Pathogenicity and Molecular Characterization of Staphylococcus aureus Strains Isolated from the Hospital Environment of CHU-Z Abomey-Calavi/Sô-Ava (Benin)

Staphylococcus aureus is a major human pathogen present on a third of the healthy population. The bacterium possesses an extensive arsenal of virulence factors. The pathogenicity is linked with S. aureus high plasticity and its exceptional ability to incorporate foreign genetic material. The aim of the present study was to perform molecular characterization of Staphylococcus aureus strains isolated from the clinical environment of the CHU-Z Abomey-Calavi/Sô-Ava. Isolation of Staphylococcus aureus bacterium was performed on Chapman agar. Toxin production by isolated S. aureus strains was investigated using the radial immunoprecipitation technique. A colorimetric assay was used to evaluate Staphylococcus aureus lipase (SA-Lipase) production. Finally, the expression of antibiotic resistance genes and genes encoding toxins production was investigated. Our data suggest that none of the isolated Staphylococcus aureus strains expressed the investigated toxin genes. Interestingly, SA-Lipase was produced by 14.28% of our isolated S. aureus strains. The mecA gene was present in 57.14% of the isolated strains, while PVL and TSST-1 genes were identified in 2.85 and 7.14% of S. aureus, respectively. Significant genetic diversity was observed along the hospital environment S. aureus strains. The present study reveals the level of virulence of S. aureus strains isolated in the different units of CHU-Z Abomey Calavi/Sô-Ava through the production of lipase, PVL, and epidermolysins. The molecular study has favored a genetic characterization within the isolated strains.


Introduction
Staphylococcus aureus bacterium is often encountered in human pathology and is the causative agent of a wide variety of infections including life-threatening toxic shock syndrome, pneumonia, and sepsis. The clinical outcome of these infections depends on numerous virulence factors that S. aureus can produce. Staphylococcal infections represent a major public health problem both because of the bacteria's virulence and the emergence of multiresistant strains [1]. Thus, S. aureus infections are challenging both in terms of diagnosis and therapeutic strategies [2]. Furthermore, Staphylococcus resistance to methicillin is mainly attributed to the acquired mec A gene [3]. In addition, a new mecC gene has recently emerged especially in S. aureus of animal origin [4]. Several studies show that multidrug-resistant S. aureus strains are involved in infections and hospital-related deaths in both industrialized and developing countries [5].
In addition, the pathogenicity of this bacterium is linked to the expression of virulence factors [6]. Indeed, S. aureus can produce various heat-resistant extracellular protein toxins that behave as virulence factors, including superantigens, hemolysins, and leukocidins. The most important of these factors is the Panton-Valentine leukocidin (PVL), which is a cytotoxin that forms pores in the membrane and has been associated with boils, skin abscesses, and severe necrotic skin infections [7,8]. Some of the pathogenic S. aureus strains also possess the gene coding for staphylococcal toxic shock toxin (TSST-1) [9]. These toxins have superantigenic activities and are capable of polyclonal T-cell activation independently of their antigenic specificity. This activation is directly linked to the clinical signs of staphylococcal toxic shock [10,11]. It has been shown that the secretion of these toxins by strains in infections constitutes a risk of prolonged hospitalization periods [12].
S. aureus can produce abundant amounts of secreted glycerol ester hydrolase (lipase) enzymes, which release free fatty acids from triglycerides [13]. Indeed, some triglycerides contain additional toxic fatty acids that appear to interfere with cell growth by altering cell permeability, cutting off oxidative phosphorylation, or blocking electron transport [14]. This broad array of pathogenic profiles is linked to the high genomic plasticity of S. aureus, which confers to the bacteria an exceptional ability to incorporate exogenic genetic material from other strains and thus to acquire new properties, including antibiotic resistance and virulence [13]. All these factors make S. aureus a predominant nosocomial pathogen among immunecompromised patients in hospital environments and health care facilities [15]. The present study is aimed at evaluating the genetic diversity and virulence capacity of S. aureus strains isolated from the hospital environment of CHU-Z Abomey-Calavi/Sô-Ava.

DNA Extraction.
A total of 42 S. aureus isolates previously isolates from the hospital environment of CHU-Z Abomey-Calavi/Sô-Ava in Benin [16] were used in this study. Those isolates were collected from five medical units of CHU-Z Abomey-Calavi/Sô-Ava: pediatrics (10 isolates), operating room (7 isolates), neonatology (10 isolates), maternity (8 isolates), and central sterilization room (7 isolates). Purification of isolates DNA was done as previously described by Rasmussen and Morrissey [17]. Briefly, 1.5 ml microcentrifuge tubes containing bacterial strains were centrifuged at 12000 rpm for 5 min, the supernatant was poured, and 500 μl of sterile distilled water was added to the bacterial pellet. The mixture was then heated in a dry bath at 95°C for 15 min. The tubes were centrifuged again at 12000 rpm for 5 min, and the obtained supernatants were recovered in new tubes. 500 μl of absolute ethanol was added, and the tubes were centrifuged at 12000 rpm for 5 min. The DNA pel-lets were suspended in 50 μl sterile distilled water and maintained at 4°C for immediate use or at -20°C for long-term storage.

Production of Leukotoxins and Epidermolysins.
The production of Panton and Valentine Leukotoxin (PVL), LukE-LukD leukotoxin, and epidermolysins (ETA, ETB) of S. aureus isolates was investigated by radial immunoprecipitation [19]. Briefly, fresh S. aureus colonies were cultured in 500 μl of Yeast Casamino-acid Pyruvate (YCP) broth in 12well culture plates and incubated at 37°C for 18-24 hours under agitation (200 rpm). The content of each well was poured into microcentrifuge tubes, and culture supernatants were collected (using a micropipette) after centrifugation at 5,000 rpm for 5 min. On a 0.6% agarose gel, 7 wells spaced 8 mm were dug in a rosette. Approximately 30 μl of each sample is placed in the corresponding outer well, followed by purified rabbit anti-leukotoxin (PVL and LukE-LukD) and antiepidermolysins (ETA and ETB) antibodies (OD = 3), placed in the rosette's central well as control antigens (OD = 0:2) in the upper and lower wells in a clockwise direction. After a 16-hour diffusion, the precipitation arcs were viewed directly with the naked eye or after staining with Coomassie blue [19].
2.4. Lipase Production Capability. The procedure described by Janda [20] was used to detect the production of lipolytic enzymes in which polyoxyethylene sorbitan monooleate (Tween 80) is used as a lipid substrate. This method is based on the precipitation of the calcium crystals salt in presence of fatty acid induced by lipase. If the bacterial strain has a lipolytic enzyme, the calcium soap crystals' precipitation will be visible as an opaque halo around the colonies. In addition, if the lipase activity is intense, the precipitation will appear as crystals visible to the naked eye.
The bacterial strains are therefore inoculated on nutrient agar supplemented with Tween 80. The substrate is added to the culture medium, previously sterilized, at a concentration of 1%. The appearance of opaque precipitation around bacterial colonies occurs after 24 to 72 hours of incubation at 37°C. 2.7. Data Analysis and Processing. The data collected was coded and processed using Microsoft Excel 2013 spreadsheet software. Graph Pad Prism 7 software was used for comparison testing of positive isolates at various collection sites. R software was used for correspondence factor analysis and then using the results of this analysis for dendrogram clustering. Significance was accepted when p < 0:05. Using the polymorphism based on the size and number of bands, the studied S. aureus isolates were classified. Thus, considering scale 1, three phylogenetic subgroups of Staphylococcus aureus were obtained (Figure 2). Notable was that the strains of S. aureus that make up subgroup 1 are related and originate from the five services studied. On the other hand, strains in subgroup 2 were from the neonatal, pediatric, and maternity wards. Furthermore, subgroup 3 comprises strains from four services (central sterilization, operating room, maternity, and neonatology).  Table 1). On the other hand, the lipase test revealed that at least one strain in each of the departments such as pediatrics, neonatology, and central sterilization has produced lipase, but in the maternity ward, 2 strains were observed to produce lipase (Table 1). However, the strains of S. aureus isolated in the operating theatre did not produce lipase. In general, 14.28% of the S. aureus strains isolated in the hospital environment were lipase-producing.

3.2.2.
Detection of the mecA, LPV, and TSST-1 Gene. After PCR, the distribution of the presence of the mecA gene by service shows that bacterium isolated in neonatology are more carriers of the mecA resistance gene, followed by maternity and pediatrics (Table 2). On the other hand, it appears that only one strain of S. aureus has the Panton and Valentine leukotoxin (PVL) gene. This strain with the PVL gene is isolated in the maternity ward. In contrast, for the TSST-1 gene, three strains of S. aureus possess this gene and are from pediatrics, maternity, and central sterilization ( Table 2).
Globally, it should be noted that 57.14% of our S. aureus strains have the methicillin resistance gene (mecA) in their genome. 2.85% and 8.57% represent the proportion expressing the PVL and Toxic Shock Toxin genes, respectively ( Figure 3).

Discussion
The molecular technique showed large importance of its use since 7 strains were not S. aureus. The 16S-23S ribosomal RNA gene can be said to be a target of choice and useful for better strain identification [22]. The reason for the genetic polymorphism (band size and number of bands) linked to the 16S-23S rRNA gene observed in our study is due to the presence of multiple copies of this gene in S. aureus strains. Several authors have shown that S. aureus was the species with the highest number of polymorphisms [18,23,24].

BioMed Research International
Furthermore, the characteristic band profile (about 447 bp and 569 bp) is also observed in the work of Mendoza et al. [25] and Akineden et al. [26].
Concerning the pathogenicity of S. aureus, one of the strategies adopted by host cells to respond to infection is the production of fatty acids and lipids, which form small holes in the bacterial membrane. In contrast, S. aureus produces enzymes called lipases that destroy these fatty acids before causing damage to the bacterial membrane [27]. It appears that 14.28% of our S. aureus strains are lipase producers. This result could be explained by the low use of fats in the composition of detergents used in surface cleaning. Also, lipase production in the test with Tween 80 is justified by the stimulating effect of Ca 2+ present in the medium in S. aureus. This is confirmed by Bornsheur et al. [28], who state that bivalent cations such as calcium often stimulate the enzymatic activity of lipase. Recent work by Cadieux et al. [29] reported lipase production by strains of S. aureus, S. hycus USA 300, and Pogaku et al. [30] in Staphylococcus sp. Lp12 strains. Therefore, this virulence factor deserves more attention because microbial lipases are highly stable to temperature, detergents, and certain proteolytic enzymes [31].       BioMed Research International The phenotypic results on the nonproduction of PVL, ETA, and ETB toxins by our S. aureus strains are partly reassuring. The same observations were made by Ahoyo et al. [32] in S. aureus strains isolated in the hospital environment in central Benin and by Sina et al. [33] in S. aureus isolated from catheters at the CNHU of Cotonou. On the other hand, many clinical strains isolated from body fluids produce PVL up to more than 50% of the bacterium isolated [33,34]. This difference could be due to the fact that strains isolated from body fluids are often confronted to the immune system and have developed this virulence factor. However, molecular PCR assays showed that only one strain had the PVL gene, and three strains of S. aureus had the TSST-1 gene. And the mere fact that these strains came from the pediatric and maternity wards is worrisome given the proximity of the two wards in the hospital and the risk of probable gene transfer. Our results on the presence of the PVL gene are different from the 7.14% obtained by Cadé et al. [35] in France. For the TSST-1 gene, the 7.14% obtained in our study is slightly higher than the 3.3% obtained by Baba-Moussa et al. [7] in S. aureus isolated from dermatoses.
This variation could be attributed to the level of pathogenicity of the strains or the geographical location but also to the difference in gene detection methods. Although the pathogenicity of these strains does not appear to be related to the production of PVL based on the immunological results obtained, it is easy to understand that the presence of the genes that can promote the expression of these different toxins is associated with an increase in virulence of certain strains of S. aureus [2]. Thus, the primary concern remains in the possibility of horizontal transfer of these genes and their possible expression within S. aureus species, which could promote the spread of virulent strains in the coming years. In addition, certain hereditary immune deficiencies are associated with severe PVL+ staphylococcus infections: chronic septic granulomatosis, leukocyte adhesion defects, congenital neutropenia, and innate immunity defects. However, these strains (PVL+) are known to be responsible, particularly in nonimmunocompromised children, for necrotizing pneumopathies, rapid progression, and poor prognosis despite appropriate antibiotic therapy [36].
However, methicillin resistance is also one of the criteria for virulence of Staphylococcus aureus. Indeed, the phenotypic expression of methicillin resistance is influenced by several parameters (mean temperature, pH, and NaCl content in the medium and even genotypic). Thus, the presence of the mecA gene by the molecular method gives a clearer idea of the MRSA strains in our samples. We deduce that at least half of the S. aureus strains isolated from neonatal, maternity, and pediatric wards are carriers of the mecA gene. The contiguous nature of these three departments would be a critical factor in bringing the three departments closer together, thus, favoring a probable transfer of the mecA gene and also the diffusion of its resistant bacterium through the movement of staff from one department to another or of patients from one department to another.
Overall, 57.14% of isolated S. aureus possess the mecA gene. These results are consistent with those of Rocchetti et al. [37], who reported that 50.6% of S. aureus isolated from blood culture bottles in Brazil had the mecA gene. On the other hand, our results are contrary to those of Sajit Khan et al. [38] in South India, who found that 94% of clinical MRSA were carriers of the mecA gene. This difference could be explained by a low transfer of the mecA gene within S. aureus strains from CHU-Z Abomey-Calavi/Sô-Ava. Furthermore, several studies show that the mecA gene is also present in coagulase-negative staphylococci [37]. It is important that this work can help improve new therapy options to limit the spread of these pathogens. Because hospitalacquired MRSA is also responsible for community-acquired infections in patients with risk factors such as a history of hospitalization [39].

Conclusion
This work allows to highlight the level of virulence of S. aureus strains isolated in the departments of CHU-Z Abomey-Calavi/Sô-Ava through the production of lipase as well as the production of PVL and epidermolysins. PCR research has shown the presence of the mecA gene and the PVL gene in our S. aureus strains. The molecular study favored a genetic characterization within our strains, which allowed a group matching. The acquisition of these virulence factors as well as the genetic background of these strains requires more attention and suggests the necessity of specific hospital measures.

Data Availability
The data used to support the findings of this work are available from the corresponding author upon request.

Disclosure
This paper has been extracted from a PhD thesis by Akim Socohou carried out in Abomey-Calavi University, Benin.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
AS, HS, CD, and LB-M conceptualized and designed this study. AS, HS, and ES participated significantly in data acquisition. ES, AT, and BB performed the statistical analysis under the supervision AA and HA-S. ES, AT, and CN drafted the final manuscript with AA and HA-S. All authors contributed to the revision of the final manuscript and approved submission.