Abstract
This study aimed to identify the risk factors for wounds and burns patients in Iraq hospitals by antibiotic resistance of Staphylococcus aureus, and relate that virulence to their associated with PVL gene. 200 clinical samples were taken from patients with burns and wound infections at Al-Yarmuk General Education and Baghdad Medical City Hospital. As a result of their examination, S. aureus was determined in 103 samples. While 50 samples were tested biochemically, the remaining 53 were identified using the VITEK2 system. Four antibiotics were used for antimicrobial susceptibility testing in the VITEK2 system. It was determined that the examined isolates were resistant to Cefoxitin (100 %), Benzylpenicillin (96.2 %), Oxacillin (94.3 %) and sensitive to Gentamicin (96.2 %), and all isolates were Methicillin-Resistant. Conventional PCR was performed for the molecular identification of S. aureus in 53 isolates for the detection of the 16S rRNA gene and the detection of the PVL virulence gene. As a result, 16S rRNA gene (100 %) and PVL gene (35.8 %) were visualized on agarose gel in tested isolates. Then, sequencing was applied to 12 bacterial isolates for the 16SrRNA gene and 5 bacterial isolates for the PVL gene, which gave positive results for two genes, and the alignment in the NCBI showed identities of 16SrRNA gene 99% and PVL gene 99% with global isolates of Staphylococcus aureus. Real-time PCR was applied for the detection of PVL genes in 53 isolates. The prevalence of the PVL gene was 41.5%. The fold gene expression of the PVL gene was 2.1 and the 16SrRNA was 0.8. In this study, it was concluded that the PVL virulence gene, which has a role in leukocyte lysis and tissue necrosis, has a higher prevalence in Methicillin Resistant Staphylococcus aureus clinical isolates compared to previous studies and increases the virulence and pathogenicity of MRSA, as these MRSA had high multidrug resistance to the tested antibiotics.
Keywords: Staphylococcus aureus, PVL gene, 16S rRNA, MRSA

Introduction

Staphylococcus aureus are Gram-positive cocci about 0.5-1.5μm that looks as a clusters of grapes under light microscopic examination (Baker and Acharya et al., 2004). Staphylococcus aureus are non-motile and do not produce spores. A few of them create capsules or pseudocapsules, making them much more virulent than unencapsulated strains, facultative anaerobes which reproduce through aerobic respiration or fermentation. It is an opportunistic pathogen (Kluytmans et al.,1997), and one of the most dangerous bacterial infections in nosocomial and community environment (Valaperta et al. ,2010). It can develop a great range of diseases, including folliculitis, impetigo (Dhawan et al., 2015), it can produce abscesses and spread through the blood stream (bacteraemia) (Fowler et al., 2006), damage heart valves (endocarditis) (Fernández Guerrero et al. 2009) pneumonia (Gillet et al., 2002), bones (osteomyelitis) (Elasri et al,. 2002 (, urinary tract infections and it can cause meningitis in extremely rare conditions (Sibbald et al., 2006). The wide variety of diseases caused by Staphylococcus aureus is due to variety of virulence factors that allow it to adhere to surfaces, penetrate or escape the immune system, and cause detrimental toxic consequences to the host)Holmes et al., 2005; Gordon and Lowy, 2008(.Compared to infections with methicillin-susceptible S. aureus, methicillin-resistant S. aureus (MRSA) infections are related to a greater death rate (Cosgrove et al. ,2003). Also it is the most prevalent cause of nosocomial infections. Multi-drug resistance of this bacteria is common in MRSA strains, and also it is the major cause of therapeutic failure and cost increases (Köck et al.,2010.(One of the main virulence factors in S.aureus is Panton-Valentine leukocidin PVL. Its a bicomponent cytolysin dependent on two produced proteins (LukF-PV and LukS-PV) that enter into the host’s cytoplasmic membrane and hetero-oligomerize to create a pore (Kaneko and Kamio, 2004). It has a high affinity for leukocytes which leads to serious necrotizing pneumonia and infectious skin infections (Foster, 2005). Also it causes calcium channel activation and gene transcription alterations (O‟Hara et al. 2008). According to the researches, luk-pv is found in 77 % of community-acquired MRSA isolates but just 4 % of hospital-acquired MRSA isolates (Yoong and Torres, 2013). PVL has gotten a lot of interest since it’s been suggested that it’s a significant S. aureus toxin and an epidemiological biomarker of acute S. aureus infections in various research (Vandenesch et al., 2003).

Aim of the study: The current study aims to identify the risk factors for wounds and burns patients in Iraq hospitals by antibiotic resistance of Staphylococcus aureus, and relate that virulence to their associated with PVL gene.

Material and methods

Collection of samples

It was collected from Two hundred samples from  patients in Al Yarmouk Hospital and Baghdad Medical City (Baghdad,Iraq), only 103 isolates diagnosed as bacteria  S. aureus, samples were included burn swab  )53 samples), and wound swabs( 50 samples(

Laboratory Staphylococcus aureus identification

Diagnostic examination depends on morphological features of bacteria growth in mannitol salt agar (MSA), blood agar, and nutrient agar, including the colony size, shape, color, pigments, odor, and edges. Then the VITEK 2 IDGP card (Reference number 21342), and AST-GP76 Antimicrobial susceptibility card (Reference Number 418424) were used to identify bacteria and check resistance to antibiotics .

Genetic detection

Extraction  and purification of DNA by Kit Genomic DNA bacteria were extracted by using Quick-DNA Bacterial Miniprep extraction kit. A primer was prepared from lyophilized primer after dissolved in nuclease-free water to make a stock solution with a concentration of 100 μM for each primer and store at -20°C. A working solution )10 μM) prepared via diluting )10μL) of stock solution in 90 μL of nuclease-free water and store at -20°C until use. PCR applied for the investigation of two genes for S. aureus which are 16s RNA ,and PVL genes .PCR reaction mixture consist of ) 1.5 μl DNA sample, 1 μl for each forward and reverse primers, 5μl of Maxime PCR PreMix and 16.5 μl of nuclease-free water to reach 25 μl as a total volume, then the PCR mixture was transported to a thermal cycler to start the reaction depending on optimum detection condition for each gene (1 and 2). Agarose gel electrophoresis was applied to detect PCR products for genes.

Table 1 :Optimum detection condition of 16S rRNA

Phase	Tm (ᵒC)	Time	No. of cycle
Initial Denaturation	94ᵒC	3 min.	1 cycle
Denaturation 2	94ᵒC	45sec	35 cycle
Annealing	56ᵒC	45sec
Extension1	72ᵒC	1min
Extension 2	72ᵒC	7 min.	1 cycle

Table 2 :Optimum detection condition of PVL gene

Phase	Tm (ᵒC)	Time	No. of cycle
Initial Denaturation	94ᵒC	5 min.	1 cycle
Denaturation 2	94ᵒC	45sec	35 cycle
Annealing	65ᵒC	1min
Extension1	72ᵒC	1min
Extension 2	72ᵒC	5 min.	1cycle

Gene Expression

Genomic RNA was extracted by using Quick-RNA TM Bacterial Miniprep Kit (Zymo, USA) (Catalog No. R2014).cDNA Synthesis using PrimeScript™ RT reagent Kit (Takara, Japan) (Catalog No. RR037A) by reverse transcription of RNA. According to the manufacturer’s instructions, 20μl was the reaction volume of the procedure as shown in table (3(. The reaction mixture incubated under the following condition involving : 37℃ 15 min (Reverse transcription), 85℃ 5 sec (Inactivation of reverse transcriptase with heat treatment) and then 4℃.

Tables 3:The reaction components and volume for cDNA Synthesis

Reagents	Volumes
5 × PrimeScriptTM mix	2 μl
PrimeScript RT Enzyme Mix I	0.5 μl
total RNA	500ng to 8 μ l
RNase Free dH2O	up to 10 μ l

qRT-PCR was carried out  by Real-time PCR (Sacace, Italy). The fold of gene expression were measuring the threshold cycle (Ct) employing KAPA SYBR FAST qPCR Master Mix (2X) Universal Kits components (Catalog No: KK4601).The technique was applied to determine expression gene of S. aureus genes by using the commercial kit of KAPA SYBR FAST qPCR Master Mix (2X) then depended on the amplification gene sequences 16s RNA, PVL.The condition of qRT PCR and reaction component of gene expression shown in Tables (4 and 5) respectively .

Table 4: Stages and temperature of qRT PCR

Component	Volume
Template DNA Sample Volume	3 µL
Forward primer	0.4 μL
Reverse primer	0.4μL
KAPA SYBR FAST qPCR Master Mix (2X) Universal	10 μL
Nuclease-free water	Up to 20 µL
Total volume	3µL

 

Table 5 :Reaction Components and volume for gene expression

Step	Temp(°C)	Time	Cycles
Enzyme activation	95 °C	5 min	Hold
Denaturation	95°C	20 second	40
Annealing	60 °C	20 second
Extension	72°C	20 second

Results and discussion

Isolation and Identification of Staphylococcus aureus

In this study, 200 different clinical samples were taken from the analysis laboratory in the city of Baghdad (Al-Yarmouk Teaching Hospital) and the analysis laboratory (Baghdad Medical City). 103 isolates of Staphylococcus aureus were identified from various clinical samples. These clinical isolates were obtained from two sources: burns (53 isolates) and wounds (50 isolates) of different ages for both genders.

All isolates were cultured on Mannitol Salt agar MSA, which is a selective and differential medium for Staphylococcus aureus as it can grow with its high salt concentration and produce yellow colonies due to its ability to ferment mannitol (Figure 1) (Moura et al., 2018). Then all isolates were cultured on blood agar and showed beta-hemolytic (Figure 2) (Thangavel et al. , 2011). The results of the cultural tests are mentioned in (Table 6).

Table 6:  The positive results of cultural tests for Staphylococcus aureus isolates

Type    of isolates	Mannitol Salt agar	Blood agar	Nutrient agar	Total
Wounds	50/50	50/50	50/50	50
Burns	53/53	53/53	53/53	53

Figure 1: Staphylococcus aureus growth on Mannitol salt agar

Figure 2: Staphylococcus aureus growth on blood agar

Microscopic examination

A microscopic test was performed for S. aureus isolates and the Gram stain indicated that Gram-positive cocci arranged in grape-like irregular clusters under a compound light microscope at 40X and 100X shown in (Figure 3) (Brooks et al., 2007).

                                    Figure 3: S. aureus identification by Gram stain (10 µm)

Identification results

 VITEK 2 GP  were used to identify S.aureus isolates.Whereas the catalase reaction gave a positive result for all 50 isolates from burns and wounds, where the positive results occurred by the formation of bubbles caused through catalyzed the degradation of H₂O₂ to oxygen ,and water which indicates the presence of catalase enzyme that distinguish these isolates from Streptococcus spp. (Al-Ugaili, 2013). The VITEK 2 GP card is based on 43 biochemical measuring tests carbon source utilization, inhibition and resistance, and enzymatic activities (figure 4) (Crowley et al., 2012). This system is designed to perform 43 standard biochemical tests for a single colony of pure S. aureus isolate. The results were positive for all 53 isolates with 100% for S. aureus

Figure 4:Identification of S. aureus isolates in VITEK 2 GP system

Antimicrobial Susceptibility Test for S. aureus Isolates

Four types of antibiotics were used to test antimicrobial susceptibility for S. aureus 53 isolates by VITEK System, table (7)and figure (5) shown the results of sensitivity and resistantce for S. aureus against four types of antibiotics.

According to the results, most of these isolates were resistant to Cefoxitin (100%), Benzylpenicillin (96.2%), Oxacillin (94.3%) and sensitive to Gentamicin (96.2%). The cefoxitin resistance test was used to determine the Methicillin-Resistant Staphylococcus aureus (MRSA) isolates. Cefoxitin resistance was detected only in MRSA with a mecA- mediated resistance mechanism (Swenson et al., 2007). All 53 S.aureus isolates in this study were Methicillin-Resistant Staphylococcus aureus (MRSA). A study found 64% of S. aureus isolates were resistant to cefoxitin and MRSA (Anand et al., 2009). Another study found 54% of S. aureus isolates were resistant to cefoxitin and MRSA (Lakshmi Priya ,2009).

Table 7:Antimicrobial susceptibility test by VITEK System results

Antibiotics	Number of bacterial isolates and their Percentage
	R	S
Cefoxitin	53(100%)	0(0%)
Benzylpenicillin	51(96.2%)	2(3.7%)
Oxacillin	50(94.3%)	3(5.6%)
Gentamicin	2(3.7%)	51(96.2%)

Figure 5: the number isolates of resistance and sensitivity to the tested antibiotics

 

Molecular Diagnosis of S. aureus

Polymerase Chain Reaction (PCR) For identification of S.aureus isolates, and detection 16S rRNA and pvl genes

1. aureus Conventional PCR were applied for S.aureus genome for 53 MRSA isolates for identification, and detection the presence of genes by using specific primers. By using gel electrophoresis, the products of PCR were detected as shown in Figures (6,7).

For the identification of S.aureus isolates, the 16S rRNA gene was used. This gene is frequently used to identify bacterial isolates because it is universally present among bacteria, is highly conserved, and can be rapidly amplified using primers (Nguyen et al., 2016).

All 53 isolate gave the positive outcomes for 16S rRNA gene which applied primer with size 750 bp, in this study the presence of gene in target isolate is 100% Figure 6 and Figure 7. The result of PCR agreed with Ali et al., who found the same result for the 16S rRNA gene in 100% of all tested isolates. The PCR is a specific and successful approach for classifying and identifying S. aureus isolates (Ali et al., 2014).

Figure 6: Electrophoresis (1% agarose/ 100Vol , 60 min) of PCR products for S.aureus 16S rRNA gene (750 bp) 1-25 : show positive results: ladder 1000 bp.

Figure 7: Electrophoresis in 1% agarose, 100Vol , 60 min of PCR products for S.aureus 16S rRNA gene (750 bp) 26-53 : show positive results: ladder 1000 bp.

 

Only 19 isolate gave the positive outcomes for the PVL gene that applied the primer with size (400 bp(. In this study, presence of the gene in target isolate was 35.8%(Figures 8). In a study, it was found that the PVL-gene- producing isolates accounted for 17.9% of all tested nosocomial isolates (Elgendy et al., 2016), which is half the percentage in this study.

 

 

 

 

Figure 8: Electrophoresis in 1% agarose, 100Vol , 60 min of PCR products for S.aureus PVL gene (400 bp), show positive results, ladder 1000 bp

 

Sequencing Results

 16s rRNA gene

Sequencing was conducted on 12 bacteria isolate of 16s rRNA gene, and the alignment by NCBI appeared identities 99% with a global isolates of S. aureus as shown in table 8 and figures 9-20.

Table 8:Analysis of alignment and locations type variations for 16S ribosomal RNA gene

No.	Type of substitution	Location	Nucleotide	Sequence ID with compare	Source	Identities
1	Transition	348	A\G	ID: MK000713.1	S. aureus	% 99
	Transition	447	T\C
	Transvertion	506	G\C
	Transition	539	A\G
2	Transvertion	248	T\G	ID: MW453043.1	S. aureus	% 99
	Transition	460	T\C
	Transvertion	479	A\T
	Transition	504	G\A
	Transvertion	545	T\G
	Transvertion	567	G\C
	Transvertion	608	G\C
3	Transition	446	T\C	ID: MK000713.1	S. aureus	% 99
4	Transition	244	A\G	ID: MK000713.1	S. aureus	% 99
	Transition	280	C\T
	Transvertion	325	AC
	Transvertion	326	G\T
	Transvertion	397	AC
	Transvertion	409	T\G
	Transition	446	T\C
	Transvertion	452	T\G
	Transvertion	539	AC
	Transvertion	551	G\T
	Transition	579	C\T
	Transvertion	588	A\T
	Transvertion	594	G\C
	Transvertion	602	T\A
5	Transition	446	T\C	ID: MK000713.1	S. aureus	% 99
	Transvertion	553	G\C
	Transition	592	T\C
6	Transition	254	A\G	ID: KP728240.1	S. aureus	% 99
	Transition	456	T\C
7	Transition	452	T\C	ID: KY971290.1	S. aureus	% 99
	Transvertion	787	AC
8	Transvertion	140	CA	ID: KY971290.1	S. aureus	% 99
	Transvertion	264	G\C
9	Transvertion	152	A\T	ID: KY971290.1	S. aureus	% 99
	Transition	205	C\T
	Transition	214	T\C
10	Transvertion	152	A\T	ID: KY971290.1	S. aureus	% 99
	Transition	214	T\C
	Transition	256	A\G
	Transvertion	654	T\G
11	Transvertion	264	G\C	ID: KY971290.1	S. aureus	% 99
	Transvertion	427	AC
	Transvertion	438	AC

Figure 9:Alignment results for isolate number 1 of 16s rRNA gene with global isolate under ID: MK000713.1

Figure 10: Alignment results for isolate number 2 of 16s rRNA gene with global isolate under ID: MW453043.1

Figure 11:Alignment results for isolate number 3 of 16s rRNA gene with global isolate under ID: MK000713.1

Figure 12: Alignment results for isolate number 4 of 16s rRNA gene with global isolate under ID:MK000713.1

 

Figure 13: Alignment results for isolate number 5 of 16s rRNA gene with global is

olate under ID: MK000713.1

Figure 14: Alignment results for isolate number 6 of 16s rRNA gene with global isolate under ID: KP728240.1

Figure 15: Alignment results for isolate number 7 of 16s rRNA gene with global isolate under ID: KY971290.1

 

Figure 16:Alignment results for isolate number 8 of 16s rRNA gene with global isolate under ID:KY971290.1

Figure 17: Alignment results for isolate number 9 of 16s rRNA gene with global isolate under ID: KY971290.

Figure 18: 16s rRNA geninin 10 numaralı izolat için hizalama sonuçları:ID KY971290.1

Figure 19:Alignment results for isolate number 11 of 16s rRNA gene with global isolate under ID: KY971290.1

Figure 20: Alignment results for isolate number 5 of 16s rRNA gene with global isolate under ID: KY971290.1

PVL gene

Sequencing was conducted on 5 isolates for the PVL gene and alignment by NCBI appeared identities of 99% with global isolate of S. aureus as shown in Table 9 and figures 21- 25.

Table 9:Analysis of alignment and locations type variations for PVL gene

No.	Substitution type	Location	Nucleotide	Sequence ID with compare	Source	Identifies
1	Transition	1924352	T\C	ID: AP023034.1	S. aureus	99%
	Transition	1886031	T\C	ID:CP038229.1	S. aureus	99%
2	Transversion	1924151	T\C
	Transition	1924352	T\C	ID: AP023034.1	S. aureus	99%
3	Transversion	1924254	T\C
	Transition	1924321	T\A	ID:AP023034.1	S. aureus	99%
4	Transversion	1924352	T\C
	Transversion	1885788	G/C	ID:CP038229.1	S. aureus	99%
5	Transition	1885999	A\T
		1886031	T\C

 

 

 

 

Figure 21:Alignment results for isolate number 1 of PVL gene with global isolate under ID: AP023034.1

Figure22: Alignment results for isolate number 2 of PVL gene with global isolate under ID: CP038229.1

Figure 23: Alignment results for isolate number 3 of PVL gene with global isolate under ID: AP023034.1

Figure 24: Alignment results for isolate number 4 of PVL gene with global isolate under ID: AP023034.1

Figure 25:Alignment results for isolate number 5 of PVL gene with global isolate under ID: CP038229.1

Estimation of PVL Gene Expression

The gene expression of PVL gene performed for 53 isolates but only 22 isolates gave positive results for gene expression and housekeeping gene was 16s rRNA gene. The mean of PVL gene expression are shown in table (10).

The amplification was measured as a Ct value using syber green, a fluorescent dye that detects any double-stranded DNA, including cDNA (cycle threshold). A lower Ct value indicates the presence of more target copies, and vice versa, in terms of gene expression, high Ct values signify low gene expression (Schmittgen and Livak, 2008).

The mean±SD of Ct value for 16s rRNA (Housekeeping gene) in S. aureus was (22.39±3.11), for 22 isolates. The gene 16s rRNA was used to the data normalize in the study. To accomplish, a 2ˆ-Ct ratio in samples and control was used (Schmittgen and Livak ,2008), as shown in table (4.7).

The main purpose of studying gene expression is to find the number of isolates that have PVL virulence gene from 53 isolates and know the expression of the PVL gene in these isolates by comparing with the expression of reference gene 16s rRNA also to know the importance of PVL gene expression in antibiotic resistance.

The gene expression compared to 16s rRNA gene expression for measure the expression gene of the PVL. The measure was conducted for 53 isolates, while the results were used to calculate the quantity of gene expression for the sample. ΔΔCt applied to measure gene expression. ΔΔ Ct obtained by subtracting (Δ Ct sample) from Δ Ct calibrator . Fold change= 2^{-ΔΔ Ct} method ( Livak and Schmittgen ,2008),

PVL gene expression was determined by using RT-qPCR to determine PVL gene in the Staphylococcus aureus. Only 22 isolates (41.5%) had PVL gene, and different expressions were detected in different isolates Figure 4.10, table 4.6 explain Ct values and fold of gene expression for PVL gene and housekeeping gene 16S rRNA.

Figure 26. PVL amplification by qPCR

Figure (26) shows the mean of Ct ±SD 28.17 ± 1.88 and fold gene expression was 2.1 for the PVL gene, while the reference gene had a mean of: Ct 22.39±3.11 and fold gene expression 0.81. This indicates the PVL gene expression increased, as well as there was a highly significant P value < 0.05

Table 10. Ct mean and housekeeping gene expression 16srRNA, and PVL for Staphylococcus aureus

genes	Mean Ct ±SD	ΔCt	ΔΔCt	Fold =(2ˆ-∆∆Ct)	Fold =2ˆ-CT
H.K.G 16srRNA	22.39 ±3.11				0.817
PVL gene	28.17 ± 1.88	5.82	-1.076	2.108	2.1

        P≤0.05 = highly significant while P > 0.05 non-significant

The 16S rRNA gene is widely used as an internal standard in quantitative research to analyze other gene expression (Vaudaux et al., 2002, Yarwood et al., 2002). housekeeping gene applied in genetic study  based on assumption that expression remains stable in cells or tissues being studied (Rebouças, 2013). Reference genes were used to reduce sample-to-sample variation. Selecting stably express reference gene was crucial, as a variations in reference genes expression could result in false positive (Dheda. et al., 2005).

The current study supports the use 16s rRNA as the reference gene of S. aureus gene expression. The gene expression fold change was calculated using relative quantification (RQ) from the delta-delta Ct value.

Methicillin-Resistant Staphylococcus aureus (MRSA) isolates pose a ٥ problem among pathogenic agents that able to cause severe infections, and when produce PVL gene become more virulence and can cause serious necrotic pneumonia (Motamedi et al., 2015), it may outcome in epidemics impacting vulnerable groups such as neonates and critical care units. (Amin et al., 2020). Examination for virulence features in patient-derived isolates is thus critical for prompt detection of individuals harboring multi-drug resistant MDR and dangerous bacterial strains, allowing for isolation in hospitals (Amin et al., 2020).

The existence of PVL gene is a genetic marker for the MRSA communities (Motamedi et al. 2015), also in a Nigerian study all PVL-producing isolates were multi-drug resistant MDR (Adebola et al. 2021).

The prevalence of PVL gene in current study by real time PCR was (41.5%) 22 from 53 MRSA isolates. This result is similar to study found thirteen out of thirty-two MRSA isolates (40.62 %) tested positive for the PVL gene from hospital isolates. Also PVL was shown to be more common in kids (<14 years old) than in adults and patients in the older age groups (Rostamzad and Rostamneia 2016). While other studies show different prevalence of PVL in MRSA isolates, it was 16% only 4 from 25 isolates ( Galia et al. 2019), 27.7% 13 from 47 (Amin et al. 2020). Whereas PVL prevalence in MRSA strains from hospitals in Turkey has been observed to range among 1.7% and 20% (Akočlu et al.,2010, Oksuz et al., 2013, Kılıç et al ,2015, Duman ve Sevimli, 2018).

A  study in England and Wales found an increasing trend in clinical samples of PVL-MRSA as part of an enhanced surveillance program (Ellington et al., 2010), and evidence of a PVL-positive MRSA epidemic at a regional neonatal unit in the UK in 2012 (Ali et al., 2012). In 2016, the total PVL positive rate in MRSA isolates was 7.8%, according to a large-scale examination of pediatric patients with invasive S. aureus infections obtained from 13 hospitals in 7 European countries (Gijón et al., 2016).

According to sources, the prevalence of the pvl within MRSA isolates at a Japanese hospital in 2019 reached 13.5 % (Funaki et al. 2019), while PVL-positivity was 39% within MRSA isolates obtained from pediatric patients in China the same year (Wang et al., 2019). Another research identified MRSA isolates from blood cultures in South Africa from 2013 to 2016, reporting a pvl-positive rate of 25% (Singh-Moodley et al., 2019).

The result of gene expression in this study was consistent with a previous study that found high expression of the PVL gene in an S. aureus strain where the observed community had inadequate health education and often used beta-lactam antibiotics. This might be the underlying cause of increased PVL gene expression (Karmakar et al., 2018), and also the high expression of the PVL gene found in another study in hospital strains of S. aureus (Darboe et al. ,2019). In a Brazilian study, the high expression of PVL in association with other virulence factors may increase the fitness of this lineage, affecting its pathogenicity and permitting entry and establishment in Brazilian hospitals. (Chamon et al., 2020).

Conclusions

The detection of the 16S rRNA gene, for molecular identification of Staphylococcus aureus isolates, showed the presence of the 16S rRNA gene in all tested isolates , as well as PVL showed the presence of this gene in only 19 isolates by PCR.

PVL gene expression was found to be high in tested isolates when compared to the reference gene16S rRNA. The use of real-time PCR to determine the gene expression of the PVL gene as a virulence factor can represent a powerful molecular tool to study the relationship between this gene and antibiotic resistance in S.aureus. The results of real-time PCR are more accurate than those of conventional PCR and detect the PVL gene in 22 S.aureus isolates.

Acknowledgment

None .

 

Conflicts of Interest

None.

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