Investigation of SCCmec Types using the Real Time PCR Method in Cefoxitin-Resistant Staphylococcus aureus Isolates

doi:10.21203/rs.3.rs-2112960/v1

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Research Article

Investigation of SCCmec Types using the Real Time PCR Method in Cefoxitin-Resistant Staphylococcus aureus Isolates

https://doi.org/10.21203/rs.3.rs-2112960/v1

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published 30 Jun, 2024

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Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that can cause many community and hospital-acquired infections. This study was conducted to investigate the SCCmec gene types responsible for methicillin resistance in MRSA isolates isolated from hospitalised patients.

Methods: MRSA isolates isolated from samples sent from various clinics to Gaziantep University Şahinbey Research and Application Hospital Microbiology Laboratory between March 2021 and January 2022 were included in the study. Bacteria were identified using conventional methods and the VITEK 2 automated system. Cefoxitin (FOX) resistance was determined by the disk diffusion method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. FOX resistance was confirmed by the (Penicillin Binding Protein 2' (PBP2') latex agglutination test. Types of mecA, mecC, coa, nuc, Panton Valentin Leukocidin (PVL), ccrC2, class A mec, SCCmec (I, II, III, IVa, IVb, IVc, IVd, IVg, IVh, IVi, IVj, V, VI, VII, VIII, IX, X, XI, XII and XIII) in isolates detected as MRSA were investigated by real time PCR.

Results: In this study 116 isolates meeting the study criteria were examined. By detecting the nuc and coa genes in all isolates by PCR, the phenotypic identification of S.aureuswas confirmed. While the mecA gene was detected in all MRSA isolates, no mecC gene was detected in any isolates. SCCmec Type I was detected in three isolates (2.6%), Type II in 33 (28.4%), Type III in 15 (12.9%), Type IVa in 13 (11.2%) 3.4) Type IVb, four (3.4%) Type IVc, 14 (12.1%) Type IVg, one (0.9%) Type V, five (4.3%) Type VII, Type VIII in 21 (18.1%), Type IX in one (0.9%), and Type XII was detected in two (1.7%) isolates. On the other hand, SCCmec Type VI, Type X, Type XI and type XIII were not found in any isolate. It was determined that four of the MRSA isolates (3.4%) carried the PVL gene.

Conclusions: Monitoring of FOX resistance is an effective and safety method in determination of MRSA isolates. The change in mec gene cause resistance should be monitored regularly with molecular methods. Our study is the first study in Turkey in this context.

S.aureus

MRSA

SCCmec

mecA

mecC

PVL

nuc

coa

PBP2'

Real Time PCR

Staphylococcus aureus is a pathogen that causes a wide variety of infections, from skin soft tissue infections to bacteraemia and endocarditis. Methicillin resistance is an important resistance mechanism that limits treatment options in S.aureus isolates. Methicillin-resistant S.aureus (MRSA) strains have higher mortality and morbidity than methicillin-susceptible S.aureus (MSSA) strains. In addition, the cost of treatment is significantly higher [1].

Methicillin, a semi-synthetic penicillin, was first used in the treatment of infections caused by β-lactamase produced by staphylococci in 1960 [2]. However, methicillin resistance was reported in infections caused only one year later [3]. Since then, methicillin-resistant S.aureus infections have become widespread worldwide [2]. MRSA isolates are isolates that are phenotypically resistant to β-lactam antibiotics such as methicillin, cefoxitin, and oxacillin [4]. Treatment of systemic MRSA infections requires treatment with drugs such as vancomycin, daptomycin and linezolid [5].

Resistance usually arises with the acquisition of the SCCmec (Staphylococcal Cassette Chromosome mec) element carrying the 2.1 kb mecA gene [6–8]. SCCmec is a group of mobile genetic elements integrated into the chromosome of MRSA [6]. According to the International Staphylococcal Cassette Chromosome Working Group (IWG-SCC), strains are classified as SCCmec type I-XIV based on the combination of the mec and ccr gene complexes [9,10]. PVL can also be found on SCCmec. This exotoxin, which is cytotoxic to leukocytes, is an important virulence factor of bacteria.

Although MRSA has been reported as a hospital-acquired (HA-MRSA) pathogen worldwide, community-acquired infections caused by methicillin-resistant S.aureus (CA-MRSA) strains have emerged in the last two decades [11]. While SCCmec types I, II and III are seen in HA-MRSA, other SCCmec types are common in CA-MRSA strains [12]. PVL is more common in CA-MRSA.

This study was carried out to determine the SCCmec types in S.aureus strains isolated from various clinical specimens of hospitalised patients, and to investigate the presence of PVL in these strains.

Aim

The aim of this study is to determine the SCCmec types in cefoxitin-resistant S.aureus strains.

Design And Setting Of The Study

Research Samples

Our study included 116 strains defined as cefoxitin-resistant S.aureus, which were sent from various clinics to Gaziantep University Şahinbey Research and Application Hospital Microbiology laboratory between March 2021 and January 2022. The first isolate in recurrent growths was included in the study. The hospital where the study was conducted is a tertiary hospital with 1100 beds located in the southeast of Turkey. Methicillin-susceptible S.aureus (MSSA) ATTC 29213 and methicillin-resistant S.aureus (MRSA) ATCC 43300 isolate were used as control strains in the study.

Identification of bacteria

Bacteria were identified using conventional methods (catalase, Gram stain, coagulase) and the VITEK 2 Compact (bioMérieux, USA) automated identification system. Methicillin resistance was determined by the disc diffusion method with cefoxitin (FOX) disc (30 µg) according to EUCAST standards. Antibiotic susceptibility tests were performed with VITEK 2. PBP2' latex agglutination (Oxoid, Japan) test was performed on all isolates.

DNA Isolation

DNA isolation was performed automatically by selecting the bacterial isolation protocol on the QIAsymphony SP/AS (Qiagen, Hilden Germany) device.

PCR

To determine the coa, nuc, PVL, SCCmec types (type I, type II, type III, type IVa, type IVb, type IVc, type IVd, type IVg, type IVh, type IVi, type IVj, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII) and genes (mecA, mecC, ccrC2, class A mec) located on SCCmec, 28 different primer pairs were used (Table 1). The PCR protocol was optimised under laboratory conditions. Ten µL of RT2 SYBR® Green qPCR Mastermix (Qiagen, Maryland, USA), 1 µL of 10 pmol of forward and reverse primer pairs (Macrogen, Seoul, Korea), 6.5 µL of DNase-RNase free H₂O and 6.5 µL of isolation product mix were used. PCR protocols were classified for amplification in Rotor Gene-Q (Qiagen, Hilden, Germany) by optimising the coupling temperatures (Table 2).

Table 1

Used primer sequeces
	Primer Name	5’ − 3’ Sequence Primer Sequence	Bp	Ref.
SCCmec I	Type I-F Type I-R	GCTTTAAAGAGTGTCGTTACAGG GTTCTCTCATAGTATGACGTCC	613	[15]
SCCmec II	Type II-F Type II-R	CGTTGAAGATGATGAAGCG CGAAATCAATGGTTAATGGACC	398	[15]
SCCmec III	Type III-F Type III-R	CCATATTGTGTACGATGCG CCTTAGTTGTCGTAACAGATCG	280	[15]
SCCmec IV	Type IVa-F Type IVa-R	GCCTTATTCGAAGAAACCG CTACTCTTCTGAAAAGCGTCG	776	[15]
	Type IVb-F Type IVb-R	TCTGGAATTACTTCAGCTGC AAACAATATTGCTCTCCCTC	493	[15]
	Type IVc-F Type IVc-R	ACAATATTTGTATTATCGGAGAGC TTGGTATGAGGTATTGCTGG	200	[15]
	Type IVd-F Type IVd-R	CTCAAAATACGGACCCCAATACA TGCTCCAGTAATTGCTAAAG	881	[15]
	Type IVg-F Type IVg-R	GCAAGCTGTTATCGGCATTT GATCGTTCGTGTTTGTGTGC	378	[15]
	Type IVh-F Type IVh-R	TTCCTCGTTTTTTCTGAACG CAAACACTGATATTGTGTCG	664	[15]
	Type IVi-F (CB18F1) Type IVi-R (CB18R3)	CCAAGAAATTAATGTCGTCG AGGCTTCAACGATTTGAGAAC	1099	[12]
	Type IVj-F (C18F1) Type IVj-R (C18R2)	ATCTGTTGACTTTGTCAACC CGCTCTTAATTGAATTCTTCC	331	[12]
SCCmec V	Type V-F Type V-R	GAACATTGTTACTTAAATGAGCG TGAAAGTTGTACCCTTGACACC	325	[15]
SCCmec VI	mecI-F mecI-R	CGTTATAAGTGTACGAATGGTTTTTG TCATCTGCAGAATGGGAAGTT	126	[16]
	ccrB4-F ccrB4-R	CGAAGTATAGACACTGGAGCGATA GCGACTCTCTTGGCGTTTA	134
	IS1272J-F IS1272J-R	GAAGCTTTGGGCGATAAAGA GCACTGTCTCGTTTAGACCAATC	98
SCCmec VII	Type VII-F Type VII-R	GTGACGTTGATATTGCAGTGGT TGAAGAAGTTTGTTCCGCGT	473	[17]
SCCmec VIII	Type VIII-F Type VIII-R	AGCGACGATGAACAACACCGCTACTTACTCAA TTGGTTGAGAATGAGAACAGTGGTAAGATC	138	[17]
SCCmec IX	Type IX-F Type IX-R	TGGCATGGTTGATAGAACAGTG TCACTAATTTTGCCTCACGTCT	642	[17]
SCCmec X	Type X-F Type X-R	ATTTACGCCGATGCGTTGAC TATGCGATTGCGCAGGTGAT	708	[17]
SCCmec XI	Type XI-F Type XI-R	GGCGATACAACGACACATCC TGTTAGTGCTTGACCGCTCTT	255	[17]
SCCmec XII	Type XII-F Type XII-R	AGAAGACGGAGGACATCGACA TCGCTTCTTCAACGCCATCTT	371	[17]
SCCmec XIII	ccrC2-F2 ccrC2-R2	ATAAGTTAAAAGCACGACTCA TTCAATCCTATTTTTCTTTGTG	257	[18]
SCCmec XIII	class A-F class A-R	CCCTTTTTATACAATCTCGTT ATATCATCTGCAGAATGGG	146	[15]
	mecA-F mecA-R	CCTAGTAAAGCTCCGGAA CTAGTCCATTCGGTCCA	331	[19]
	mecC-F mecC-R	ACTAGTATCTCGCCTTGG -ATCCCGAGTGATTATCCC	102	[17]
	coaA-F coaA-R	ATAGAGATGCTGGTACAGG GCTTCCGATTGTTCGATGC	673	[20]
	nuc F nuc R	GCGATTGATGGTGATACGGTT AGCCAAGCCTTGAACGAACTAAAGC	270	[21]
PVL	LukS-F LukF-R	CAG GAG GTA ATG GTT CAT TT GCATCAAGTGTATTGGATAGCAAAAGC	560	[22]

Table 2

PCR Protocol
mecA, mecC, coa				95°C	10 dk	95°C	15 sec	55°C	60 sec
						40 cycler
PVL, nuc				95°C	10 dk	95°C	30 sec	58°C	60 sec
						40 cycler
SCCmec type I-V				95°C	10 dk	95°C	15 sec	54°C	60 sec
						45 cycler
SCCmec type VI-X				95°C	10 dk	95°C	30 sec	58°C	60 sec
						45 cycler
SCCmec type XI-XII				95°C	10 dk	95°C	30 sec	59°C	60 sec
						45 cycler
SCCmec type XIII	ccrC2	50°C	2 dk	95°C	10 dk	95°C	30 sec	59°C	60 sec
						45 cycler
	class A			95°C	10 dk	95°C	30 sec	55°C	60 sec
						40 cycler

Statistical Analysis

The data obtained from the study were evaluated with the SPSS 21.0 package program. In the study, mean, standard deviation (±) and percentile distributions were given as descriptive statistics. Whether there was a difference in the distribution of SCCmec types according to age groups was tested by chi-square analysis. Obtained results were evaluated at the 95% (p < 0.05) significance level.

Of the 116 strains included in the study, 48 (41.4%) were isolated from female and 68 from (58.6%) male patients. The mean age of these patients was 25.70 ± 23.38 in women and 29.13 ± 23.56 in men. The age and gender characteristics of the groups were statistically similar (p > 0.005). The distribution of MRSA isolates according to the clinics in which they were detected is shown in Table 3.

Table 3

Distribution of samples according to clinics
			Type of Sample						Total
			Sputum	Throat	Blood Culture	Tracheal Aspirate	Wound	Other*	Total
Department	Pediatrics	n (%)	11 (21.6)	33 (64.7)	3 (5.9)	1 (2)	1 (2)	2 (3.9)	51 (100)
	Chest Diseases		8 (66.7)	0 (0)	1 (8.3)	0 (0)	0 (0)	3 (25)	12 (100)
	Internal medicine		2 (8.6)	0 (0)	0 (0)	0 (0)	3 (42.9)	2 (28.6)	7 (100)
	Orthopedics		0 (0)	0 (0)	0 (0)	0 (0)	9 (75)	3 (25)	12 (100)
	Intensive Care Unit		0 (0)	0 (0)	1 (25)	3 (75)	0 (0)	0(0)	4 (100)
	Oncology		4 (80)	0 (0)	0 (0)	0 (0)	1 (20)	0 (0)	5 (100)
	Hematology		2 (50)	1 (25)	1 (25)	0 (0)	0 (0)	0 (0)	4 (100)
	Surgical Units		0 (0)	0 (0)	1 (14.3)	0 (0)	5 (71.4)	1 (14.3)	7 (100)
	Other**		2 (14.3)	0 (0)	4 (28.6)	2 (14.3)	5 (35.7)	1 (7.1)	14 (100)
Total			29 (25)	34 (29.3)	11 (9.5)	6 (5.2)	24 (20.7)	12 (10.3)	116 (100)
* Surgical material, BAL, Nose, Urine culture, Peritoneal culture
** Endocrinology, Otorhinolaryngology, Dermatology, Anesthesiology, Psychiatry and Outpatient Clinics

Of the isolates included in the study, 44% (51 isolates) were isolated from the paediatric clinic. It was found that 33 of the isolates identified in the paediatric clinic were isolated from throat swab samples of cystic fibrosis patients.

Antibiotic resistance rates of MRSA isolates are given in Table 4.

Table 4

Antibiotic resistance rates of MRSA isolates
Antibiotic	Resistance n (%)
Cefoxitin	116 (100)
Benzylpenicillin	116 (100)
Gentamicin	10 (8.6)
Ciprofloxacin	20 (7.2)
Levofloxacin	15 (12.9)
Inducible clindamycin resistance	57 (49.1)
Erythromycin	76 (65.5)
Clindamycin	72 (62.1)
Daptomycin	3 (2.6)
Tetracycline	56 (48.3)
Tigecycline	3 (2.6)
Fosfomycin	25 (21.6 )
Fusidic acid	8 (6.9)
Mupirocin	1 (0.9)
Trimethoprim/sulfamethoxazole	6 (5.2)

All isolates were susceptible to linezolid, teicoplanin and vancomycin.

The nuc gene we used to identify S.aureus strains was found to be positive (100%) for all strains. When we investigated the presence of the coa gene by PCR, it was found to be compatible with the coagulase latex agglutination method. The phenotypic tests used for identification of bacteria and determination of cefoxitin resistance were found to be fully compatible with PCR.

All strains and the ATCC 43300 control strain carried the mecA gene, while the ATCC 29213 control strain was negative for the mecA gene. No mecC gene was found in any of the strains in our study.

The SCCmec types detected for all isolates and control strains in the study are given in Table 5).

Table 5

Distribution of PVL in SCCmec types
		Total n (%)	PVL (n)
SCCmec	I	3 (2.6)	-
	II	33 (28.4)	-
	III	15 (12.9)	-
	IVa	13 (11.2)	1
	IVb	4 (3.4)	-
	IVc	4 (3.4)	-
	IVg	14 (12.1)	1
	V	1 (0.9)	-
	VII	5 (4.3)	-
	VIII	21 (18.1)	2
	IX	1 (0.9)	-
	XII	2 (1.7)	-
Total		116 (100)	4

SCCmec Type VI, SCCmec Type X, and SCCmec Type XI were not detected in any isolates. In addition, SCCmec type XIII, which we can determine by looking at the ccrC2 and class A gene regions, was not found in any isolate. SCCmec type IV was found most frequently in 35 isolates, and types V and IX were detected least, in one isolate each (Fig. 1, 2).

The MSSA ATCC 29213 control strain was found to be SCCmec gene-negative, while our MRSA ATCC 43300 control strain was detected as SCCmec type II. The PVL gene was detected in four (3.4%) of the MRSA isolates (Table 6).

Table 6

Characteristics of patients with isolated PVL gene bacteria
	IVa	IVg	VIII
Age	38	26	54	31
Gender	K	K	E	E
Sample	Woung	Wound	Surgical material	Blood
Service	Orthopedics	General Surgery	Orthopedics	Dermatology

The distribution of SCCmec types to the clinics where bacteria were isolated is shown in Table 7.

Table 7

Distribution of SCCmec types according to clinics
		Department									Total
		Pediatrics	Chest Diseases	Internal Medicine	Orthopedics	Intensive Care Units	Oncology	Haematology	Surgical Departments	Other	Total
SCCmec I-XII n (%)	I	0 (0)	1 (8.3)	1 (14.3)	1 (8.3)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	3 (2.6)
	II	10 (19.6)	4 (33.3)	2 (28.6)	3 (25)	2 (50)	2 (40)	2 (50)	2 (28.6)	6 (42.9)	33 (28.4)
	III	7 (13.7)	1 (8.3)	0 (0)	2 (16.7)	0 (0)	1 (20)	0 (0)	2 (28.6)	2 (14.3)	15 (12.9)
	IV	9 (17.6)	0 (0)	0 (0)	3 (25)	0 (0)	0 (0)	0 (0)	1 (14.3)	0 (0)	13 (11.2)
	IVb	2 (3.9)	1 (8.3)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	0 (0)	0 (0)	4 (3.4)
	IVc	2 (3.9)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (14.3)	4 (3.4)
	IVg	9 (17.6)	2 (16.7)	0 (0)	1 (8.3)	0 (0)	1 (20)	0 (0)	1 (14.3)	0 (0)	14 (12.1)
	V	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (7.1)	1 (0.9)
	VII	3 (5.9)	0 (0)	0 (0)	1 (8.3)	0 (0)	1 (20)	0 (0)	0 (0)	0 (0)	5 (4.3)
	VIII	8 (15.7)	3 (25)	3 (42.9)	1 (8.3)	1 (25)	0 (0)	2 (50)	0 (0)	3 (21.4)	21 (18.1)
	IX	0 (0)	0 (0)	1 (14.3)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (0.9)
	XII	1 (2)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (0)	0 (0)	2 (1.7)
Total		51 (100)	12 (100)	7 (100)	12 (100)	4 (100)	5 (100)	4 (100)	7 (100)	14 (100)	116 (100)

Type IV was found to a significantly higher extent in MRSA isolates isolated from the paediatric clinic compared to other clinics (p < 0.05). It was found that Type II, which was detected with the second highest frequency, was also most frequently isolated from the paediatric clinic.

The distribution of SCCmec types according to the age groups is shown in Table 8.

Table 8

Distribution of SCCmec types by age groups
Age	SCCmec									Total	p
Age	I	II	III	IV	V	VII	VIII	IX	XII	Total	p
< 18	0 (0)	10 (18.5)	10 (18.5)	21 (38.9)	0 (0)	4 (7.4)	8 (14.8)	0 (0)	1 (1.9)	54 (100)	0.047
≥ 18	3 (4.8)	23 (37.1)	5 (8.1)	14 (22.6)	1 (1.6)	1 (1.6)	13 (21)	1 (1.6)	1 (1.6)	62 (100)
Total	3 (2.6)	33 (28.4)	15 (12.9)	35 (30.2)	1 (0.9)	5 (4.3)	21 (18.1)	1 (0.9)	2 (1.7)	116 (100)

Of the MRSA isolates, 54 (46.6%) were obtained from paediatric patients, and SCCmec type IV was dominant in 21 (38.9%) of these samples, while SCCmec type II was dominant in 23 (37.1%) of the samples obtained from 62 (53.4%) adult patients (Table 8). It was found that SCCmec type IV occurred to a significantly higher extent in MRSA isolates isolated from paediatric patients, while SCCmec type II occurred to a significantly higher extent in MRSA isolates isolated from adult patients (p < 0.05).

A comparison of the antibiotic resistance profiles of SCCmec types of the samples included in our study is given in Table 9.

Table 9

Antibiotic resistance profiles of SCCmec types (%)
	BP	GEN	CIP	LEV	MLSBi	ERY	CLI	DAP	TET	TGC	FOS	FA	MUP	SXT
I	100	33.3	33.3	33.3	66.7	100	100	0	100	0	33.3	0	0	0
II	100	15.2	30.3	21.2	39.4	51.5	48.5	0	48.5	6.1	18.2	9.1	3	6.1
III	100	0	0	0	73.3	100	86.7	0	15	0	13.3	6.7	0	0
IVa	100	7.7	7.7	0	38.5	53.8	46.2	7.7	38.5	0	15.4	0	0	0
IVb	100	0	25	25	50	75	75	0	75	0	50	0	0	0
IVc	100	0	0	0	50	75	75	0	50	0	25	50	0	0
IVg	100	0	14.3	14.3	35.7	50	50	0	21.4	0	14.3	0	0	14.3
V	100	0	0	0	100	100	100	0	100	0	0	0	0	0
VII	100	0	0	0	100	100	100	0	100	0	20	0	0	0
VIII	100	9.5	19	14.3	47.6	61.9	57.1	4.8	33.3	4.8	23.8	9.5	0	9.5
IX	100	0	0	0	0	0	100	0	0	0	100	0	0	0
XII	100	50	50	50	50	100	100	50	100	0	100	0	0	0
*BP; benzylpenicillin, GEN; gentamicin, CIP; ciprofloxacin, LEV; levofloxacin, MLSBi; inducible clindamycin resistance, ERY; erythromycin, CLI; clindamycin, DAP; daptomycin, TET; tetracycline, TGC; tigecycline, FOS; fosfomycin, FA; fusidic acid, MUP; mupirocin, SXT; trimethoprim/sulfamethoxazole

Type II was found to be resistant against all antibiotics except daptomycin, which was the most common type, and resistance was found in only two antibiotic groups in Type IX.

Methicillin-resistant S.aureus strains cause infections with high morbidity and mortality, especially in hospitalised patients. This is due to the transfer of genes that provide resistance to various antibiotics, including anti-staphylococcal drugs [13]. With the genetic structure that causes resistance to cefoxitin, it has been observed that resistance develops against many antibiotics such as clindamycin, aminoglycosides, fluoroquinolones, macrolides, co-trimoxazole and chloramphenicol [14].

In this study, which was conducted to investigate the SCCmec types, primer sequences obtained from the literature were used [12,15–22].

All of the isolates were found susceptible to linezolid, teicoplanin, and vancomycin, and 65.5% was resistant to erythromycin, and they were similar to the literature [23–26].

In the study, SCCmec type IV, including its subtypes, was found in 35 samples, while SCCmec types V and IX were found in only one sample each. Huang et al. [27] reported that the prevalence of SCCmec type IV was 3–20% among the isolates they obtained in the period 1994–2004, the dominant type was SCCmec Type III, and this rate increased to 43% in 2005, and the dominant type was again SCCmec. Similarly, other studies have reported that SCCmec type IV and its subtypes are dominant compared to other types [11,28–33].

It has been reported that children may be at higher risk for infections caused by isolates carrying SCCmec type IV compared to adults [34]. It was found that 21 (61.8%) of 35 Type IV SCCmec types detected in our study were in the paediatric age group.

In our study, SCCmec type II was the second most dominant type, with 33 samples. This type was found to be significantly more common in the adult age group. Motallebi et al. [35] found 33 (64.7%) of 51 MRSA isolates to be the most common type of SCCmec type II in their study. Takata et al. [36] found SCCmec type II in 47 of 50 MRSA strains and SCCmec type IV in three of them.

In our study, SCCmec types V and IX were the least common types, and both were detected from one sample each. In a similar study conducted in our country, it was reported that Type V was detected in two samples [37].

Baig et al. [38] found a new type of SCCmec from the Danish ST152 strain from a 30-year-old male patient with bacteraemia during a study on the evolution of the CA-MRSA CC152 strain reported from Central Africa and suggested that it be named SCCmec type XIII. For SCCmec type XIII, we tried to determine the type through the ccrC2 and class A mec regions that we designed based on reference sources. In our study, SCCmec type XIII could not be detected because these two primer pairs were not found together in any patient sample.

Within the scope of primers belonging to SCCmec type XIII, the ccrC2 region was found in only two patient samples in our study. The ccrC2 gene region is a common region with SCCmec type XII [39,40]. With the primers we designed for SCCmec type XII, positivity was detected in two patient samples, and we type-verified these patient samples with the ccrC2 primer site.

We found that four of the MRSA isolates in our study carried the PVL gene. Of these patient samples carrying the PVL gene, two carry the mobile genetic element SCCmec type VIII, the others carry SCCmec IV, SCCmec type IVg. Bağsever [41] investigated the PVL gene in 207 patient samples and detected it in five (2.4%) of these samples. They determined that all of the samples that they detected as PVL-positive were also SCCmec type IV. Funaki et al. [11] and Song et al. [42] obtained similar results in their study. The low PVL gene positivity in our study may be due to the isolation of our study isolates from hospitalised patients and the fact that the isolates carrying the PVL gene are isolates that cause more community-acquired infections. In order to prevent the rapid spread of MRSA strains carrying the PVL gene in the hospital environment by gaining resistance to other antibiotics, it has been reported that it will be beneficial both clinically and epidemiologically to monitor the detected strains by conducting surveillance studies [43].

Considering the increase in MRSA frequency and the possibility of developing resistance to vancomycin, rapid and reliable characterisation of isolates and identification of clonal spread in hospitals are important in terms of controlling the infection and preventing an epidemic [44]. This study is one of the few studies in the literature that have investigated all identified SCCmec types. It is thought to be a source for monitoring the change epidemiologically.

Cefoxitin-resistant S.aureus strains are resistant against all beta-lactam antibiotics. However, it has the potential to rapidly develop resistance against other antibiotic classes. Therefore, typing the mec genes that cause this resistance and determining the regions where the antibiotic resistance genes are transferred will guide the treatment.

ATCC: American Type Culture Collection; BAL: Bronchoalveolar Lavage; BP: Benzylpenicillin; CA-MRSA: Community- acquired MRSA; ccrC2: Cassette Chromosome Recombinase C2; CIP: Ciprofloxacin; CLI: Clindamycin; DAP: Daptomycin; ERY: Erythromycin; EUCAST: European Committee on Antimicrobial Susceptibility Testing; FA: Fusidic acid; FOS: Fosfomycin; FOX: Cefoxitin; GEN: Gentamicin; HA-MRSA: hospital-acquired MRSA; IWG-SCC: International Staphylococcal Cassette Chromosome Working Group; LEV: Levofloxacin; MLSBi: Inducible clindamycin resistance; MRSA: Methicillin-resistant Staphylococcus aureus; MSSA: Methicillin-susceptible S.aureus; MUP: Mupirocin; PBP2': Penicillin Binding Protein 2'; PCR: Polymerase Chain Reaction; PVL: Panton Valentin Leukocidin; Ref: Reference; SCCmec: Staphylococcal cassette chromosome mec; Sec: Second; SPSS: Statistical Package for Social Sciences; SXT: Trimethoprim/sulfamethoxazole; TET: Tetracycline; TGC: Tigecycline

Ethics approval and consent to participate

Local ethics committee approval was obtained with protocol number 2021/106 before the study. All the Cefoxitin-resistant S.aureus strains were obtained from microbiology culture stock in our laboratory. In this context voluntary consent form was not required. Ethics approval was taken from the local ethics committee indicating the mentioned situations.

Informed Consent

Not applicable

Competing interests

The authors declare that they have no competing interests.

Authors’ Contributions

MS wrote the draft paper, collected the samples, planned and carried out the experiments, scanned the related literature. IHK and YZ carried out the statistical analyses of the data and literature discussion. All authors read and approved the final manuscript.

Funding

This study was supported by Gaziantep University Scientific Research Projects Management Unit (SRPMU) with project number FEF.DT.21.19.

Acknowledgements

We thank to Gaziantep University Scientific Research Projects Management Unit due to their funding.

Consent for publication

Not applicable

Author Details

¹Gaziantep University Institute of Natural and Applied Sciences, Department of Biology, Gaziantep Turkey, e-mail: [email protected], Phone: +90532 5501033

²Gaziantep University Faculty of Arts and Science, Department of Biology, Gaziantep, Turkey, e-mail: [email protected], Phone: +90505 7069116

³Gaziantep University Faculty of Medicine Department of Medical Microbiology, Gaziantep, Turkey, e-mail: [email protected], Phone: +90532 6760837

* Corresponding Author: Mustafa Sağlam

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Investigation of SCCmec Types using the Real Time PCR Method in Cefoxitin-Resistant Staphylococcus aureus Isolates

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Introduction

Material And Methods

Aim

Design And Setting Of The Study

Research Samples

Identification of bacteria

DNA Isolation

PCR

Statistical Analysis

Results

Discussion

Conclusions

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