vancomycin.When the ratio of the AUC of the isolate to the hVISA control (Mu3) is 0.9 to 1.3, the isolate is reported as hVISA 131 .

Clinical relevance
Despite of clinical impact, hVISA phenotype is not definitively enlightened 134,135 , some studies suggest that the presence of hVISA is commonly associated with failure in vancomycin treatment, persistent bacteremia, prolonged hospitalization, and adverse clinical outcomes [14][15][16]138,139 . In along with the irrational use of antibiotics has led to the emergence of S. aureus strains that present resistance mechanisms to different classes of antimicrobial agents, which makes treatment more difficult and aggravates the infectious process 10 .Methicillin-resistant S. aureus (MRSA) arose from the acquisition of genes that encode altered penicillin binding proteins (PBP2a), mecA or mecC, found in the mobile genetic element named mec staphylococcal cassette chromosome (SCCmec) 11 .With the advance of infections caused by MRSA, vancomycin has become the main therapeutic option.However, the constant use of this glycopeptide has increased the selective pressure among MRSA strains, leading to consequences, such as MRSA with reduced susceptibility to vancomycin (hVISA and VISA) 12,13 .These strains are related to failures in vancomycin treatments, persistent bacteremia, prolonged hospitalization and adverse clinical outcomes 12,14-16.Treatment of staphylococcal infections has become a major challenge, due to S. aureus high virulence potential and the current narrow therapeutics options since penicillin, methicillin and recently vancomycin resistance rates are increasing.In this context, it is important to understand phenotypic and molecular characteristics of MRSA and strains with reduced vancomycin susceptibility to control its dissemination, as well as assist antibiotic therapy in cases related to this multiresistant microorganism.The purpose of this review is to summarize the current knowledge on pathogenicity, antimicrobial resistance, and clinical implications of the infections caused by Staphylococcus aureus.

Figure 1 .
Figure 1.Structure of the agr system in Staphylococcus aureus.

aureus.
Goudarzi et al. (2016) 41 report that S. aureus from agr group I was prevalent in noninvasive infections and those from agr group II in invasive infections.In according to these finds,Rasmussen et al. (2013) 42 had shown a significant association between agr group III and staphylococcal invasive infections.Cotar et al. (2012) 35 found a prevalence of agr group III in respiratory tract infections, while the agr group IV was related to staphylococcal scalded skin syndrome by Lamand et al. (2012) 43 .Despite the importance of agr for staphylococcal virulence, some studies have suggested that agr dysfunctions confer survival advantages for microorganism and worst clinical outcomes in patients infected by S. aureus.In study performed by Chong et al. (2013) 44 agr dysfunction was associated with persistent bacteremia caused by MRSA and MRSA with vancomycin heteroresistance.In 2014, Viedma et al. (2014) 45 evaluated the relation between agr dysfunction and vancomycin reduced susceptibility (VRS) and the results showed a significant association between S. aureus with VRS and dysfunctions in the agr locus.Corroborating with this study, Schweizer et al.(2011)46 evaluated 814 patients with bacteremia caused by S. aureus and observed a significant association between agr dysfunctions and mortality in patients with staphylococcal infections.Beyond agr importance for staphylococcal pathogenesis many studies have shown an important relation between agr groups polymorphisms and vancomycin reduced susceptibility in S. aureus.The findings show that agrII is often associated with vancomycinreduced susceptibility and with treatment failures with vancomycin.Moreover, Cechinel et al. (2016) 47 reported that death risk increases by 12.6 times in patients with bacteremia caused by MRSA expressing agrII when compared to those that express other agr group type.In study conducted by Cázares-Domínguez et al. (2015b) 40 the agr type II polymorphism was the most prevalent among multidrug MRSA isolates.In 2015, Park et al. 48evaluated 188 MRSA from blood culture and observed that among the isolates presenting MIC of 2 μg/mL the presence of agrII was significant when compared to other agr types.ANTIMICROBIAL RESISTANCE Methicillin-resistant Staphylococcus aureus Penicillin was discovered in 1928 by Alexander Fleming, being the first choice for staphylococcal infections treatment in early 1940.However, in 1942 were reported the first cases of penicillin resistant S. aureus due production of a beta-lactamase enzyme (penicillinase) encoded by the blaZ gene.

Figure 2 .
Figure 2. General structure of the SCCmec element.
-MRSA, HA-MRSA and LA-MRSA MRSA emerged in the 1960s, after introduction of methicillin in clinical practice and rapidly spread in nosocomial environments.In the 1980s, specific MRSA lineages were found outside the hospital environment, furthermore called Community-Acquired MRSA (CA-MRSA).These strains present increased susceptibility to antimicrobials and increased virulence as major characteristics, when compared to nosocomial lineages (Hospital-Acquired MRSA; HA-MRSA) 63,77,78 .MRSA, according to Centers for Disease Control and Prevention (CDC) is considered CA-MRSA when recovered from patients coming from community or up to 48h after hospital admission that do not present history of infections or colonization by MRSA, previous hospitalization or invasive procedures in past year.Normally, CA-MRSA are associated with skin infections in healthy young patients, resistance to betalactam and SCCmec types IV or V. Some cases are associated with the production of Panton-Valentine leukocidin, that confers more virulence to CA-MRSA Enterococcus faecalis.Tn1546 contains the vanA genes, which causes the D-alanyl-D-alanine (D-ala-D-ala) fragment to change to D-alanyl-D-lactate (D-ala-D-lac),

Figure 1 .
Figure 1.Characteristics of Staphylococcus aureus with heterogeneous vancomycin resistance a,b The significant discrepancy of the epidemiological data, despite reflecting the geographic variation, is largely due to the methodological inconsistency of the detection process of this phenotype; the absence of standardization and, also, due to the biological characteristics and the mechanism of resistance of these isolates.Factors such as the site of the clinical sample, the population of patients tested and the number of samples analyzed can also interfere at the rates found 2,129 .Currently, the most commonly used screening methods for the detection of the hVISA phenotype are: (a) Macro Etest, which associates dense inoculum, prolonged incubation and nutrient medium with vancomycin Etest strips; (b) Glycopeptide Resistance Detection (GRD), which uses a vancomycin and teicoplanin doublesided gradient in a single strip for detection of hVISA and VISA; and (c) vancomycinsupplemented agar, where more resistant colonies are selected from the growth in BHI agar containing 6 μg/mL vancomycin (BHIA-6V) 12,108,125 .

Table 1 .
. The first SCCmec type was identified in 1999, Japan, using MRSA N315.In short time lapse, other two were described, SCCmec II and III 69 .Since then, various SCCmec were discovered around the world: SCCmec IV 70 , Specifications of the SCCmec types a .
Table adapted from Wu et al. 66 and IWG-SCC 76 .