The Prevalence of ACME-arcA and PVL Genes Among Staphylococcus Aureus Isolates in a Student Population from North-West of Iran

Objectives: Staphylococcus aureus (S. aureus) is the most prevalent cause of skin infections, especially in colonized individuals. Panton–Valentine leukocidin (PVL) and Arginine catabolic mobile element (ACME) are known as the most common virulence factors of S. aureus. This cross-sectional study was conducted to examine the prevalence of ACME-arcA and PVL genes among S.aureus isolates in the student population. Nasal swab samples were randomly collected from 400 healthy students from Tabriz, Iran. The antibiotic resistance pattern of S.aureus isolates was examined by the disk diffusion method. The presence of ACME-arcA, PVL, and mecA genes was detected by PCR reaction. Results: overall, 15% (60/400) students were nasal carriage of S. aureus and 2.75 % (11/400) were MRSA carriage. The frequency of mecA, ACME-arcA, and PVL genes was 54.54% (36/60), 46.66% (28/60), and 16.66% (10/60) respectively. The prevalence of ACME-arcA and PVL genes was independent of gender (P =0.142, P=0.337, respectively). A notable association was observed between the existence of ACME-arcA gene and the frequency of mecA gene (P <0.05), while the incidence of PVL was independent on mecA. These ndings highlight the necessity of monitoring nasal carriers in a healthy community to prevent subsequent infections.


Introduction
Staphylococcus aureus (S. aureus) is the most prevalent cause of skin infections, especially in colonized individuals [1][2][3]. The S. aureus colonization is not often the leading cause of infection, but may act as the main reservoir of clinical infections in the carriage persons [4]. Based on reports, S.aureus colonization occurres in 30 to 50% of the healthy population [5,6].
At present, the incidence of Methicillin-resistant S. aureus (MRSA) strains has become problematic in the clinical settings. The resistance to methicillin and beta-lactam antibiotics is related to the existence of mecA, a gene encoding low-a nity penicillin -binding protein (PBP2a) [7]. Although, MRSA colonization is considered as the leading cause of subsequent infections, the escalating MRSA is related to the occurrence of factors promoting colonization such as Panton-Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) [8]. In this respect, S.aureus pathogenesis is dependent on virulence factors such as PVL and ACME that facilitate adherence and attachment of pathogen to host cells [9]. ACME was rst identi ed downstream of the cassette chromosome mec (SCCmec) type IVa in MRSA USA300 strain as the ACME-SCCmec composite island [10]. This gene is known as a factor enhancing colonization of S.aureus in the skin and mucous membranes, which act through neutralization of acidic pH and enhancement of the acid tolerance of pathogen. As regards, antimicrobial fatty acids and low pH can protect human skin against bacterial pathogens [11]. Moreover, PVL as a pore -forming toxin plays a crucial role in the occurrence of skin and soft tissue infections in the community-associated (CA-MRSA) stains so that it is known as a diagnostic marker of community-acquired (CA) strains [12].
This study was aimed to examine the frequency of ACME-arcA and PVL genes in the nasal carriage of S.aureus among students.

Bacteria Identi cation
A total of 400 students aged 16 to 17 years from high schools (mean age: 16.5) of Tabriz city e participated in a cross-sectional study from January 1, 2018 to March 1, 2018. The healthy students without previous antibiotic consumption (during the last three months) were included in this study. The nasal swab samples were randomly obtained from students and transferred into tryptic soy broth media and incubated overnight at 37 °C. The S. aureus isolates were recognized via conventional biochemical and microbiological [13].

Primer Designing
The design of speci c primers for ACME-arcA, PVL, and mecA genes were performed on the S. aureus genome sequence available in the Gene Bank database using Gene Runner software. Primer-BLAST was used to con rm the speci city of designed primers. PCR ampli cation for detection of mecA, ACME-arcA and PVL positive genes The bacterial DNA was extracted from isolates by the boiling method through TE buffer (10 mM Tris, 1 mM EDTA). The quality and quantity of DNA were assessed by the ratio of the absorbance at 260 nm and 280 nm wavelength. After that, PCR reaction was carried out to detect mecA, ACME-arcA and PVL genes using speci c primers (Table 1) in a 25-µL reaction for 35 cycles (94 °C for 1 min, 49 °C for 1 min, 72 °C for 1 min) after an initial denaturation at 94 °C for 4 min. The nal extension was carried out at PCR products were analyzed by sequencing. Results:

Antimicrobial Susceptibility
Out of 400 students, 60 (15%) S. aureus strains were isolated that 9.5% (38/400) of the isolates were related to male students and 5.5% (22/400) of the isolates were from female students. Based on statistical analysis results, the prevalence of S.aureus among students was dependent on gender (p = 0.025, p < 0.05) ( Table 2). Also, the highest resistance rate was against penicillin antibiotics (98.33%). Totally, 31.66% (19/60) of the S. aureus isolates were multiple drug resistance (MDR) based on resistance to 3 or more classes of antibiotics and 18.33% (11/60) of the isolates were resistant to methicillin (MRSA nasal carriage) which overall 2.75% (11/400) of the students were MRSA nasal carriage (Fig. 1). Identi cation of mecA, ACME-arcA and PVL positive isolates Based on PCR results, the mecA gene fragment was revealed as a single band of 305 bp in 54.54% (36/60) of the isolates (FigureS1A). ACME-arcA gene was identi ed in 46.66% (28/60) of the isolates as an expected band of 577 bp ( Figure S1B). Also, results indicated that 16.66% (10/60) cases were positive for the PVL gene (FigureS1C), which among them 11.66% (7/60) of the isolates was positive for both PVL and ACME-arcA genes. A signi cant correlation was observed between the presence of the ACME-arcA gene and resistance to methicillin (p < 0.05), while 90% (9/10) of the PVL positive isolates were sensitive to methicillin (p < 0.05). According to statistical analysis, the prevalence rate of ACME-arcA and PVL genes among S.aureus isolates was independent gender (P = 0.142, P = 0.337, respectively) ( Table 2).

Sequencing analysis
For con rmation of the accuracy of PCR, ACME-arcA/PVL positive isolates were sequenced and then analyzed by NCBI BLAST. Based on results, ACME-arcA/PVL positive isolates with 99% identity were related to USA300 strain (Sequence ID: CP027476.1).

Discussion:
In this study, we rst surveyed the MRSA carriage rate (2.75%) in healthy students in North West of Iran, which results were indicating a higher rate of MRSA in student than children (1.3%) in IRAN [15].
The outbreak of MRSA in this study was similar to a study performed in Belgium, which 2.1% of nonhospitalized patients were MRSA carriage (16). Our results were almost identical to the MRSA rate in health care workers, 3.4% (7/204) from Western Nepal [17].
However, the MRSA carriage rate in our region was less than the results obtained from farmworkers (8.7%) in Turkey [18].
In addition to, the high resistance to penicillin and ampicillin, the highest resistance rate was observed to amoxicillin/clavulanic acid (33.84%), cefoxitin (18.40%) and erythromycin (16.44%), respectively. These results were not consistent with a similar study done in Nigeria that 25% of cases were resistant to amoxicillin-clavulanic acid and 23% to erythromycin [19]. The difference observed between resistance rate to cefoxitin (11/60), and frequency of mecA (36/60) was related to the spread of the cefoxitin/oxacillin susceptible mecA positive OS-MRSA isolates in healthy population consistent with previous studies [20,21].
Based on results, the frequency of the MRSA colonization in this study was dependent on gender consistent with research carried out by Humphreys H, in 2015 regarding higher prevalence of MRSA carriage in men [22]. In a similar survey done in 2019, the outbreak of MRSA in males was more than female [23]. In the present study, 31.66% of the isolates were positive MDR, similar to a research done in Kashan, IRAN, with a prevalence of 29.3% MDR [24].
According to our ndings, 46.66% of the isolates were positive for the ACME-arcA gene, and 16.66% % of the cases were positive for the PVL gene. In this respect, 11.66% of the PVL positive isolates were positive for the ACME-arcA gene. These ndings were not consistent with a study done in central IRAN, with a prevalence rate of 17% and 20% for ACME-arcA and PVL genes, respectively [25]. Consistent with the previous researches [26], in this study, there is a signi cant relationship between the presence of ACME-arcA gene and the frequency of mecA positive strains (MRSA). In contrast 85.71% PVL positive isolates were MSSA indicating lack of association between the occurrence of PVL and rate of MRSA.

Conclusion:
The results of this study is indicating the prevalence of ACME positive MRSA strains on a healthy population as the leading cause of skin infections; hence there is an essential need for continuous monitoring of nasal carriers in a healthy community to prevent subsequent infections.

Limitations:
However, there were limitations to our study. First, the period of the study was short. Moreover, samples were only obtained from students of high school. Despite these limitations, the incidence of PVL/ ACME-arcA positive MRSA isolates indicates the necessity of control of MRSA colonization in a healthy population.

Consent for publication
Not applicable.

Figure 1
Antibiotic susceptibility test of the S.aureus isolates in a healthy student population in Tabriz.

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