Phenotypic and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Clones Carrying the Panton-Valentine Leukocidin Genes Disseminating in Iranian Hospitals

Abstract

The spread of Panton-Valentine leukocidin (PVL)-carrying Staphylococcus aureus strains in both hospital and the community is a significant worldwide problem. The aim of the study was to investigate the clonal dissemination pattern of PVL-producing S. aureus strains isolated from hospitalized patients in Tehran, Iran. In this cross-sectional study, 70 PVL-carrying S. aureus strains were recovered from 240 clinical specimens and characterized by antibiotic susceptibility testing, agr typing, SCCmec typing, spa typing, multilocus sequence typing, and virulence and adhesion gene profiling. All the PVL-carrying S. aureus strains were confirmed as methicillin-resistant S. aureus (MRSA) and recovered from wounds (48.6%), blood (25.7%), exudate/pus (11.4%), sputum (8.6%), and body fluid (5.7%) samples. Among the 70 PVL-carrying S. aureus strains tested, 38 (54.3%) were positive for ant (4′)-Ia gene, 27 (38.6%) for aac (6′)-Ie/aph (2″), 13 (18.6%) for msr(A), 13 (18.6%) for erm(C), 13 (18.6%) for tet(M), 11 (15.7%) for erm(A), 10(14.3%) for msr(B), 9 (12.9%) for aph (3′)-IIIa, 5 (7.1%) for mupA, and 2 (2.9%) for erm(B) genes. Five clonal complexes (CC) and nine different clones were detected in this study. The most frequent CC was CC22 (ST22) (42.8%) followed by CC30 (ST30) (21.5%), CC8 (ST8) (17.2%), CC1 (ST772) (11.4%), and CC80 (ST80) (7.1%). In this study, ST22-SCCmec IV/t852 was the predominant PVL-positive MRSA clone (20%), followed by ST8-SCCmec IV/t008 (17.2%), ST30-SCCmec IV/t019 (12.9%), ST22-SCCmec IV/t790 (11.4%), ST22-SCCmec IV/t005 (11.4%), ST30-SCCmec IV/t021 (8.6%), ST80-SCCmec IV/t044 (7.1%), ST772-SCCmec V/t657 (7.1%), and ST772-SCCmec V/t10795 (4.3%). Diversity in clonal types of PVL-carrying MRSA strains in our study supports the need to perform a systematic surveillance of PVL-positive MRSA strains.

Introduction

Staphylococcus aureus is one of the most prevalent pathogens of both nosocomial and community-acquired infections worldwide.^1,2 Unfortunately, recent reports described a worldwide increase in the prevalence of this organism as well as the high rates of mortality and morbidity in healthcare settings, so currently, S. aureus has become a major worldwide concern.³ The pathogenesis of S. aureus strains is related to the expression of different virulence factors, including cell surface components (e.g., collagen binding protein, clumping factor, fibronectin binding protein, and elastin binding protein), and secreted factors (e.g., staphylokinase, toxic shock syndrome toxin-1, hemolysin, Panton-Valentine leukocidin (PVL), exfoliative toxins (eta and etb), staphylococcal enterotoxins (SEs), and lipase.^1,3,4

Although the carriage of the PVL genes is more common among community-onset (CO) S. aureus strains, both in methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA), there are reports that show the carriage of these genes among hospital-onset (HO) MRSA strains that indicate a change in the epidemiology of PVL-positive S. aureus isolates.^5,6

PVL is a bicomponent beta-barrel, pore-forming cytotoxin that reduces immune resistance by leukocyte lysis or apoptosis, and targets complement receptors.⁷ According to the literature, its toxic effect results from the synergistic action of two adjacent, co-transcribed genes, namely lukF-PV and lukS-PV, which are carried out on a variety of lysogenic bacteriophages.^6–8 Epidemiological studies of PVL-positive S. aureus (mainly MRSA) have revealed that PVL-carrying strains are primarily associated with recurrent, chronic, or particularly severe skin and soft tissue infections, and rapidly fatal pneumonia.^5,9

Although the prevalence of S. aureus strains producing PVL have been reported to vary around the world, there are reports that indicate S. aureus clones, which combine PVL production with methicillin resistance, are rapidly emerging and becoming an important public health challenge.^5,10 In this regard, the Health Protection Agency Staphylococcus Reference Unit (HPASRU) reported a twofold increase in the number of PVL-producing S. aureus cases annually identified in England.⁵ It is well established that S. aureus isolates carrying PVL genes are predominantly genetically distinct from each other.¹¹

In our country, one of the indispensable public health concerns as regard to S. aureus infections is the growing frequency of MRSA strains that carried PVL genes.^1,12,13 According to the published data in Iran, the prevalence of resistance to methicillin among S. aureus strains isolated from hospitalized patients is steadily increasing.^2,3 Dadashi et al. performed a systematic review and meta-analysis of the literature to determine the prevalence of MRSA in Iran. They estimated that the prevalence of MRSA in healthcare settings was 43.0%.⁸ Although there has been no comprehensive study to determine the prevalence and clonal dissemination of hospital PVL-producing S. aureus in Iran, it is reported that PVL-producing S. aureus strains identified in several studies belong to CC22-MRSA-IV,^1,13 which also have frequently been reported in the Middle East and other regions, such as Western Europe.^11,14 In the United States, one of the most prevalent PVL-positive MRSA types belongs to ST8-SCCmecIVa (USA300) clone and ST1-SCCmecIVa (USA400), while in Europe and some Asian countries, the majority of PVL-MRSA types circulating belong to the ST80-type IVa SCCmec and ST59-SCCmecV clones, and have been predominant, respectively.^14,15

However, the prevalence and clonal dissemination of PVL-producing S. aureus, isolated from patients in Iran, are unknown and there have been no published data on the molecular epidemiological characterization of the PVL-positive MRSA population in Iran. With regard to the increasing prevalence of multidrug-resistant (MDR)-PVL-positive MRSA and their spread into hospitals, this article aims to provide insights into the molecular characteristics of PVL-producing S. aureus strains and antibiotic resistance phenotypes. To the best of our knowledge, this is the first report of the molecular typing of PVL-producing S. aureus from Iran.

Materials and Methods

Study design and bacterial isolation

We conducted a cross-sectional study over the period of 2 years from July 2015 to June 2017 in five university hospitals in Tehran, Iran. Seventy PVL-positive S. aureus strains obtained from 240 S. aureus clinical isolates were included in this study. The frequency of S. aureus isolated from studied hospitals in Tehran was as following; 73 S. aureus isolates (30.4%) from hospital D, 58 isolates from hospital C (24.2%), 49 isolates from hospital A (20.4%), 32 isolates from hospital B (13.3%), and 28 isolates from hospital E (11.7%). One isolate per patient was included in the study and duplicate samples were excluded. Bacterial isolation and species identification were performed by conventional microbiological methods, including colony morphology, Gram staining, growth on mannitol salt agar, and the production of catalase, coagulase, and DNase. All the presumptive S. aureus colonies were confirmed using a polymerase chain reaction (PCR) for the nucA gene.¹³

All the strains were also analyzed for the presence of lukS-PV and lukF-PV gene-encoding components of PVL by the PCR technique. The study protocol was approved by the ethics committee of the Shahid Beheshti University of Medical Sciences (IR.SBMU. MSP.REC.1395.181). The PVL-positive S. aureus strains were stored in tryptic soy broth (Merck, Germany) containing 20% glycerol at −70°C and they were recovered when they were analyzed.¹²

If a positive culture of S. aureus was obtained on or after 96 h of admission to a hospital, the S. aureus strain was considered being HO; if the culture was obtained before the fourth calendar day of hospitalization, however, it was considered CO.¹

Antimicrobial susceptibility testing

Susceptibility to a range of antimicrobial agents was determined by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines¹⁶ for the following antibiotics: penicillin, ceftriaxone, kanamycin, ciprofloxacin, rifampicin, clindamycin, quinupristin-dalfopristin, tetracycline, erythromycin, linezolid, teicoplanin, fusidic acid, amikacin, tobramycin, gentamicin, and trimethoprim–sulfamethoxazole. All the antibiotic disks used in this study were supplied by Mast Co., United Kingdom. S. aureus ATCC25923 and ATCC29213 were used as quality control strains. It is worth mentioning that in this study, the selection of various antibiotics was based on pharmacy records in our hospitals and general guidelines for treating PVL-positive MRSA infections in Iran. Inducible macrolide, lincosamide, and streptogramin B (iMLSB) resistance were defined for the isolates that were susceptible to clindamycin and resistant toward erythromycin, detected by the D-zone test and the broth microdilution method according to the CLSI procedure.¹⁶ The constitutive MLS_B (cMLS_B) phenotype was defined for the isolates that were resistant to both erythromycin and clindamycin. The minimum inhibitory concentration (MIC) for vancomycin, mupirocin, and fusidic acid was determined by the broth microdilution test.^12,17,18 According to the CLSI guidelines,¹⁶ the MIC breakpoints for vancomycin were defined as follows: susceptible, ≤2 μg/mL; intermediate, 4–8 μg/mL; and resistant, ≥16 μg/mL. The testing MIC range of fusidic acid was 0.125–2 μg/mL and in accordance with the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines,¹⁹ MIC values ≤1 μg/mL were considered susceptible and MIC values >1 μg/mL were considered resistant. Strains were considered low-level mupirocin resistance (LLMUPR) if the MIC value was between 8 and 256 μg/mL and high-LMUPR (HLMUPR) if the MIC value was ≥512 μg/mL.¹⁶ MDR was defined as resistance to three or more unique antibiotic classes, in addition to beta-lactams.¹ The MIC₅₀ and MIC₉₀ represent the concentrations required to inhibit 50% and 90% of the strains, respectively.

Extraction of genomic DNA

Bacterial DNA was prepared from cultured colonies on tryptic soy agar (Merck, Germany) using the commercial kit InstaGene Matrix (BioRad, Hercules Co., CA) according to the manufacturer's instructions. Briefly, for each sample, we added lysostaphin (Sigma–Aldrich Co.) at a final concentration of 30 μg/mL for the cell wall lysis.¹⁷ After extraction, the purity of DNA was assessed using a spectrophotometer.

Detection of PVL genes

All the S. aureus isolates were subjected to the PCR method to determine the presence of PVL genes according to the primers and protocols described by Lina et al.²⁰ The S. aureus strain ATCC49775 was chosen as positive control.

MRSA screening

The S. aureus isolates were screened with a cefoxitin disc (30 μg) and an oxacillin disc (1 μg) on Mueller Hinton agar plates supplemented with 4% NaCl in accordance to the CLSI guideline.¹⁶ Isolates with phenotypic resistance to oxacillin were confirmed to harbor the mecA gene by PCR, as described elsewhere.

Adhesion, resistance, and toxin-encoding gene detection

The presence or absence of genes that encode for adhesion (cna, bbp, ebp, fnbB, fnbA, clfB, and clfA),⁴ resistance (mecA, vanA, vanB, mupB, mupA, erm(A), erm(B), erm(C), msr(A), msr(B), tet(M), ant (4′)-Ia, aac (6′)-Ie/aph (2″), and aph (3′)-IIIa),¹⁷ toxin (etb, eta, and tst),¹ SEs (sea, seb, sec, sed, see, seg, seh, sei, and sej)²¹ and arcA genes as an indicator of the arginine catabolic mobile element (ACME)²² were investigated by conventional and multiplex PCR. Resistance genes were selected for inclusion in the study based on the phenotypic results.

Multiplex PCR amplification for SCCmec typing

Different SCCmec types were determined by multiplex PCR using previously published primers and conditions according to the method of Boye et al.²³ SCCmec types were identified by comparing the banding patterns of MRSA to ATCC 10442 (SCCmec type I), N315 (SCCmec type II), 85/2082 (SCCmec type III), MW2 (SCCmec type IVa), and WIS (SCCmec type V) as reference strains.

spa typing

All the isolates were subjected to molecular epidemiological analysis by spa typing as described by Harmsen et al.²⁴ The spa gene PCR products were purified using the QIAGEN PCR purification kit and were subjected to DNA sequence analysis, and their nucleotide sequences on both strands were determined using an ABI Prism 377 automated sequencer (Applied Biosystems, Perkin-Elmer Co., Foster City, CA). Sequence editing was performed using the Chromas software (Version 1.45, Australia). The edited sequences were assigned to particular spa types according to the guidelines described by a Ridom SpaServer database (www.spaserver.ridom.de).

Identification of agr alleles using multiplex PCR

Multiplex PCR was performed for agr typing as described by Gilot et al.²⁵ These oligonucleotide primers were designed to amplify a 441-bp fragment of the agr group I strains, a 575-bp fragment of the agr group II strains, a 323-bp fragment of the agr group III strains, and a 659-bp fragment of the agr group IV strains.

Multilocus sequence typing

All the isolates were subjected to multilocus sequence typing (MLST) according to the method of Enright et al.²⁶ by sequencing an internal fragment of seven unlinked housekeeping genes to determine the following allelic profiles: carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glp), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyl-coenzyme A acetyltransferase (yqiL). The PCR products were purified and sequenced as previously described in spa typing method. The isolates were assigned a specific allelic profile and sequence type (ST) by comparing the sequences with the S. aureus database maintained on the MLST website (http://saureus.mlst.net/). Isolates were divided into clonal complex (CC) by using the program BURST (Based Upon Related Sequence Types).

Results

Sampling and antibiotic susceptibility

This study sampled 240 S. aureus clinical strains isolated from patients, in which PVL-encoding genes were found in 29.2% (n = 70) of the tested isolates. They were isolated from 41 male (58.6%) and 29 female (41.4%) patients with an average age of 35.4 year (range, 5–63 years). In this study, the PVL-positive S. aureus strains were recovered from wounds (n = 34, 48.6%), blood (n = 18, 25.7%), exudate/pus (n = 8, 11.4%), sputum (n = 6, 8.6%), and body fluids (bronchoalveolar lavage and cerebrospinal fluid; n = 4, 5.7%). Out of 70 PVL-positive S. aureus isolates, 25 isolates were obtained from hospital D (35.7%), 17 isolates from hospital C (24.3%), 12 isolates from hospital B (17.1%), 9 isolates from hospital E (12.9%), and 7 isolates from hospital A (10%).

All the strains harbored the mecA gene and were confirmed as MRSA. Among the PVL-positive isolates, 13 (18.6%) and 57 (81.4%) isolates were classified as CO-MRSA and HO-MRSA, respectively. As listed in Table 1, the isolates carrying the PVL-encoding genes were resistant to penicillin (84.3%), erythromycin (72.9%), gentamicin (71.4%), tetracycline (70%), ceftriaxone (68.6%), ciprofloxacin (60%), amikacin (55.7%), kanamycin (51.4%), tobramycin (48.6%), clindamycin (44.3%), rifampicin (42.9%), trimethoprim–sulfamethoxazole (27.1%), quinupristin–dalfopristin (18.6%), and mupirocin (17.1%). The rate of resistance was higher among the HO cases than the CO cases. All the strains were susceptible to vancomycin, linezolid, teicoplanin, and fusidic acid. None of the isolates was susceptible to all the antibiotics tested. All the strains were inhibited by fusidic acid at similar MIC₅₀ and MIC₉₀, 0.25 μg/mL. Out of 12 isolates resistant to mupirocin, 5 (41.7%) and 7 (58.3%) isolates exhibited the HLMUPR and LLMUPR phenotypes. All the five HLMUPR strains harbored the mupA gene. All the MUPR-MRSA strains belonged to wound samples. No mupirocin-resistant isolate harbored the mupB gene. Among the 70 PVL-carrying MRSA clinical isolates examined, iMLS_B and cMLS_B were detected in 20 (28.6%) and 31 (44.3%) MRSA isolates, respectively. The results showed that all the isolates were MDR. Three antibiotic resistance patterns were identified among the tested isolates. The predominant resistance pattern included resistance to seven antibiotics (81.4%, 57/70), followed by nine antibiotics (14.3%, 10/70) and eight antibiotics (4.3%, 3/70), simultaneously. More detail regarding the antibiotic resistance profiles is presented in Table 2.

Table 1.

Antibiotic Resistance Pattern of 70 MRSA Panton-Valentine Leukocidin-Positive Strains

	No. (%) resistance
Type of antibiotic	Community onset	Hospital onset	Total resistance no. (%)
Penicillin	4 (6.8)	55 (93.2)	59 (84.3)
Ceftriaxone	8 (16.7)	40 (83.3)	48 (68.6)
Gentamicin	2 (4)	48 (96)	50 (71.4)
Kanamycin	12 (33.3)	24 (66.7)	36 (51.4)
Amikacin	6 (15.4)	33 (84.6)	39 (55.7)
Tobramycin	12 (35.3)	22 (64.7)	34 (48.6)
Clindamycin	1 (3.2)	30 (96.8)	31 (44.3)
Erythromycin	7 (13.7)	44 (86.3)	51 (72.9)
Tetracycline	8 (16.3)	41 (83.7)	49 (70)
Ciprofloxacin	7 (16.7)	35 (83.3)	42 (60)
Rifampicin	3 (10)	27 (90)	30 (42.9)
Mupirocin	0 (0)	12 (100)	12 (17.1)
Quinupristin–dalfopristin	3 (23.1)	10 (76.9)	13 (18.6)
Trimethoprim–sulfamethoxazole	7 (36.8)	12 (63.2)	19 (27.1)

MRSA, methicillin-resistant Staphylococcus aureus.

Table 2.

Characteristics of the MRSA Panton-Valentine Leukocidin-Positive Clones Isolated from Patients

CC	PVL-positive MRSA clones	Virulence/adhesion genes (no. %)	Antibiotic resistance profile (no. %)	Antibiotic resistance genes (no. %)	CO N (%)	HO N (%)	Total N (%)
CC22	ST22-SCCmec IV/t852	sea(6; 42.9), sec(10; 71.4), sed(11; 78.6)/clfB(12; 85.7), clfA(10; 71.4), fnbB(12; 85.7), fnbA(9; 64.3), ebp(5; 35.7), bbp(6; 42.9) cna(10; 71.4)	PG, GM, T, CRO, E, CD, K (6; 42.9)	MecA(14; 100), erm(C) (3; 21.4), msr(A) (10; 71.4), ant(4′)-Ia(5; 35.7), aac(6′)-Ie/aph(2″) (7; 50)	2 (14.3)	12 (85.7)	14 (20)
			PG, CRO, AK, TN, CIP, E, RP (3; 21.4)
			PG, GM, AK, TN, T, CIP, RP, SYN, TS (3; 21.4)
			CRO, K, AK, E, T, RP, CIP (2; 14.3)
	ST22-SCCmec IV/t790	sec (7; 87.5), seg (8; 100), see (7; 87.5), sei (6; 75), arc (7; 87.5), tst (2; 25)/clfB(3; 37.5), clfA(8; 100), fnbB(6; 75), fnbA(5; 62.5), bbp (4,50) cna(2; 25)	PG, GM, AK, CIP, MUP, TN, TS (5; 62.5)	MecA (8; 100), erm(A) (6; 75) msr(B) (5; 62.5), ant(4′)-Ia (3; 37.5), aac(6′)-Ie/aph(2″) (5; 62.5), aph(3′)-IIIa(3; 37.5)	1 (12.5)	7 (87.5)	8 (11.4)
	ST22-SCCmec IV/t790		PG, GM, T, CRO, E, CD, K (3; 37.5)		1 (12.5)	7 (87.5)	8 (11.4)
	ST22-SCCmec IV/t005	sec (5; 62.5), etb (3; 37.5)/clfA(7; 87.5), fnbB(6; 75), fnbA(2; 25), ebp(3; 37.5), cna(4; 50)	PG, CRO, AK, TN, CIP, E, RP (3; 37.5)	MecA (8; 100), erm(B) (2; 25), msr(A) (3; 37.5), tet(M) (1; 12.5), ant(4′)-Ia (4; 50), aac(6′)-Ie/aph(2″) (4; 50)	0 (0)	8 (100)	8 (11.4)
			PG, GM, AK, TN, T, CIP, RP, SYN, TS (4; 50)
			CRO, K, AK, E, T, RP, CIP (1; 12.5)
CC30	ST30-SCCmec IV/t019	seg (7; 77.8), sei (5; 55.6)/clfB(8; 88.9), clfA(9; 100), fnbB(5; 55.6), ebp(3; 33.3), bbp (6; 66.7) cna(9; 100)	PG, GM, T, CRO, E, CD, K (4; 44.5)	MecA (9; 100), erm(A) (3; 33.3) erm(C) (2; 22.2), tet(M) (3; 33.3), ant(4′)-Ia (5; 55.6), aac(6′)-Ie/aph(2″) (4; 44.5), aph(3′)-IIIa (4; 44.5)	4 (44.5)	5 (55.5)	9 (12.9)
			CRO, K, AK, E, T, RP, CIP (3; 33.3)
			PG, GM, AK, TN, T, CIP, RP, SYN, TS (2; 22.2)
	ST30-SCCmec IV/t021	seb(3; 50), seg(4; 66.7), eta (2; 33.3)/clfB(3; 50), clfA(6; 100), fnbB(4; 66.7), fnbA(5; 83.3), ebp(3; 50), cna(4; 66.7)	PG, GM, T, CRO, E, CD, K (3; 50)	MecA (6; 100), msr(B) (2; 33.3), tet(M) (1; 16.7), ant(4′)-Ia (3; 50), aac(6′)-Ie/aph(2″) (3; 50)	2 (33.3)	4 (66.7)	6 (8.6)
	ST30-SCCmec IV/t021		PG, CRO, AK, TN, CIP, E, RP (3; 50)		2 (33.3)	4 (66.7)	6 (8.6)
CC8	ST8-SCCmec IV/t008	sea (5; 41.7), arc (12; 100)/clfA(11; 91.7), fnbB(12; 100), bbp (5; 41.7) cna(9; 75)	GM, TN, CD, E, T, CIP, MUP, SYN (3; 25)	MecA (12; 100), mupA (5; 41.7), erm(A) (2; 16.7) erm(C) (2; 16.7), tet(M) (3; 25), ant(4′)-Ia (8; 66.7),	3 (25)	9 (75)	12 (17.2)
			PG, GM, AK, CIP, MUP, TN, TS (4; 33.3)
			PG, GM, T, CRO, E, CD, K (4; 33.3)
			PG, GM, AK, TN, T, CIP, RP, SYN, TS (1; 8.4)
CC1	ST772-SCCmec V/t657	sea (5; 100), sec (4; 80), sed (5; 100), seg (4; 80), sei (5; 100)/clfB(2; 40), clfA(3; 60), fnbB(4; 80), ebp(1; 20), bbp (2; 40) cna(4; 80)	PG, GM, T, CRO, E, CD, K (4; 80)	MecA (5; 100), erm(C) (4; 80), msr(B) (3; 60), tet(M) (3; 60), ant(4′)-Ia (5; 100), aac(6′)-Ie/aph(2″) (3; 60).	0 (0)	5 (100)	5 (7.1)
	ST772-SCCmec V/t657		CRO, K, AK, E, T, RP, CIP (1; 20)		0 (0)	5 (100)	5 (7.1)
	ST772-SCCmec V/t10795	sea (2; 66.7), sec (3; 100), seg (1; 33.3)/clfB(3; 100), fnbB(2; 66.7), bbp (2,66.7) cna(3; 100)	PG, GM, T, CRO, E, CD, K (3; 100)	MecA (3; 100), ant(4′)-Ia (3; 100),	0 (0)	3 (100)	3 (4.3)
CC80	ST80-SCCmec IV/t044	clfB(5; 100), clfA(1; 20), fnbB(3; 60), fnbA(4; 80), ebp(1; 20), bbp (3; 60) cna(2; 40)	PG, CRO, AK, TN, CIP, E, RP (3; 60)	MecA (5; 100), erm(C) (2; 40), tet(M) (2; 40), ant(4′)-Ia (2; 40), aac(6′)-Ie/aph(2″) (1; 20), aph(3′)-IIIa(2; 40)	1 (20)	4 (80)	5 (7.1)
			PG, GM, T, CRO, E, CD, K (1; 20)
			CRO, K, AK, E, T, RP, CIP (1; 20)

PG, penicillin; CRO, ceftriaxone; CD, clindamycin; E, erythromycin; GM, gentamicin; RI, rifampicin; T, tetracycline; CIP, ciprofloxacin; K, kanamycin; SYN, quinupristin–dalfopristin; AK, amikacin; TN, tobramycin; TS, trimethoprim– sulfamethoxazole; MUP, mupirocin; CC, clonal complex; PVL, Panton-Valentine Leukocidin; CO, community onset; HO, hospital onset.

Detection of resistance-, toxin-, and adhesion-encoding genes

Among the isolates under study, the prevalence of ant (4′)-Ia, aac (6′)-Ie/aph (2″), msr(A), erm(C), tet(M), erm(A), msr(B), aph (3′)-IIIa, mupA, and erm(B) genes was found in 38 (54.3%), 27 (38.6%), 13 (18.6%), 13 (18.6%), 13 (18.6%), 11 (15.7%), 10(14.3%), 9 (12.9%), 5 (7.1%), and 2 (2.9%) isolates, respectively. The erm(C) gene was the most common gene detected among the isolates with the constitutive phenotype (10; 14.3%), followed by erm(A) (9; 12.9%), msr(A) (6; 8.6%), and msr(B) (6; 8.6%), while msr(A) (8; 11.4%), erm(C) (3; 4.3%), erm(B) (1; 1.4%), and msr(B) (1; 1.4%) genes were distributed in isolates with an inducible phenotype. It is worth noting that none of isolates carried vanA and vanB genes. Among the adhesion genes tested, the most prevalent was the clfA gene (55; 78.6%), followed by fnbB (54; 77.1%), cna (47; 67.1%), clfB (36; 51.4%), bbp (28; 40%), fnbA (25; 35.7%), and ebp (16; 22.9%) genes.

Among the isolates under study, three isolates (4.3%) were found to carry the etb gene, two isolates (2.9%) carried the eta gene, and two isolates (2.9%) carried the tst gene. Analyzing the SE genes in the MRSA strains carrying PVL-encoding genes revealed that the most prevalent gene was sec (n = 29; 41.4%); this was followed by seg (n = 24; 34.3%), sea (n = 18; 25.7%), sed (n = 16; 22.9%), sei (n = 16; 22.9%), see (n = 7; 10%), and seb (n = 3; 4.3%). None of the isolates carries seh and sej genes.

ST and spa, agr, and SCCmec elements identified in the MRSA strains carrying the PVL-encoding genes

According to the MLST method, our isolates were distributed in five STs. The most frequent CC was CC22 (ST22) (42.8%); this was followed by CC30 (ST30) (21.5%), CC8 (ST8) (17.2%), CC1 (ST772) (11.4%), and CC80 (ST80) (7.1%). In total, nine spa types were identified, with the most common being spa type t852, which accounted for 20% of the isolates, followed by t008 (17.2%), t019 (12.9%), t790 (11.4%), t005 (11.4%), t021 (8.6%), t044 (7.1%), t657 (7.1%), and t10795 (4.3%). Three agr specificity groups were revealed by agr typing. Forty-two isolates (60%) were agr type I, 20 isolates (28.6%) were agr type III, and 8 isolates (11.4%) were agr type II. agr-type IV was not detected among the isolates. According to the SCCmec type analysis, SCCmec type IV was the predominant PVL-positive MRSA type (n = 62; 88.6%), followed by small numbers of SCCmec type V (n = 8; 11.4%).

Molecular epidemiology of MRSA PVL-positive strains

By the combination of different typing assays, the tested isolates were clustered into five CCs and nine different clones. The characteristics related to the 70 PVL-positive MRSA isolates are presented in Table 2.

CC22. The majority of CC/ST22-MRSA-IV isolates were assigned to the spa type t852 (46.6% [14/30]) and was followed by the spa type t790 (26.7% [8/30]) and t005 (26.7% [8/30]). Among the CC22-MRSA-IV isolates, resistances to aminoglycosides encoded by aac (6′)-Ie/aph (2″), ant (4′)-Ia, and erythromycin encoded by msr(A) were common. Sixty percent of the isolates were found to be ciprofloxacin resistant. CC/ST22-MRSA-IV isolates carried the greatest range of toxin genes, including sec (73.3% [22/30]), sed (36.7% [11/30]), seg (26.7% [8/30]), see (23.3% [7/30]), arc (23.3% [7/30]), sea (20% [6/30]), sei (20% [6/30]), etb (10% [3/30]), and tst (6.7% [2/30]). The iMLSB and cMLSB phenotypes, among the CC/ST22-MRSA-IV strains, exhibited similar frequency (30% [9/30]). However, the spa types showed wide differences in the carriage of adhesions. A low-level resistance to mupirocin was shown in five isolates.

CC30. Among the CC/ST30-MRSA-IV isolate spa types, t019 (60% [9/15]) and t021 (40% [6/15]) predominated. Fewer than half of the isolates carried resistance genes, with erythromycin resistances encoded by erm(A) (20% [3/15]), erm(C) (13.3% [2/15]), and msr(B) (13.3% [2/15]), as well as tetracycline resistances encoded by tet(M) (26.7% [4/15]). Aminoglycoside resistance encoded by ant (4′)-Ia (53.3% [8/15]), aac (6′)-Ie/aph (2″) (46.7% [7/15]), and aph (3′)-IIIa (26.7% [4/15]) was also common among these isolates. Inducible and constitutive resistance were observed among 6 (40%) and 7 (46.7%) CC/ST30-MRSA-IV isolates, respectively. Among the CC30 MRSA spa types, the distribution of adhesion genes was various, with the exception of the clfA gene, which was carried by all the strains. The majority of the isolates carried the seg gene (73.3% [11/15]).

CC8. All the ST8-MRSA-IV isolates exhibited a single spa type, t008, and the majority of the isolates exhibited resistances to aminoglycosides encoded by ant (4′)-Ia (66.7% [8/12]). All the five HLMUPR strains, which harbored the mupA gene, belonged to this CC. Two strains displayed the LLMUPR phenotype. The enterotoxin gene sea was the only toxin gene detected among the ST8-IV MRSA isolates. ACME was identified in all the isolates. The frequency of the cMLSB phenotype was found to be 58.3% (7/12).

CC1. CC1/ST772-MRSA-V isolates exhibited the spa types t657 (62.5% [5/8]) and t10795 (37.5% [3/8]). Of the eight CC1/ST772-MRSA-V isolates, seven isolates and one isolate exhibited constitutive and inducible resistance, respectively. The ant (4′)-Ia gene was identified in all the ST772-MRSA-V isolates. The sea and sec genes accounted for 87.5% of isolates, while sed, seg, and sei genes were detected in 62.5% of the isolates.

CC80. All the CC/ST80-MRSA-IV isolates were assigned to the spa type t044. None of the isolates harbored enterotoxin genes. The majority of the CC/ST80-MRSA-IV isolates exhibited resistances to kanamycin, amikacin, and gentamicin encoded by ant (4′)-Ia, aac (6′)-Ie/aph (2″), and aph (3′)-IIIa, and fewer than half of the isolates were resistant to erythromycin encoded by erm(C) (40% [2/5]) and tetracycline encoded by tet(M) (40% [2/5]).

As a result, ST22-SCCmec IV/t852 was the predominant PVL-positive MRSA clone (20%), followed by ST8-SCCmec IV/t008 (17.2%), ST30-SCCmec IV/t019 (12.9%), ST22-SCCmec IV/t790 (11.4%), ST22-SCCmec IV/t005 (11.4%), ST30-SCCmec IV/t021 (8.6%), ST80-SCCmec IV/t044 (7.1%), ST772-SCCmec V/t657 (7.1%), and ST772-SCCmec V/t10795 (4.3%). The result showed that circulating clones in hospital D included ST8-SCCmec IV/t008 (10 isolates), ST22-SCCmec IV/t852 (6 isolates), ST30-SCCmec IV/t019 (5 isolates), and ST22-SCCmec IV/t005 (4 isolates). Hospital C included ST22-SCCmec IV/t852 (7 isolates), ST22-SCCmec IV/t790 (5 isolates), ST80-SCCmec IV/t044 (3 isolates), and ST8-SCCmec IV/t008 (2 isolates) clones. Circulating clones in hospital B included ST30-SCCmec IV/t021 (3 isolates), ST30-SCCmec IV/t019 (3 isolates), ST772-SCCmec V/t10795 (2 isolates), ST22-SCCmec IV/t790 (2 isolates), and ST22-SCCmec IV/t005 (2 isolates). In hospital E, ST772-SCCmec V/t657 clone (5 isolates), ST22-SCCmec IV/t005 clone (2 isolates), ST30-SCCmec IV/t019 (1 isolate), and ST22-SCCmec IV/t852 clone (1 isolate) were detected. ST30-SCCmec IV/t021 (3 isolates), ST80-SCCmec IV/t044 (2 isolates), ST772-SCCmec V/t10795 (1 isolate) and ST22-SCCmec IV/t790 (1 isolate) clones were detected in hospital A. An inducible resistance to clindamycin was observed in ST22-SCCmec IV/t852 (n = 5; 7.2%), ST22-SCCmec IV/t005 (n = 4; 5.7%), ST80-SCCmec IV/t044 (n = 4; 5.7%), ST30-SCCmec IV/t019 (n = 3; 4.3%), ST30-SCCmec IV/t021 (n = 3; 4.3%), and ST772-SCCmec V/t657 clones (n = 1; 1.4%). In this study, five isolates (7.1%) expressed high-level resistance to mupirocin and carried the mupA gene, all of which belonged to the ST8-SCCmec IV/t008 clone. The LLMUPR phenotype was detected in seven isolates (10%) that belonged to the ST22-SCCmec IV/t790 (7.1%) and the ST8-SCCmec IV/t008 (2.9%) clones. None of these strains carried the mupA gene.

Discussion

This study showed several striking findings, including a small increase in the prevalence of PVL and a high diversity of PVL-positive MRSA clones. In this study, the prevalence of PVL was found to be 29.2%. This finding was much higher than the reported rate in an international study performed in England and the United States (1.6%).⁶ However, the high prevalence of PVL was reported by several investigators from around the world.^1,11,14 We previously reported a PVL-MRSA carriage rate of 21.4% in 2016 and 19.5% in 2017¹³ from Iran. Overall, our data clearly indicated a relatively low, but increasing prevalence of PVL-positive MRSA, which is in accordance to reports from Ireland during the last decade.¹⁸ Although this discrepancy may be due to the origin of isolates and the type of sample, it may also reflect the fact that susceptibility to infection with PVL-converting phages among S. aureus strains is increasing. Our study showed that infection with MRSA was higher among HO cases than CO cases that are in agreement with this global trend and with our previous report of HO-PVL-positive MRSA infections that are increasing in investigated hospitals.¹

Concerning the antimicrobial resistance pattern in this study, the differences in the pattern of resistance were shown among isolates, with a high resistance to β-lactams and to other therapeutic options, such as macrolides, lincosamides, and aminoglycosides, which were similar to the previously described studies in Iran.^1,13 In this study, 17.1% of the isolates were resistant to mupirocin, of which 7.1% and 10% exhibited the HLMUPR and the LLMUPR phenotypes, respectively. It is significantly higher than the reported prevalence in France (2.2%)²⁷ and Jordan (2.6%).²⁸ This finding suggests that unrestricted policies that allow an inappropriate use of mupirocin for long periods can be a possible reason for resistance toward mupirocin. Although the resistance rates to fusidic acid among MRSA isolates are gradually increasing, in line with the studies conducted by Aschbacher et al.²⁹ in Italy and Otokunefor et al.³⁰ in the United Kingdom, this study showed that none of the tested isolates was resistant to fusidic acid.

The distribution of the spa types varies from one geographical region to another. An analysis of spa typing showed that nine spa types were found in our collection of PVL-positive MRSA; the spa type t852 was the most common spa type found (20%). A Kuwait-based study of the clonal distribution of MRSA has replicated these findings.¹⁴ Similar to many Europeans countries, spa type t852 isolates have been reported among MRSA isolates in Saudi Arabia,³¹ Qatar,³² and Oman,³³ suggesting an increasing transmission of this variant in the Gulf Persian Cooperative Council countries. To the best of our knowledge, this is the first report of the spa type t852 in Iran.

Regarding the distribution of the SCCmec types, the study shows that the majority of the tested isolates belong to the SCCmec type IV (88.6%), followed by the SCCmec type V (11.4%). This finding is in agreement with the study by Chamon et al.³⁴ from Brazil, which reported a high frequency of SCCmec IV (62%). Similar to the earlier report from the United Kingdom,⁶ which showed a high prevalence of agr type I among the PVL-carrying HO-MRSA isolates, among our PVL-carrying MRSA isolates, the most predominant agr group was Group I (60%), followed by type III (28.6%) and then type II (11.4%).

In this study, the second most frequent spa type was found to be t008 (17.2%). This spa type was previously reported from Kuwait, the United States, and many European countries.^11,14 Considering the literature, the spa type t019 PVL-positive MRSA is irregularly disseminated in the United States, and certain Asian and European countries like Egypt, Japan, Poland, and Taiwan.¹¹ In our study, 12.9% of the tested isolates belonged to t019 as the third most common spa type detected. In a multicenter study by Tristan et al.¹⁵ on 469 PVL-positive CA-MRSA isolates from 17 countries, spa type t019 accounted for 3.6% of the isolates. In contrast to many studies that reported t790 as the predominant spa type, we reported the prevalence of this spa type in 11.4% of the isolates. A low frequency of these spa types in this study was in accordance with the findings by Boswihi et al.¹⁴ from Kuwait and a study made by Goudarzi et al.¹ in Iran. In contrast with a study conducted in the United Kingdom, which reported spa type t005 as the most frequent spa type (47.4%) detected among PVL-MSSA strains,³⁰ in this study, we observed a low frequency of the t005 spa type (11.4%) among the PVL-MRSA strains. In line with our findings, several earlier researchers in other countries reported a low frequency of the t021, t044, t657, and t10795 spa types among PVL-positive MRSA clinical isolates in comparison to other spa types, but not to the same extent.^5,6,11,14

This survey exhibited diversity in the numbers and types of PVL-positive MRSA clones obtained in our hospitals. Although several clones of the PVL-positive MRSA population were identified, the majority (42.9%) corresponded to the well-established hospital-associated ST22-IV MRSA clone. CC22-MRSA-IV-PVL-positive populations were reported from different parts of the world, including Iran, England, Saudi Arabia, Germany, Ireland, Australia, Nepal, and Kuwait.^{1,5,11,14,31,35,36} The results of this study showed that the ST22-IV MRSA corresponded to three clones, including ST22-SCCmec IV/t852, ST22-SCCmec IV/t790, and ST22-SCCmec IV/t005. Although ST22-MRSA-IV is currently predominant in Iranian hospitals, these isolates are genetically distinct from each other in this survey. Increasing reports of this clone in Iran highlights the ability of these strains to spread in Iranian hospitals. Diversity of the ST22-IV variants circulating in Iranian hospitals may reflect the independent evolution of these strains and the diverse sources of their acquisition.

The ST30-IV MRSA clone, which is known also as the Southwest Pacific or the USA1100 clone, was the second common clone among our PVL-positive MRSA clinical isolates. This MRSA clone was also reported in a study conducted by Boswihi et al. in Kuwait. They reported the prevalence of 30% and 22% of ST30-IV-MRSA from 2001 to 2003 and 2006, respectively, which decreased to 2.9% in 2010.¹⁴ Similar to other studies from other countries,^6,11 our ST30-IV MRSA isolates belong to agr type III, but possesses different toxin and antimicrobial resistance patterns. Moreover, in line with our result, the emergence of the PVL-positive ST30-IV MRSA lineage has been reported in Italy,²⁹ Ireland,¹⁸ and Brazil.³⁴

Based on the literature, ST30 isolates differed in their carriage of antibiotic resistance-, virulence-, and enterotoxin-encoding genes.^11,14 Virulence gene analysis of the ST30-IV MRSA isolates showed that the clfA gene was present in all the isolates and the majority of the isolates carried the seg gene (73.3% [11/15]). Fewer than half of the isolates carried the bbp gene (40% [6/15]). Data obtained from a study conducted by Chamon et al. in Brazil³⁴ showed that the isolates were distributed in 15 lineages, with a majority of USA1100/ST30/CC30 clones (25 isolates). They also revealed a correlation between the ST30 and the bbp gene as a possible marker of this lineage. In contrast to the findings of our study, Shore et al.¹⁸ showed that all the CC/ST30-MRSA-IV isolates carried egc and fewer than half of the isolates were resistant to fusidic acid encoded by fusC. The results of this study showed that the majority of ST30-IV MRSA isolates carried seg (73.3% [11/15]), and followed sei (33.3% [5/15]) and seb (20% [3/15]), which conform to the results of a study performed in Italy by Aschbacher et al.,²⁹ who reported that all the PVL-positive ST30-IV had identical toxin profiles and carried seg and sei genes simultaneously.

USA300, as a major international epidemic clone, has been remarkable for the rapidity of its dissemination within the community and hospital.³⁷ In this study, USA300 accounted for 17.2% of the MRSA isolates, which was higher among the HO cases (75%) than the CO cases (25%). These are in agreement with a study conducted in Colombia.³⁸ ACME was identified in all the isolates of this clone. In line with our result, the ACME-positive ST8-IV MRSA isolates were also reported in Iran,¹⁰ Kuwait,¹⁴ and Ireland.¹⁸ Although the resistance genes are subjected to high, but variable, selective pressure, the resistance genetic pattern in these isolates is highly variable. In our study, all the five HLMUPR strains, which harbored the mupA gene, belonged to this clone. Interestingly, the emergence of the USA300 clones harboring mupA has been previously reported in the United States.³⁹ As stated in the literature, the drug resistance pattern in the ST8-IV isolates varies. A high resistance rate to aminoglycosides and ciprofloxacin and a low resistance rate to tetracycline in the ST8-IV MRSA isolates have been reported by several investigators,^5,18 which is in accordance with this study. In addition, similar to the detection of mupirocin, clindamycin, erythromycin, and/or tetracycline resistance in the ST8-IV MRSA isolates investigated in this study, the increasing incidence of resistances to these antibiotics in the USA300 strains has been reported from Spain.⁹ The enterotoxin gene sea was the only toxin gene detected among the ST8-IV isolates. Variability in the enterotoxin genes within USA300 was reported by several investigators.^5,18 This emergence of ST8-IV isolates in our study may be attributed to the high transmissibility and rapidity of its spread within the community and/or its increase in volume through international travel.

ST772-MRSA-V is generally synonymous with the Bengal Bay clone, which emerged in Bangladesh, and was previously reported in Italy, United Kingdom, Ireland, New Zealand, Nepal, India, Australia, Kuwait, Malaysia, Saudi Arabia, UAE, and the Middle East.^{5,11,14,18,29,35} This MRSA lineage was identified in 11.4% of the investigated strains. With regard to the antibiotic resistance pattern, our findings support a recent study conducted by Shore et al.,¹⁸ which reported that all the CC1/ST772-MRSA-V isolates were resistant to multiple antimicrobial agents and carried multiple resistance genes, including ant (4′)-Ia, aac (6′)-Ie/aph (2″), and tet(M). Taken together, the results from this study reflected that the genetic characteristics of our ST772-MRSA-V isolates share similarities in their susceptibility to antibiotics and toxin-encoding genes with the isolates recently reported in the Middle East,¹⁴ which can be related to travel history to or from these countries. Our study demonstrated the presence of ST772-MRSA-V for the first time in Iran, although at a low level. Given the propensity of these clones to spread worldwide, therefore, these clones might quickly spread through the community and within our hospitals.

ST80-MRSA-IV was another clone detected among PVL-positive MRSA strains. In contrast to the results of several studies, which showed that PVL-positive ST80 was the dominant clone in many European countries and the Middle East,^5,11 this study indicated a low prevalence rate of this clone (7.1%). Our ST80 isolates contained genes for ant (4′)-Ia, aph (3′)-IIIa, erm(C), tet(M), and aac (6′)-Ie/aph (2″), and they belonged to the agr type III and spa type t044. The ST80 MRSA IV clone in this study was similar to the ST80 isolates reported from the United Kingdom in their resistance to clindamycin, gentamicin, tetracycline, and ciprofloxacin.⁵ This study showed that all the ST80 isolates were susceptible to fusidic acid. This finding is in contrast to the results of a study in Irish by Shore et al.,¹⁸ who reported that resistances to fusidic acid encoded by fusB were common among ST80-MRSA-IV isolates. Although ST80-MRSA-MRSA isolates harboring the PVL gene have been detected in certain Asian countries, including Kuwait, Malaysia, and Singapore,^11,14 the presence of ST80-MRSA in our area seems to be quite sporadic and, perhaps, associated with travel histories to or from the Mediterranean and Middle Eastern countries.

In summary, this study demonstrates the first report on the clonal dissemination of PVL-positive MRSA strains in Iran. The PVL-carrying MRSA isolates belonged to diverse genetic backgrounds with a predominance of ST22-MRSA-IV, followed by ST30-MRSA-IV, ST8-MRSA-IV, ST772-MRSA-V, and ST80-MRSA-IV. These data highlight the special attention for the ongoing and the systematic surveillance of PVL-positive MRSA strains to detect changes in their clonal composition and dissemination. Antimicrobial stewardship programs may be necessary to help curb the emergence and the dissemination of MDR-PVL-MRSA clones as a global threat to public health.

Footnotes

Acknowledgments

The authors thank the Research Deputy of Shahid Beheshti University of Medical Sciences for funding this study (grant no. 9067). The funder had no role in study design, data collection, analysis, decision to publish, or preparation of the article.

Disclosure Statement

No competing financial interests exist.

References

Goudarzi

, Goudarzi

, Figueiredo

A.M.S.

, Udo

E.E.

, Fazeli

, Asadzadeh

, and Seyedjavadi

S.S.

. 2016. Molecular characterization of methicillin resistant Staphylococcus aureus strains isolated from intensive care units in Iran: ST22-SCCmec IV/t790 emerges as the major clone. PLoS One, 11:e0155529.

Goudarzi

, Seyedjavadi

S.S.

, Azad

, Goudarzi

, and Azimi

. 2016. Distribution of spa types, integrons and associated gene cassettes in Staphylococcus aureus strains isolated from intensive care units of hospitals in Tehran, Iran. Arch. Clin. Infect. Dis., 11:e38813.

Nezhad

R.R.

, Meybodi

S.M.

, Rezaee

, Goudarzi

, and Fazeli

. 2017. Molecular characterization and resistance profile of methicillin resistant Staphylococcus aureus strains isolated from hospitalized patients in Intensive Care Unit, Tehran-Iran. Jundishapur. J. Microbiol., 10:e41666.

Eftekhar

, Rezaee

, Azad

, Azimi

, Goudarzi

, and Goudarzi

. 2017. Distribution of adhesion and toxin genes in Staphylococcus aureus strains recovered from hospitalized patients admitted to the ICU. Arch. Ped. Infect. Dis., 5:e39349.

Ellington

M.J.

, Ganner

, Warner

, Cookson

B.D.

, and Kearns

A.M.

. 2009. Polyclonal multiply antibiotic-resistant methicillin-resistant Staphylococcus aureus with Panton–Valentine leucocidin in England. J. Antimicrob. Chemother., 65:46–50.

Holmes

, Ganner

, McGuane

, Pitt

, Cookson

, and Kearns

. 2005. Staphylococcus aureus isolates carrying Panton-Valentine leucocidin genes in England and Wales: frequency, characterization, and association with clinical disease. J. Clin. Microbiol., 43:2384–2390.

Shallcross

L.J.

, Fragaszy

, Johnson

A.M.

, and Hayward

A.C.

. 2013. The role of the Panton-Valentine leucocidin toxin in staphylococcal disease: a systematic review and meta-analysis. Lancet. Infect. Dis., 13:43–54.

Dadashi

, Nasiri

M.J.

, Fallah

, Owlia

, Hajikhani

, Emaneini

, Mirpour

. 2017. Methicillin-Resistant Staphylococcus aureus (MRSA) in Iran: a Systematic Review and Meta-analysis. J. Glob. Antimicrob. Resist., 12:96–103.

Cañas-Pedrosa

A.M.

, Vindel

, Artiles

, Colino

, and Lafarga

. 2012. Antimicrobial resistance and molecular epidemiology of Panton-Valentine leukocidin–positive community-associated methicillin-resistant Staphylococcus aureus from Gran Canaria (Canary Islands, Spain). Diagn. Microbiol. Infect. Dis. 74:432–434.

10.

Azimian

, Havaei

S.A.

, Khosrojerdi

, Naderi

, and Samiee

. 2014. Isolation of PVL/ACME-positive, community acquired, methicillin-resistant Staphylococcus aureus (USA300) from Iran. J. Med. Microbiol. Infect. Dis., 2:100–104.

11.

Monecke

, Coombs

, Shore

A.C.

, Coleman

D.C.

, Akpaka

, Borg

, Chow

, Ip

, Jatzwauk

, and Jonas

. 2011. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One, 6:e17936.

12.

Goudarzi

, Seyedjavadi

S.S.

, Nasiri

M.J.

, Goudarzi

, Nia

R.S.

, and Dabiri

. 2017. Molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients with bacteremia based on MLST, SCCmec, spa, and agr locus types analysis. Microb. Pathog., 104:328–335.

13.

Goudarzi

, Bahramian

, Tabrizi

M.S.

, Udo

E.E.

, Figueiredo

A.M.S.

, Fazeli

and Goudarzi

. 2017. Genetic diversity of methicillin resistant Staphylococcus aureus strains isolated from burn patients in Iran: ST239-SCCmec III/t037 emerges as the major clone. Microb. Pathog., 105:1–7.

14.

Boswihi

S.S.

, Udo

E.E.

, and Al-Sweih

. 2016. Shifts in the clonal distribution of methicillin-resistant Staphylococcus aureus in Kuwait Hospitals: 1992–2010. PLoS One, 11:e0162744.

15.

Tristan

, Bes

, Meugnier

, Lina

, Bozdogan

, Courvalin

, Reverdy

M.-E.

, Enright

M.C.

, Vandenesch

, and Etienne

. 2007. Global distribution of Panton-Valentine leukocidin–positive methicillin-resistant Staphylococcus aureus, 2006. Emerg. Infect. Dis., 13:594.

16.

Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 27th Informational Supplement; 2017, 31:M100–S122.

17.

Shekarabi

, Hajikhani

, Chirani

A.S.

, Fazeli

, and Goudarzi

. 2017. Molecular characterization of vancomycin-resistant Staphylococcus aureus strains isolated from clinical samples: a three year study in Tehran, Iran. PLoS One., 12:e0183607.

18.

Shore

A.C.

, Tecklenborg

S.C.

, Brennan

G.I.

, Ehricht

, Monecke

, and Coleman

D.C.

. 2014. Panton-Valentine leukocidin-positive Staphylococcus aureus in Ireland from 2002 to 2011: 21 clones, frequent importation of clones, temporal shifts of predominant methicillin-resistant S. aureus clones, and increasing multiresistance. J. Clin. Microbiol., 52:859–870.

19.

The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 8.0, 2018.

20.

Lina

, Piémont

, Godail-Gamot

, Bes

, Peter

M.-O.

, Gauduchon

, Vandenesch

, and Etienne

. 1999. Involvement of panton-valentine leukocidin—producing Staphylococcus aureus in primary skin infections and pneumonia. Clinl. Infect. Dis., 29:1128–1132.

21.

Monday

S.R.

, and Bohach

G.A.

. 1999. Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J. Clin. Microbiol., 37:3411–3414.

22.

Goering

R.V.

, McDougal

L.K.

, Fosheim

G.E.

, Bonnstetter

K.K.

, Wolter

D.J.

, and Tenover

F.C.

. 2007. Epidemiologic distribution of the arginine catabolic mobile element among selected methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates. J. Clin. Microbiol., 45:1981–1984.

23.

Boye

, Bartels

M.D.

, Andersen

I.S.

, Moeller

J.A.

, and Westh

. 2007. A new multiplex PCR for easy screening of methicillin-resistant Staphylococcus aureus SCCmec types I–V. Clin. Microbiol. Infect., 13:725–727.

24.

Harmsen

, Claus

, Witte

, Rothgänger

, Claus

, Turnwald

, and Vogel

. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol., 41:5442–5448.

25.

Gilot

, Lina

, Cochard

, and Poutrel

. 2002. Analysis of the genetic variability of genes encoding the RNA III-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis. J. Clin. Microbiol., 40:4060–4067.

26.

Enright

M.C.

, Day

N.P.

, Davies

C.E.

, Peacock

S.J.

, and Spratt

B.G.

. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol., 38:1008–1015.

27.

Desroches

, Potier

, Laurent

, Bourrel

A.-S.

, Doucet-Populaire

, Decousser

J.-W.

, Archambaud

, Aubert

, Biendo

, and Blanchard-Marche

. 2013. Prevalence of mupirocin resistance among invasive coagulase-negative staphylococci and methicillin-resistant Staphylococcus aureus (MRSA) in France: emergence of a mupirocin-resistant MRSA clone harbouring mupA. J. Antimicrob. Chemother., 68:1714–1717.

28.

Aqel

, Ibrahim

, and Shehabi

. 2012. Rare occurrence of mupirocin resistance among clinical Staphylococcus isolates in Jordan. Acta. Microbiol. Immunol. Hung., 59:239–247.

29.

Aschbacher

, Pichon

, Spoladore

, Pagani

, Innocenti

, Moroder

, Ganner

, Hill

, Pike

, and Ganthaler

. 2012. High clonal heterogeneity of Panton–Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus strains from skin and soft-tissue infections in the Province of Bolzano, Northern Italy. Int. J. Antimicrob. Agents, 39:522–525.

30.

Otokunefor

, Sloan

, Kearns

A.M.

, and James

. 2012. Molecular characterization and Panton-Valentine leucocidin typing of community-acquired methicillin-sensitive Staphylococcus aureus clinical isolates. J. Clin. Microbiol., 50:3069–3072.

31.

Monecke

, Skakni

, Hasan

, Ruppelt

, Ghazal

S.S.

, Hakawi

, Slickers

, and Ehricht

. 2012. Characterisation of MRSA strains isolated from patients in a hospital in Riyadh, Kingdom of Saudi Arabia. BMC Microbiol., 12:146.

32.

El-Mahdy

, El-Ahmady

, and Goering

. 2013. Molecular characterization of MRSA isolated over a two year period in a Qatari hospital from multinational patients. Clin. Microbiol. Infect., 20:169–173.

33.

Udo

E.E.

, Al-Lawati

B.-H.

, Al-Muharmi

, and Thukral

. 2014. Genotyping of methicillin-resistant Staphylococcus aureus in the Sultan Qaboos University Hospital, Oman reveals the dominance of Panton–Valentine leucocidin-negative ST6-IV/t304 clone. New Microbes. New Infect., 2:100–105.

34.

Chamon

R.C.

, Iorio

N.L.P.

, da Silva Ribeiro

, Cavalcante

F.S.

, and K.R.N. dos Santos. 2015. Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro hospitals. Diagn. Microbiol. Infect. Dis., 83:331–334.

35.

Pokhrel

R.H.

, Aung

M.S.

, Thapa

, Chaudhary

, Mishra

, Kawaguchiya

, Urushibara

, and Kobayashi

. 2016. Detection of ST772 Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (Bengal Bay clone) and ST22 S. aureus isolates with a genetic variant of elastin binding protein in Nepal. New Microbes. New Infect., 11:20–27.

36.

Linde

, Wagenlehner

, Strommenger

, Drubel

, Tanzer

, Reischl

, Raab

, Höller

, Naber

, and Witte

. 2005. Healthcare-associated outbreaks and community-acquired infections due to MRSA carrying the Panton-Valentine leucocidin gene in southeastern Germany. Eur. J. Clin. Microbiol. Infect. Dis., 24:419–422.

37.

Nimmo

. 2012. USA300 abroad: global spread of a virulent strain of community-associated methicillin-resistant Staphylococcus aureus. Clin. Microbiol. Infect., 18:725–734.

38.

Alvarez

C.A.

, Yomayusa

, Leal

A.L.

, Moreno

, Mendez-Alvarez

, Ibañez

, and Vanegas

. 2010. Nosocomial infections caused by community-associated methicillin-resistant Staphylococcus aureus in Colombia. Am. J. Infect. Control., 38:315–318.

39.

McDougal

L.K.

, Fosheim

G.E.

, Nicholson

, Bulens

S.N.

, Limbago

B.M.

, Shearer

J.E.

, Summers

A.O.

, and Patel

J.B.

. 2010. Emergence of resistance among USA300 methicillin-resistant Staphylococcus aureus isolates causing invasive disease in the United States. Antimicrob. Agents. Chemother., 54:3804–3811.