Diversity and Distribution of Staphylococcal Chromosomal Cassettes Mec (SCCmec) Types I, II and III in Coagulase-Negative Staphylococcal Strains Isolated from Surfaces and Medico-Technical Materials of the University Hospital of Abomey-Calavi/Sô-Ava

The coagulase-negative staphylococci (CoNS) have long been considered to be low pathogenicity. The possibility of a horizontal transfer of resistance and virulence genes from S. aureus to CoNS could increase the pathogenicity of these bacteria. showed resistance to methicillin. However, only 28.5% (20/70) carried the mecA gene. SCCmec was identified in only 17.14% (12/70) of these strains. Four strains carried mecA gene as well as one of the three types of SCCmec searched. SCCmec types I, II and III were identified in CoNS strains studied. SCCmec type I was the most frequent chromosomal cassette in mecA + strains, only or in association with another SCCmec. The study also revealed methicillin-resistant strains carrying SCCmec lacking the mecA gene. Finally, 60% (12/20) of the strains were found to be non-typeable. Our results show that CoNS strains present a high resistance to methicillin and the source of this resistance in the CoNS of our study is not only the mecA gene. There is also a high diversity of SCCmec, justified by a large number of non-typeable CoNS strains. The mecA − SCCmec + methicillin-resistant strains deserve to be sequenced for further studies.


Introduction
The coagulase-negative staphylococci (CoNS) have long been considered to be low in pathogenicity, but are increasingly implicated in many healthcare-associated infections [1]. If nowadays, these bacteria occupy an important place in human pathology [2] it's because, since their appearance, they have constantly developed resistance mechanisms to antibiotics in order to ensure their survival [3].
This resistance is linked to their great genomic plasticity which can be acquired or contributed by a plasmid or other mobile genetic elements such as staphylococcal chromosomal cassettes, transposons, bacteriophages or insertion sequences [4].
Thus, after the emergence of methicillin-resistant S. aureus strains, a consequence of the excessive use of this antibiotic; we observe the appearance of coagulasenegative staphylococci resistant to methicillin (MRCoNS), an antibiotic of choice used in the staphylococcal infection's treatment [5]. However, in S. aureus the resistance to methicillin (MRSA) leads to resistance to almost all beta-lactam antibiotics [6]. This makes the management of patients a challenge for clinicians.
The main mechanism of the resistance to methicillin is related to beta-lactam target modification [6] [7]. Resistance to this semi-synthetic antibiotic is under the control of the mecA gene located in a mobile genetic element called the staphylococcal chromosome cassette mec (SCCmec) [8]. SCCmec harbors the mec genes, which confer resistance not only to methicillin but also to almost all other beta-lactams [9]. SCCmec has two components, it is about: the mec gene complex and the cassette chromosome recombinase (ccr) gene complex. The mec gene complex which consists of mecA, the regulatory genes, and the associated insertion sequences; has been classified into six different classes (A, B, C1, C2, D,  [10]. This implies that coagulase-positive and coagulase-negative strains of staphylococci exchange staphylococcal chromosomal cassettesmec (SCCmec) by horizontal transfer, thus promoting the acquisition of antibiotic resistance genes by the latter [11].
The aim of this study is therefore to investigate the diversity and distribution of staphylococcal chromosome cassettes mec (CCSmec) types I, II and III in coagulase-negative staphylococcal strains isolated from surfaces and medicotechnical materials from the University Hospital of Abomey-Calavi/Sô-Ava (South Benin) from January to June 2019 in the Departments of Neonatology, Pediatrics, Maternity Ward, Operating Room and Central Sterilization.

Susceptibility of CoNS Strains to Methicillin
The susceptibility of the CoNS isolate was investigated by cefoxitin disk diffusion method on the Muller Hinton agar medium according to the EUCAST [13].
The bacterial suspension was standardized using the 0.5 McFarland control.

DNA Extraction
DNA extraction of the different SCN strains was done according to the method adapted from Rasmussen and Morrissey [14] [15]: after inoculating a colony of the strain to be tested on MH agar medium poured into petri dishes, the dishes were incubated at 37˚C for 18 h. Then, the preculture of the different colonies obtained on the petri dishes was done in 1 mL of liquid MH in an eppendorf tube. The tubes were incubated at 37˚C for 18 h. The obtained bacterial cultures were centrifuged at 12,000 rpm for 5 minutes. The supernatant was poured off and 500 µL of sterile distilled water was added to the bacterial pellet and heated in a dry bath at 95˚C for 15 minutes. Then, the heated pellet was centrifuged at N. Chimene et al. American Journal of Molecular Biology 12,000 rpm for 5 minutes. The resulting supernatant was emptied into another eppendorf tube and 500 µL of 70% (v/v) alcohol was added. Centrifugation was performed again at 12000 rpm for 5 minutes. The resulting supernatant was again emptied and the eppendorf tubes were left for 24 hours in a laminar flow hood for drying. The DNA pellets were suspended in 50 µL of EDS and stored at 4˚C for immediate use or at −20˚C for long-term storage.

mecA Gene Detection
The detection of the mecA gene in methicillin-resistant CoNS (MR-CoNS) strains was performed by monoplex PCR using specific primers ( Table 1)

SCCmec Typing
The typing of CCSmec present in CoNSRM was done via multiplex PCR using specific primer pairs (Table 1) capable of detecting CCSmec type I, II and III.
The sequences of the different primers used are shown in Table 1

Data Analysis
The Excel 2019 workbook was used to make graphs to better analyze data. The ± signs were used to reflect the presence or absence of the desired gene and staphylococcal chromosomal cassettes.

SCCmec Typing
To assess the distribution of SCCmec in CoNS, Multiplex PCR was done. Analysis revealed the presence of one of the three SCCmec in twelve (17.14%) of the seventy CoNS strains studied (Figure 3, Table 2). Among the mecA + strains, four (40%) were found to carry one of the three SCCmec. Thus, SCCmec types I, II and III alone or in combination with another SCCmec were distributed among twelve CoNS strains. However, it was observed that among the strains carrying at least one of the three SCCmec type, two CoNS strains (S. cohnii and S. sciuri) carry SCCmec lacking the mecA gene. Our results also show that SCCmec type I, either alone or in combination, is the most frequent SCCmec in the CoNS strains studied. Indeed, it was identified alone and in combination with SCCmec type III (n = 4). The other type of SCCmec identified was SCCmec type II and it was identified as many times as SCCmec type III. This study also shows a high genetic diversity of SCCmec within the MR-CoNS since 60% of Non-typeables mecA + strains could not be typed ( Figure 3, Table 2).

Discussion
CoNS are ubiquitous bacteria with a strong adaptive capacity and are among the most common germs isolated in hospitals. For a long time considered not very pathogenic, they are, as shown by recent studies, more and more incriminated in health care associated infections [14]. The increase in antibiotic-resistant strains of CoNS increases the pathogenicity of these strains and complicates the treatment of patients with infections caused by these bacteria [15].  [20]. This result could be explained by the heterogeneity of the expression of methicillin resistance in CoNS strains. In addition, several factors are susceptible to influence the expression of this resistance in these strains [22] [23]. In fact, Ph, temperature and salt concentration can affect the expression of methicillin resistance in CoNS strains. In addition, there are other mechanisms of methicillin resistance in coagulase-negative staphylococci [24]. Also, according to the CNRS CoNS strains with true methicillin resistance and with the mecA gene in their genome are quite rare [25]. Although having high scores, methods for phenotypic detection of methicillin resistance would not be as reliable as genotypic methods for detecting resistance to this class of antibiotics. In this regard, the work of Asante et al. shows that 05 strains of CoNS with the mecA gene were not detected as mecA + after their resistance to methicillin was assessed phenotypically [16].
SCCmec are mobile genetic elements and also genetic carriers the mecA gene,  [31]. In such cases, the SCCmec although retained within the CoNS strain would lack the mecA gene and this strain would be mecA − after PCR although carrying a SCCmec. This may be the case in this study as the strains studied were stored at low temperature for a significant period of time.
In view of the significant number of CoNS with the methicillin resistance gene and staphylococcal chromosomal cassette in this study, it's clear that this group of bacteria shouldn't be neglected in daily practice as multidrug resistance is a major constraint in the treatment of infections caused by these bacteria. It's therefore necessary not only to carry out more in-depth studies on this bacterial group but also to adopt measures to improve hygiene in hospitals in order to limit possible contamination that could lead to infections.

Conclusion
Often neglected because their pathogenicity is much weaker than that of S. aureus strains, CoNS are nowadays incriminated in many cases of nosocomial infections. Our results show that CoNS strains present a high resistance to methicillin and the source of this resistance in the CoNS of our study is not only the mecA gene. It's therefore essential to evaluate their antibiotic susceptibility profiles and to determine the source of their resistance to these antibiotics in order to prescribe appropriate antibiotic therapy. This study also shows a high diversity of SCCmec in CoNS strains isolated from surfaces and medico-technical materials of the University Hospital of Abomey-Calavi/Sô-Ava (South Benin), as shown by the large number of non-typeable CoNS strains. The mecA − SCCmec + meticillin resistant strains deserve to be sequenced for further studies. All three types of SCCmec sought were identified in this study but a large number of mecA + CoNS strains could not be typed for the I, II and III SCCmec. Then, these strains should be typed for one of the thirteen other SCCmec identified to date. These staphylococcal chromosomal cassettes, which carry the mecA gene, may also be the genetic carriers of many other antibiotic resistance genes. The mobility of these genetic elements could then favor their movement between the different CoNS strains by horizontal transfer, a hypothesis that was verified since one of the SCCmec studied was detected in the CoNS strains. More attention should also be paid to CoNS, their antibiotic resistance profile, the genes that mediate antibiotic resistance and their genetic backbone. Meticillin-resistant strains with mecA − SCCmec + genotype deserve to be sequenced for further study. We also plan to search for other types of staphylococcal chromosome cassette in nontypeable CoNS. On the other hand, whole genome sequencing will help to understand the mechanism of acquisition of the mecA gene in strains that do not carry the staphylococcal chromosome cassette mec.