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Virulence , biofilm formation ability and antimicrobial resistance of Staphylococcus aureus isolated from cell phones of university students

Introduction: Contamination of cell phones can contribute to the dissemination of pathogens in the community and/or hospital environment. Objective: To characterize Staphylococcus aureus strains isolated from cell phones of university students. Methods: Samples were collected from 100 cell phones. Detection of genes associated with virulence factors such as biofilm formation (icaA and icaD), enterotoxins production (SEA, SEB, SEC, and SED), and resistance to methicillin (mecA and mecC) was performed in S. aureus isolates by PCR. Typing mecA gene performed by multiplex PCR. Susceptibility to antimicrobials and biofilm formation rate also evaluated by using disk diffusion test and crystal violet staining. Results: S. aureus was present in 40% of the total samples and about 70% of them belonged to Nursing students. Of the isolates, 85% presented resistance to penicillin and 50% were classified as moderate biofilm producers. In addition, 92.5% of isolates contained the gene icaA and 60% of the gene icaD. Approximately 25% of the isolates presented the mecA gene. Typing of the mecA gene showed the presence of staphylococcal chromosome cassette SCCmec I and c III respectively in 20% and 10% of the isolates. 70% of the samples could not be typed by the technique. Regarding the enterotoxins, the most prevalent gene was SEA (30%) followed by the SEC gene (2.5%). The presence of SED and SEB genes not observed in any of the isolates. Conclusion: The cleaning and periodic disinfection of cell phones can contribute to the reduction of the risk of nosocomial infection.


INTRODUCTION
Cell phones are devices necessary for both personal and professional life. For health professionals, they can improve communication, promoting collaboration and information sharing 1,2 .
Cell phones have been frequently used in hospitals and healthcare settings. That fact raises major concerns about nosocomial infections as they may be involved in the transmission of bacteria harboring genes of virulence and resistance, especially in areas requiring the highest standards of hygiene 2 . More than 50% of health professionals admit to the use of cell phones in the clinical setting, including during physical contact with patients, while bacterial contamination was observed in up to 25% of the devices 3 .
Among the possible contaminants, the presence of S. aureus is highlighted. S. aureus is a common cause of hospital and community-based infections, and methicillin-resistant S. aureus (MRSA) is considered an important nosocomial pathogen 4 . The presence of these microorganisms on cell devices is a crucial point due to the possible dissemination of antimicrobial-resistant bacteria 5,6 . In addition, these bacteria can produce biofilm, a structure known as a set of bacteria adhered to a surface and which multiply surrounded by a matrix 7 .
Students in the health field can be potential disseminators of contaminants since practical classes and clinical stages make possible direct contact with fomites and pathogens 8 . Several studies have already demonstrated that students in the health area are potential transmitters of pathogenic bacteria due to the use of contaminated objects and lack of hygiene. Garcia et al. 9 observed the presence of bacteria and fungi in ballpoint pens used by university students. Margarido et al. 10 demonstrated bacterial contamination on the coats of nursing students after their use in health care practices. Zadai et al. 11 Souza et al. Virulence, biofilm formation ability and antimicrobial resistance of Staphylococcus aureus isolated from cell phones of university students. reported the presence of pathogenic bacteria on the surfaces of cell phones of medical students.
In this context, cell phones could harbor a diverse range of species of microorganisms including antibiotic-resistant organisms known to cause nosocomial infections.
This study aimed to evaluate the presence of S aureus on the cell phones of university students, as well as to evaluate the antimicrobial resistance, formation of biofilms, and presence of virulence and antimicrobial resistance genes of these microorganisms.

Antimicrobial disc-diffusion technique in Agar
The antimicrobial susceptibility test was performed using the agar diffusion technique, as recommended by the Clinical Laboratory Standards Institute-CLSI 14 . The disks used for the evaluation of antimicrobial resistance were oxacillin, cefoxitin, penicillin, clindamycin, erythromycin, and levofloxacin. For the assessment of multidrug resistance, the MAR index (multiple antibiotic resistance) was determined according to Magiorakos et al. 15 .

Evaluation of biofilm formation
The isolates were cultured in BHI broth at 37°C for 24 hours.

Detection of virulence and resistance genes by PCR
The phenol-chloroform technique was used to extract DNA from microorganisms 17  It was then centrifuged at 9,000 rpm for 10 min at 4 °C, the supernatant decanted, and the pellet washed with cold 70% ethanol. Finally, it centrifuged, the supernatant decanted, the remaining contents allowed to dry, and these resuspended in 50 μl TE buffer.
DNA concentration was evaluated with a nano-spectrophotometer (ND-1000 Nanodrop ® Thermo Fisher Scientific, Waltham, MA, USA), using the 260/230 and 260/280 wavelength ranges. Quality was determined by electrophoresis in 1% gel to measure sample contamination and degradation. Subsequently, the DNA was quantified, evaluated for purity and quality and maintained at a temperature of -20˚C. The genotypic analysis of the strains was based on the genetic amplification from the PCR technique (polymerase chain reaction) by using the amplification protocol and the oligonucleotides (Table 1) used for the detection of genes sa442, IcaA, IcaD, mecA, mecC, SEA, SEB, SEC, and SED as described according to Martineau et al. 13 .
As described by Milheiriço et al. 18 Table 2. In the present work, the antimicrobial susceptibility of S. aureus isolated from the cell devices was also evaluated. A high percentage of resistance to Penicillin (85%) was observed. However, low levels of resistance (p<0.05) were found for Levofloxacin (2.5%) and Clindamycin (7.5%) Figure 1. The presence of multiresistant isolates was also evaluated. Approximately 57.5% of the isolates were considered multiresistant since they demonstrated resistance to two or more of the antimicrobials evaluated. It highlighted a particular sample, isolated from a student's cell phone in the nursing course, which presented resistance to 5 antimicrobials (Table 3), presenting a MAR index of 0.83.
The ability of S. aureus isolates to form biofilms was evaluated. Among the isolates, the following were observed: 10% non-adherent, 32.5% weak adherence, 50% moderate adherence, and 7.5% strong adherence ( Figure 2). Despite the presence of microorganisms considered to be strong biofilm formers in the cell phones of students of the pharmacy and nursing students, the presence of microorganisms with weak adherence or non-adherence was also observed, demonstrating the great diversity of the samples obtained (p<0.05). Polysaccharide intercellular adhesion (PIA) is an important aspect of biofilm production and is encoded by the chromosomal intercellular adhesion (ica) locus, consisting of the icaADBC. Among them, the icaA and icaD genes have been reported to play a significant role in biofilm production 25 . All samples that demonstrated a positive biofilm adhesion profile presented one of the analyzed genes. The strains that presented two genes simultaneously (53%) were classified with moderate to strong adherence, in this way, it is possible to correlate that the two genes together play a significant role in the formation of biofilm. A high percentage of non-adherent isolates with single ica locus genes was observed, generating great concern since these genes can be expressed under the effect of a stimulus and thus highlight the need for its expression by quantitative severe morbidity and mortality worldwide. MRSA strains are endemic in many countries and may be present in more than 50% of clinical isolates 29 . The homolog of the mecA gene, called the mecC gene, is also located in the SCCmec and produces a similar phenotypic profile of antimicrobial resistance 29 . In the present study, it was possible to observe that 25% of the isolates presented the mecA gene, however none of the samples were positive for the mecC gene. The low number of samples could justify the absence of mecC gene in samples, which was a major limitation in our study.
According to the combination of ccr allotypes with the mec gene complex, 11 types (I-XI) of SCCmec have already been reported 29 . The results demonstrated the presence of SCCmecI, SCCmecIII. SCCmec I is the chromosomal cassette that carries no transposons or plasmid that confer resistance to drugs other than methicillin and heavy metals. SCCmec III carries genes like mecA and mecRI, together with transposons and plasmids that form resistance to metals such as cadmium and mercury and also to antimicrobials such as tetracycline and oxacillin, being considered the oldest truly pandemic MRSA strain 30 .
In the present work, staphylococcal enterotoxins (SEs) also investigated, since enterotoxigenic S. aureus is considered the second most prevalent pathogen in foodborne diseases in Brazil 31 . The most prevalent gene was the SEA toxin, commonly related to cases of intoxication, corresponding to 75% of outbreaks, and also correlated with human food contamination 32 .