"Isolation and Molecular Characterization of Methicillin – Resistant Staphylococcus Aureus (MRSA) In Hospital Patients"

Keyword: Staphylococcus; Staphylococcus Aureus; Staphylococcal Protein A; Staphylococcal Cassette Chromosome Mec (SCCmec); Mec Gene Complex; MECA; Multilocus Sequence Typing; Polymerase Chain Reaction; Sequencing; Resistance; Epidemiology Staphylococcus spp. it is one of the genera most frequently implicated in the etiology of hospital infections, especially in nosocomial wards. Staphylococcus aureus is one of the most frequently isolated pathogenic microorganisms in hospital infections, capable of causing septicemia and infections of the skin, respiratory system, and soft tissues. Furthermore, the spread of infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) species is constantly increasing and is reaching worrying levels in various countries of the world, including Italy, in continuous and rapid expansion, even outside hospitals. In this study, strains of Staphylococcus with methicillin resistance in hospitalized patients were identified and characterized through a phenotypic and genotypic approach. In all the methicillin resistant strains analyzed, a high resistance to other classes of antibiotics tested was found, in accordance with the findings of the European Center for Disease Prevention and Control (CDC) and numerous studies at national and world level. On some isolated MRSA strains, a molecular epidemiological study was conducted to understand the origin and spread of circulating clones. All these have been identified by molecular approach aimed at genetic research and identification, by means of Multi Locus Sequence Typing (MLST), typing of the Staphylococcal Cassette Chromosome mec (SCCmec complex) and spa typing, typing of the repeated variable region of protein A.

It is a toxin capable of detaching the superficial layers of the skin and by the toxic shock syndrome, TSST-1, also deriving from action of a toxin that involves symptoms such as: fever, hypotension, desquamative erythroderma and organ symptoms [1,4,5]. The main factors that increase susceptibility to infections are the prolonged or inefficient antibiotic or corticosteroid therapies, the use of invasive procedures (vascular and bladder catheterization, tracheal intubation, etc.), prolonged hospitalization and surgical interventions [6,7]. S. aureus is also responsible for food poisoning, due to the multiplication in foods of strains of S. aureus producing toxins resistant to cooking temperatures and the action of digestive proteolytic enzymes [8,9]. S. aureus is provided with a polysaccharide capsule, with phagocytic power, neutralized by specific antibodies.
On the cell surface there are proteins that are able to cooperate with those of the host, such as fibronectin and fibrinogen, playing the role of adhesions. Among these, the clumping factor is a protein which, interacting with fibrinogen, forms aggregates that can be highlighted on the slide. Another important surface protein of S. aureus is protein A. This is involved in complement activation, inhibits the phagocytosis of the bacterium by polymorphonuclear leukocytes, invokes hypersensitization and stimulation of lymphocyte production, contributing significantly to increase the virulence of S. aureus [3,10]. Furthermore, S. aureus has always been an absolute protagonist of acquired antibiotic resistance. Of particular importance and interest was the evolution of the resistance of S. aureus to β-lactam antibiotics, characterized by two distinct periods of hospital infections. A first hospital infection, which developed early (around the early fifties of the last century) and rapidly spread all over the world, was sustained by penicillinresistant strains, which became such having acquired the ability to produce penicillinase [11]. The end after 10 years thanks to the advent of new antibiotics (such as penicillinase-resistant penicillin and the first cephalosporin's), even if the phenotypic and genotypic characteristic of β-lactamase production remained definitively acquired by most of both hospital community. A second hospital infection, still ongoing today, is that sustained by methicillinresistant strains (internationally known with the acronym MRSA, methicillin-resistant S. aureus), that is, competent of resisting methicillin, the progenitor of penicillinase-resistant penicillins [4].
Methicillin is characterized by an acyl group in 6 'which sterically prevents attachment to the β-lactam ring, thus preserving its activity even in the presence of β-lactamase [12,13]. In addition, an epidemiological-molecular study was performed on some MRSA isolates from various departments, applying MLST, to understand the origin and spread of circulating clones.

Bacterial Isolates
Eighty-one Staphylococcus spp. strains were isolated and

Detention of S. aureus using PCR Amplification
Validation of S. aureus species identification was performed by PCR using the species-specific primers [37]. Primers were as follows:

Multilocus Sequence Typing
MLST with standard primers introduced by the MLST database was performed on 7 MRSA isolates based on seven housekeeping genes (arcC, aroE, glpF, gmK, pta, tpiA and yqiL) as described by Enright et al. (2000). The following seven housekeeping genes were used in the final MLST scheme, and the fragments were amplified by using the primers shown in (Table 1)

Statistical Analysis
Statistical analysis was performed using Statgraphics Centurion® XV for Windows.

Results
In this study, 81 strains of Staphylococcus spp. were recovered  On the other hand, the previous genes (spa and pvl) were not found in 14 CoNS strains.

MLST
According to the MLST method, isolates were assigned to five different sequence types (STs) (ST5 in 1 strain, ST8 in 1 strain, ST10 in 1 strain, ST22 in 2 strains, and ST228 in 2 strains). Furthermore, the 3 MRSA of care unit were belonged to ST8 (n = 1) and ST228 (n = 2), the strain isolated from the Surgical Clinic showed ST5, from hematology the ST10, while the isolates of Infectious Diseases (n = 1) and of Pneumology (n = 1) were ST22.

Discussion
S. aureus is one of the species most frequently implicated in the etiology of hospital infections in different parts of the world, especially in the intensive care, pneumology, hematology, and surgery departments [39,40]. Although with lower percentages, CoNS are also emerging as important opportunistic pathogens, and are often involved in hospital epidemics [41,42]. This study, in agreement with these studies, highlighted beyond the isolation of S. aureus, a high percentage of CoNS from clinical samples from acutely patients, confirming the growing involvement of these problems in nosocomial infections. The MRSA spread infections is increasing and is achieving worrying levels in several countries, including Italy. Since Staphylococcus spp., in particular MRSA is transmitted through infected people, or vehicles, the first strategy to contain this spread may therefore concern the implementation of prevention, as suggested by the guidelines [43,44]. In this work, all methicillin resistant strains were found to have high resistance to other classes of tested, in accordance with what was reported by the European Center for Disease Prevention and Control (CDC) [45].
The mecA gene was considered the "golden standard" for detecting methicillin resistance in MRSA, however, recently methicillinresistant mecA negative strains have been found, in which the presence is associated with the mecC analogue (mecALGA251).
In this work 97% of methicillin-resistant staphylococci had showed the presence of the mecA gene. Instead, in two isolates, despite being resistant to methicillin from the analysis with Vitek2, they did not possess the mecA and cC genes, highlighting, as reported by other authors, the limits of the phenotypic systems [46,47]. The data confirmed that HA-MRSA showed the virulence gene of Protein A (spa) but not the Leukocidin Panton -Valentine (pvl) gene, usually associated with CA-MRSA a community circulation [48]. Through the MLST profile have been identified 5 different clones of S. aureus, 4 of which ST5, ST8, ST22 and ST228 already circulating in Italy and worldwide, while the ST10 was not yet reported in Italy, was present only at community and veterinary level, confirming the trend of diffusion and exchange between CA-MRSA and HA-MRSA [49]. The ST5 profile strain from surgical clinic, linked to the type of sequence of a HA-MRSA widespread throughout the world and responsible for nosocomial, tract, mucosal and wound complications. Strains of ST8 and ST228 were identified in the intensive care unit isolates, detecting the circulation of at least two different clones in this unit. The presence of strains with characteristics such as to be included in ST8 and ST228, found to be circulating in both hospital and community settings, has been reported throughout the world [3,31,43].
Furthermore, MRSA with ST22 type sequence had been isolated from different types of samples from infectious disease and pneumology department, clone was found mainly in hospital and outpatient clinics, but also in communities and in animals in close contact with humans (dogs and cats) [3,46]. Finally, in this work, a type of ST10 sequence never reported in Italy was found coming from a nasal swab of the hematology department.

Conclusion
In conclusion, this study demonstrated the importance of constant supervision of the clones circulating in the several hospital departments, colonization, and the probable, but already possible, diffusion and exchange of strains found in the hospital and then in the community. This study was conducted on clinical samples that were chosen to represent the reality nosocomial situation.
Although conducted on a restricted number of samples, it provides a database for the design of targeted screening and preventive molecular diagnostics.