Genetic Diversity Analysis of Mupirocin-Resistant Staphylococcus aureus Clinical Isolates in Tehran Hospitals,Iran

Abstract

Few studies describe the molecular characterization of mupirocin resistance in Staphylococcus aureus strains. In this study, we evaluated the characteristics of mupirocin resistance in S. aureus isolates for 1-year period in Tehran, Iran. In a cross-sectional study, we collected 51 unique mupirocin-resistant strains obtained from 648 S. aureus isolates. High- and low-level resistance were determined by minimum inhibitory concentration with broth microdilution method. Presence of the genes for resistance to antibiotics and toxins was detected by polymerase chain reaction assays. Genotyping was performed by S. aureus protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) typing, whereas clones were determined by multilocus sequence typing. Among mupirocin-resistant isolates, methicillin-resistant S. aureus (MRSA) 23 (45.1%) and 28 (54.9%) isolates were classified as high- and low-level mupirocin resistance, respectively. Among the 51 tested strains 25 (49%) isolates were positive for mupA genes. Seven different clones were detected in this study. High-level mupirocin-resistant (HLMUPR) strains belonged to ST8-SCCmec IV/t064 (n = 10, 19.6%), ST5-SCCmec IV/t002 (n = 5, 9.8%), ST8-SCCmec IV/t008 (n = 4, 7.8%), and ST239-SCCmec III/t631 (n = 4, 7.8%) clones, while low-level mupirocin-resistant (LLMUPR) strains belonged to ST22-SCCmec IV/t790 (n = 11, 21.6%), ST239-SCCmec III/t860 (n = 9, 17.7%), and ST15-SCCmec IV/t084 (n = 8, 15.7%) clones. Two strains belonged to ST22-SCCmec IV/t790 clone, despite carrying mupA gene, and demonstrated LLMUPR phenotype. One ST8-SCCmecIV/t008 strain with HLMUPR was confirmed as Vancomycin-intermediate S. aureus strain. Our data demonstrated the need for thorough epidemiological monitoring and a routine mupirocin testing program to prevent and detect mupirocin resistance in MRSA.

Introduction

Staphylococcus aureus is one of the most common pathogens causing community and hospital infections. It is reported that a considerable portion of S. aureus isolates in many parts of the world is resistant to many classes of antibiotics. Therefore, increasing prevalence of Methicillin-resistant S. aureus (MRSA) is one of the major public health concerns regarding S. aureus infections.¹ Dissemination of MRSA with multiresistance genes has significantly limited the choice of therapeutic options available for treatment of staphylococcal infections and their worse clinical outcome.²

Mupirocin, also known as pseudomonic acid A, is a topical antibiotic that is used for treatment of skin and soft tissue infections associated with MRSA and eradication of MRSA nasal carriage in patients and healthcare workers. Thus, this antibiotic serves as an important agent in the control of MRSA outbreaks.^3,4 Mupirocin was introduced into clinical practice in 1985. In this regard, resistance to mupirocin emerged shortly after it started being used clinically. Since then, an increase in mupirocin resistance has been reported widely in various countries to the extent that mupirocin-resistant S. aureus has emerged as a major public health concern.⁴ However, wide usage of mupirocin as the only approved antibiotic for topical use against MRSA in patients and healthcare staffs, and prolonged use of the antibiotic in some instances have caused the efficacy of this antibiotic for such usage to remain controversial.⁵

Mupirocin is an analog of isoleucyl that competitively binds to bacterial isoleucyl-tRNA synthetase (IRS). This analog is encoded by the iles gene and blocks the formation of isoleucyl tRNA, which in turn disrupts bacterial protein synthesis.⁴ Mupirocin-resistant phenotypes are grouped into two distinct categories: (i) low-level resistance (minimum inhibitory concentrations [MICs] ranging from 8 to 256 μg/mL), which results from point mutations in the native IRS gene (iles) and may be associated with higher rates of recolonization after efforts to eradicate S. aureus carriage; and (ii) high-level resistance (MIC ≥512 μg/mL), which occurs with acquisition of a plasmid-borne gene mupA or ileS2 that encodes an additional modified IRS with reduced affinity for mupirocin. A related gene, mupB, has also been shown to confer high-level resistance.^3,4

Although clinical use of mupirocin in the treatment of skin infection has become well documented, the use of this antibiotic must be limited due to concerns about the emergence of resistance. Moreover, control measures should also be taken into consideration when high-level resistance strains are detected.⁶ Given that resistance to mupirocin often is not tested routinely in our healthcare settings in Iran, information about resistance to mupirocin among S. aureus clinical isolates and the molecular characterization of these strains are sparse. However, the prevalence of 70%, 25%, 7.8%, and 6% of resistance to mupirocin among S. aureus strains isolated from personnel and patients has been previously reported in Iran.^7,8 With this background, this study was conducted to determine the molecular characterization of mupirocin resistance among S. aureus isolates for a 1-year period in Tehran (capital city of Iran).

Materials and Methods

Bacterial isolation and identification

This study was carried out on 648 S. aureus isolates for a 1-year period from June 2016 to May 2017 in five university hospitals (A–E). The strains were collected from various clinical specimens, including wound, blood, pus, urine, catheters, and body fluids (bronchoalveolar lavage and cerebrospinal fluid). Duplicate isolates from the same patients were excluded from this study. The study protocol was approved by the ethics committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1395.357). Identification of the clinical isolates of S. aureus was performed by traditional biochemical methods. Polymerase chain reaction (PCR) amplification of the nucA gene was used for confirmation.^9,10 Isolates of S. aureus were stored in Tryptic Soy Broth (Merck, Germany) containing 20% glycerol at −70°C for further investigation.

Detection of mupirocin-resistant S. aureus isolates

All strains were phenotypically screened based on Kirby-Bauer disc diffusion method for mupirocin susceptibility using a 5 μg mupirocin disc as the first step.⁴ Susceptible strains were defined as those showing zone diameters equal or more than 14 mm around 5 μg mupirocin discs and were excluded from our study. Strains presenting diameters <13 mm were considered mupirocin resistant and were subjected to broth microdilution method to determine the MIC.¹¹ Strains were considered to demonstrate low-level mupirocin resistance (LLMUPR) if the MIC value was between 8 and 256 μg/mL and high-level mupirocin resistance (HLMUPR) if the MIC value was ≥512 μg/mL.¹²

Antimicrobial susceptibility testing

Disc diffusion test was carried out, according to Clinical and Laboratory Standards Institute (CLSI) criteria by the following discs (Mast; United Kingdom): kanamycin, ciprofloxacin, rifampicin, clindamycin, quinupristin–dalfopristin, tetracycline, erythromycin, linezolid, teicoplanin, amikacin, tobramycin, gentamicin, and trimethoprim–sulfamethoxazole. Resistance to methicillin was determined by cefoxitin disc (30 μg) on Mueller Hinton agar plates supplemented with 4% NaCl according to the guidelines provided by the CLSI.¹¹

The MIC of vancomycin and fusidic acid was determined by the broth microdilution method. According to the CLSI guidelines, MIC breakpoints for vancomycin were defined as follows: susceptible: ≤2 μg/mL, intermediate: 4–8 μg/mL, and resistant: ≥16 μg/mL. In accordance with the European Committee for Antimicrobial Susceptibility Testing guidelines, the MIC breakpoints for fusidic acid were defined as follows: MIC values ≤1 μg/mL was considered susceptible and MIC values >1 μg/mL was considered resistant.¹³

Inducible macrolide, lincosamide, and streptogramin B (iMLSB) resistances were defined for the isolates that were susceptible to clindamycin and resistant against erythromycin, detected by D-zone test and broth microdilution method according to the CLSI procedure.¹¹ Constitutive MLS_B (cMLS_B) phenotype was defined for the isolates that were resistant to both erythromycin and clindamycin. The standard reference strains S. aureus ATCC 25923 and ATCC 29213 were used as quality control strains in antimicrobial susceptibility testing (AST) experiments.

Molecular analysis

DNA extraction was carried out on 24-hour cultures of mupirocin-resistant strains using the commercial kit InstaGene Matrix (BioRad, Hercules Co., CA). Based on the manufacturer's protocol for bacterial cells, we added lysostaphin (Sigma-Aldrich Co.) at a final concentration of 30 μg/mL for cell wall lysis. After extraction, the purity of DNA was assessed using a spectrophotometer.

Detection of resistance- and toxin-encoding genes

Amplification of PCR was performed on each isolate to detect resistance [vanA, vanB, mecA, mupA, mupB, erm(A), erm(B), erm(C), msr(A), msr(B), tet(M), ant (4′)-Ia, aac (6′)-Ie/aph (2′′), and aph (3′)-IIIa]- and toxin (etb, eta, pvl, and tst)-encoding genes. The primer sequences are presented in Table 1.

Table 1.

Oligonucleotide Primers Used in this Study

Target	Primer	Primer sequence (5′→3′)	Product size (bp)	Reference
nucA	F	GCGATTGATGGTGATACGGTT	270	¹
nucA	R	AGCCAAGCCTTGACGAACTAAAGC	270	¹
mecA	F	AGAAGATGGTATGTGGAAGTTAG	583	¹
mecA	R	ATGTATGTGCGATTGTATTGC	583	¹
luk-PV	F	TTCACTATTTGTAAAAGTGTCAGACCCACT	180	¹⁴
luk-PV	R	TACTAATGAATTTTTTTATCGTAAGCCCTT	180	¹⁴
tst-1	F	TTATCGTAAGCCCTTTGTTG	398	⁹
tst-1	R	TAAAGGTAGTTCTATTGGAGTAGG	398	⁹
eta	F	GCAGGTGTTGATTTAGCATT	93	¹
eta	R	AGATGTCCCTATTTTTGCTG	93	¹
etb	F	ACAAGCAAAAGAATACAGCG	226	¹
etb	R	GTTTTTGGCTGCTTCTCTTG	226	¹
ant(4′)-Ia	F	AATCGGTAGAAGCCCAA	135	¹⁴
ant(4′)-Ia	R	GCACCTGCCATTGCTA	135	¹⁴
aac(6′)-Ie/aph(2′′)	F	CCAAGAGCAATAAGGGCATACC	222	¹⁴
aac(6′)-Ie/aph(2′′)	R	CACACTATCATAACCACT	222	¹⁴
aph(3′)-IIIa	F	CTTGATCGAAAAATACCGCTGC	269	¹⁴
aph(3′)-IIIa	R	TCATACTCTTCCGAGCAAA	269	¹⁴
erm(A)	F	TATCTTATCGTTGAGAAGGGATT	139	¹⁵
erm(A)	R	CTACACTTGGCTGATGAAA	139	¹⁵
erm(B)	F	CTATCTGATTGTTGAAGAAGCATT	141	¹⁵
erm(B)	R	GTTTACTCTTGGTTTAGGATCAAA	141	¹⁵
erm(C)	F	AATCGTCAATTCCTGCATGT	299	¹⁵
erm(C)	R	TAATCGTGGAATACGGGTTTG	299	¹⁵
msr(A)	F	GGCACAATAAGAGTG TTTAAAGG	940	¹⁶
msr(A)	R	AAGTTATATCATGAATAGATTGTCCTGTT	940	¹⁶
msr(B)	F	TATGATATCCATAATAATTATCCAATC	595	¹⁶
msr(B)	R	AAGTTATATCATGAATAGATTGTCCTGTT	595	¹⁶
mupA	F	CCCATGGCTTACCAGTTGA	1158	¹⁷
mupA	R	CCATGGAGCACTATCCGA	1158	¹⁷
tet(M)	F	AGTGGAGCGATTACAGAA	158	¹⁴
tet(M)	R	CATATGTCCTGGCGTGTCTA	158	¹⁴
vanA	F	GGCAAGTCAGGTGAAGATG	713	¹⁸
vanA	R	ATCAAGCGGTCAATCAGTTC	713	¹⁸
vanB	F	GTGACAAACCGGAGGCGAGGA	430	¹⁸
vanB	R	CCGCCATCCTCCTGCAAAAAA	430	¹⁸
mupB	F	CTAGAAGTCGATTTTGGAGTAG	647	¹⁷
mupB	R	AGTGTCTAAAATGATAAGACGATC	647	¹⁷

Multiplex PCR amplification for SCCmec typing

Different staphylococcal cassette chromosome mec (SCCmec) types were determined by specific primers described by Boye et al.¹⁹ 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

The S. aureus protein A (spa) typing was performed as described by Harmsen et al.²⁰ The spa gene PCR products 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 done using Chromas software (version 1.45; Australia). Edited sequences were assigned to particular spa types according to the guidelines described by a Ridom SpaServer database (http://spaserver.ridom.de).

Multilocus sequence typing

Multilocus sequence typing (MLST) with standard primers introduced by the MLST database was performed on all isolates based on seven housekeeping genes (arcC, aroE, glpF, gmK, pta, tpiA, and yqiL) as described by Enright et al.²¹ Isolates were assigned a sequence type (ST) according to the MLST website (http://mlst.net/).

Results

In this study, we included 648 S. aureus strains from the hospitals affiliated to Shahid Beheshti University of Medical Sciences, which were uniformly distributed throughout Tehran. The frequency of mupirocin-resistant S. aureus isolated from studied hospitals in Tehran was as follows: 27 isolates (52.9%) from hospital D, 8 isolates from hospital A, 7 isolates from hospital E, 6 isolates from hospital B, and 3 isolates from hospital C. In this study, mupirocin-resistant S. aureus isolates were obtained from hospitalized patients in burn (27; 52.9%), intensive care unit (ICU) (12; 23.5%), surgery (6; 11.8%), oncology (3; 5.9%), internal medicine (2; 3.9), and infectious (1; 2%) wards.

The overall prevalence of mupirocin resistance was calculated to be 7.9% by MIC, out of which 23 (45.1%) and 28 (54.9%) were HLMUPR and LLMUPR resistant, respectively. Strains of HLMUPR were isolated from hospitals D (18 isolates, 78.3%), A (two isolates, 8.7%), E (two isolates, 8.7%), and B (one isolate, 4.3%). All 51 mupirocin-resistant S. aureus strains were resistant to methicillin according to the phenotypic method and confirmed as MRSA based on mecA gene detection. The majority of strains were obtained from wound (n = 23; 45.1%), pus (n = 10; 19.6%), blood (n = 8; 15.7%), sputum (n = 7; 13.7%), and catheter (n = 3; 5.9%). Broth microdilution results showed that among the LLMUPR strains analyzed, 11 (39.3%), 8 (28.6%), and 9 (32.1%) strains had MIC values of 16, 64, and 128 μg/mL, respectively. In this study, mupA was identified in 25 strains (49%). All of the 23 HLMUPR-MRSA strains harbored the mupA gene. The results of mupirocin MIC were as follows: 15 (65.2%) had MIC ≥512 μg/mL and 8 (34.8%) had MIC ≥1,024 μg/mL. Two remaining mupA-positive strain displayed the LLMUPR phenotype.

No mupirocin-resistant isolate harbored the mupB gene. Based on the disc diffusion test, the higher resistance rate was observed for erythromycin (96.1%) followed by gentamicin (80.4%), tetracycline (80.4%), kanamycin (80.4%), amikacin (76.5%), ciprofloxacin (60.8%), clindamycin (54.9%), trimethoprim–sulfamethoxazole (43.1%), rifampicin (35.3%), tobramycin (27.4%), and quinupristin–dalfopristin (27.4%). The results of vancomycin MIC were as follows: 10 (19.6%) isolates had MIC of 0.5 μg/mL, 21 (41.2%) had MIC of 1 μg/mL, 19 (37.2%) had MIC of 2 μg/mL, and 1 (2%) had MIC of 8 μg/mL. All the strains were inhibited by fusidic acid at similar MIC₅₀ and MIC₉₀ 0.25 μg/mL. Of 18 isolates with iMLS_B phenotype (35.3%), 15 isolates belonged to HLMUPR-MRSA isolates (83.3%) and 3 isolates (16.7%) related to LLMUPR-MRSA isolates. A total of 28 isolates with cMLS_B phenotype were distributed among HLMUPR-MRSA (8; 28.6%) and LLMUPR-MRSA (20; 71.4%) isolates. AST showed five resistance phenotypes among the tested isolates. The predominant resistance profiles among mupirocin-resistant MRSA isolates included a resistance profile to five antibiotics (56.9%) followed by nine antibiotics (37.3%), seven antibiotics (3.9%), and six antibiotics (1.9%) simultaneously.

Regarding virulence factors, 12 strains (23.5%) had pvl gene, 6 strains (11.8%) had eta gene, 1 strain (2%) had tst gene, and 1 strain had etb gene (2%). Analysis of the resistance genes in mupirocin-resistant MRSA strains revealed that the most prevalent gene was ant (4′)-Ia gene (48; 94.1%), followed by aac (6′)-Ie/aph (2′′) (41; 80.4%), tet(M) (40; 78.4%), erm(C) (40; 78.4%), aph (3′)-IIIa (39; 76.5%), msr(A) (39; 76.5%), erm(B) (35; 68.6%), erm(A) (32; 62.7%), msr(B) (31; 60.8%), and mupA (25; 49%) genes. All HLMUPR-MRSA strains carried SCCmec type IV, except four strains that contained SCCmec type III. Of 28 LLMUPR-MRSA strains, 19 strains belonged to SCCmec type IV (67.9%) and 9 strains (32.1%) belonged to SCCmec type III. Seven different spa types were identified, of which t064 (10 isolates; 43.5%), t002 (five isolates; 21.7%), t008 (four isolates; 17.4%), and t631 (four isolates; 17.4%) were observed among HLMUPR-MRSA strains and t790 (11 isolates; 39.3%), t860 (nine isolates; 32.1%), and t084 (eight isolates; 28.6%) were observed among LLMUPR-MRSA strains.

According to the MLST technique, the strains were assigned to five different STs, including ST8 in 14 strains, ST239 in 13 strains, ST22 in 11 strains, ST15 in 8 strains, and ST5 in 5 strains. Among the HLMUPR-MRSA strains, 60.9% of them belonged to ST8, 21.7% to ST5, and 17.4% to ST239, while ST22 was the predominant ST observed among LLMUPR-MRSA strains (39.3%) followed by ST239 (32.1%) and ST15 (28.6%). Molecular typing, as defined by the combination of spa CC, MLST, and SCCmec types, showed that the strains were clustered into seven different clones with ST22-SCCmec IV/t790 (n = 11, 21.6%) as the most prevalent genotype, followed by ST8-SCCmec IV/t064 (n = 10, 19.6%), ST239-SCCmec III/t860 (n = 9, 17.7%), ST15-SCCmec IV/t084 (n = 8, 15.7%), ST5-SCCmec IV/t002 (n = 5, 9.8%), ST8-SCCmec IV/t008 (n = 4, 7.8%), and ST239-SCCmec III/t631 (n = 4, 7.8%). Two strains belonging to ST22-SCCmec IV/t790 clone, despite carrying the mupA gene, demonstrated LLMUPR phenotype. All ST8-SCCmec IV/t008 and ST15-SCCmec IV/t084 strains, 90% of ST8-SCCmec IV/t064 strains, and half of ST239-SCCmec III/t631 strains were isolated from hospital D. The result showed that circulating clones in hospital A included ST22-SCCmec IV/t790 (five isolates), ST8-SCCmec IV/t064 (one isolate), ST5-SCCmec IV/t002 (one isolate), and ST239-SCCmec III/t860 (one isolate), while in hospital B, they included ST239-SCCmec III/t860 (five isolates) and ST5-SCCmec IV/t002 (one isolate). Circulating clones in hospital C included ST22-SCCmec IV/t790 (two isolates) and ST239-SCCmec III/t860 (one isolate). In hospital E, ST22-SCCmec IV/t790 clone (four isolates), ST239-SCCmec III/t631 clone (two isolates), and ST239-SCCmec III/t860 clone (one isolate) were detected. Distribution and characterization of MUPR-MRSA molecular types are presented in Table 2.

Table 2.

Molecular Characterization of Mupirocin-Resistant, Methicillin-Resistant Staphylococcus aureus Strains

Mupirocin-resistant MRSA	MUPR-MRSA clones	Toxin profile (No, %)	Antibiotic resistance profile (No, %)	Genetic resistance profile (No, %)	Hospitals (No, %)	Wards	No (%)
HLMUPR-MRSA	ST8-SCCmec IV/t064	—	CLI, ERY, AMK, GEN, TET, SXT, CIP, KAN, RIF (3, 30)	MecA (10, 100), mupA (10, 100), erm(A) (8, 80) erm(B) (10, 100), erm(C) (10, 100), msr(A) (8, 80), msr(B) (9, 90), tet(M) (10, 100), ant (4′)-Ia (10, 100), aac (6′)-Ie/aph (2′′) (10, 100), aph (3′)-IIIa (10, 100)	D (9, 90), A (1, 10)	Burn (9, 90), surgery (1, 10)	10 (19.6)
			ERY, GEN, TET, AMK, KAN (5, 50)
			GM, TET, SYN, SXT, TOB, CIP, KAN (2, 20)
	ST5-SCCmec IV/t002	—	CLI, ERY, AMK, GEN, TET, SXT, CIP, KAN, RIF (2, 40)	MecA (5, 100), mupA (5, 100), erm(A) (5, 100) erm(C) (4, 80), msr(A) (5, 100), msr(B) (5, 100), tet(M) (2, 40), ant (4′)-Ia (5, 100), aac (6′)-Ie/aph (2′′) (4, 80), aph (3′)-IIIa (4, 80)	D (3, 60), A (1, 20), B (1, 20)	Burn (3, 60), ICU (1, 20), surgery (1, 20)	5 (9.8)
	ST5-SCCmec IV/t002	—	CLI, ERY, SYN, TOB, CIP (3, 60)		D (3, 60), A (1, 20), B (1, 20)	Burn (3, 60), ICU (1, 20), surgery (1, 20)	5 (9.8)
	ST8-SCCmec IV/t008	pvl (4, 100)	ERY, AK, GEN, TET, SYN, SXT, TOB, CIP, KAN (1, 25)	MecA (4, 100), mupA (4, 100), erm(A) (1, 25) erm(B) (4, 100), erm(C) (4, 100), msr(A) (2, 50), msr(B) (3, 75), tet(M) (1, 25), ant (4′)-Ia (4, 100), aac (6′)-Ie/aph (2′′) (4, 100), aph (3′)-IIIa (4, 100)	D (4, 100)	Burn (4, 100)	4 (7.8)
			CLI, ERY, SYN, TN, CIP (2, 50)
			CLI, ERY, SYN, TOB, CIP, VAN (1, 25)
	ST239-SCCmec III/t631	eta (2, 50), etb (1, 25)	ERY, GEN, TET, AMK, KAN (4, 100)	MecA (4, 100), mupA (4, 100), erm(A) (3, 75) erm(B) (4, 100), erm(C) (4, 100), msr(A) (1, 25), msr(B) (2, 50), tet(M) (4, 100), ant (4′)-Ia (3, 75), aac (6′)-Ie/aph (2′′) (4, 100), aph (3′)-IIIa (3, 75)	E (2, 50), D (2, 50)	Oncology (1, 25), ICU (1, 25), burn (2, 50)	4 (7.8)
LLMUPR-MRSA	ST22-SCCmec IV/t790	pvl (2, 18.2), tst (1, 9.1)	ERY, GEN, TET, AMK, KAN (4, 36.4)	MecA (11, 100), mupA (2, 18.2), erm(A) (10, 90.9) erm(B) (9, 81.8), erm(C) (9, 81.8), msr(A) (11, 100), msr(B) (6, 54.5), tet(M) (7, 63.6), ant (4′)-Ia (11, 100), aac (6′)-Ie/aph (2′′) (9, 81.8), aph (3′)-IIIa (10, 90.9)	E (4, 36.4), C (2, 18.2), A (5, 45.4)	ICU (6, 54.6), surgery (3, 27.2), internal (2, 18.2)	11 (21.6)
			CLI, ERY, SYN, TOB, CIP (4, 36.4)
			CLI, ERY, AMK, GEN, TET, SXT, CIP, KAN, RIF (3, 27.2)
	ST239-SCCmec III/t860	eta (4, 44.4)	CLI, ERY, AMK, GEN, TET, SXT, CIP, KAN, RIF (6, 66.7)	MecA (9, 100), erm(A) (3, 33.3) erm(B) (7, 77.8), erm(C) (9, 100), msr(A) (7, 77.8), msr(B) (6, 66.7), tet(M) (9, 100), ant (4′)-Ia (9, 100), aac (6′)-Ie/aph (2′′) (5, 55.6), aph (3′)-IIIa (6, 66.7)	B (5, 55.6), A (1, 11.1), C (1, 11.1), D (1, 11.1), E (1, 11.1)	ICU (4, 44.5), oncology (2, 22.2), burn (1, 11.1), infectious (1, 11.1), surgery (1, 11.1)	9 (17.7)
	ST239-SCCmec III/t860	eta (4, 44.4)	ERY, GEN, TET, AMK, KAN (3, 33.3)				9 (17.7)
	ST15-SCCmec IV/t084	pvl (6, 75)	CLI, ERY, AMK, GEN, TET, SXT, CIP, KAN, RIF (4, 50)	MecA (8, 100), erm(A) (2, 25) erm(B) (1, 12.5), msr(A) (5, 62.5), tet(M) (7, 87.5), ant (4′)-Ia (6, 75), aac (6′)-Ie/aph (2′′) (5, 62.5), aph (3′)-IIIa (2, 25)	D (8, 100)	Burn (8, 100)	8 (15.7)
	ST15-SCCmec IV/t084	pvl (6, 75)	ERY, GEN, TET, AMK, KAN (4, 50)		D (8, 100)	Burn (8, 100)	8 (15.7)

CLI, clindamycin; ERY, erythromycin; GEN, gentamicin; RIF, rifampicin; TET, tetracycline; CIP, ciprofloxacin; KAN, kanamycin; SYN, quinupristin–dalfopristin; AMK, amikacin; TOB, tobramycin; SXT, trimethoprim–sulfamethoxazole; VAN, vancomycin; ICU, intensive care unit; MRSA, methicillin-resistant Staphylococcus aureus; ST, sequence type; MUPR, mupirocin resistant; HLMUPR, high-level mupirocin resistant; LLMUPR, low-level mupirocin resistant; spa, S. aureus protein A; SCCmec, staphylococcal cassette chromosome mec.

Discussion

Mupirocin as a cornerstone of decolonization regimens is widely used for eradication of S. aureus nasal colonization and control of MRSA transmission in healthcare settings.⁴ Unfortunately, during the last few years, the use of topical application mupirocin to prevent colonization and subsequent infection due to increasing number of MRSA has been increased worldwide.³ Therefore, it is important to maintain awareness about the level of mupirocin resistance among MRSA isolates, identification of molecular characteristics, and susceptibilities pattern of MRSA to antimicrobial agents.^3,4

The rate of mupirocin resistance among S. aureus clinical isolates varies according to geographic regions and/or patient population.^4,17 In this study, the prevalence of mupirocin resistance among MRSA strains was found to be 7.9%, which was lower than our previous reports from burn patients (28.3%),¹⁰ patients hospitalized in ICU (30.5%), and patients with bacteremia (24.2%) in Iran.⁹ Other studies in different areas of the world reported different rates of mupirocin resistance, including 1.6% in Greece,²² 2.2% in France,⁵ 2.6% in Jordan,²³ 5% in India,²⁴ and 13.3% in the United States of America.³ The low prevalence of mupirocin resistance among tested isolates can be attributed to the type of our clinical samples that were various and not limited to a specific sample (wound, blood, etc.) or ward. Geographic or socioeconomic factors may also contribute to the development of resistance. In this study, among the MUPR-MRSA isolates, 23 and 28 MRSA isolates were HLMUPR and LLMUPR, representing 45.1% and 54.9% of the studied isolates. This result is consistent with findings of Barakat and Nabil in the period of 2 years from 2013 to 2105 in Egypt. These researchers reported HLMUPR-MRSA and LLMUPR-MRSA isolates in 61.5% and 38.5% of MUPR-MRSA isolates, respectively.²⁵ The overall prevalence of HLMUPR and LLMUPR in this study was 3.5% and 4.3%. The percentage of HLMUPR-MRSA in our study (3.5%) was higher than the rate reported in France (0.8%),⁵ but lower than those in Canada and China (7%).^26,27 Mupirocin emerges easily in healthcare settings with unrestricted policies that allow the inappropriate use of mupirocin for long periods. Therefore, mupirocin therapy was necessarily used after susceptibility testing of MRSA isolates, not only based on a specific ward in hospitals.

In line with the findings of other studies, the mupA gene was identified in all of the isolates exhibiting a high level of resistance to mupirocin.^5,28 In this study, the mupA gene was detected in two isolates with LLMUPR phenotype. In a report, isolates with low-level mupirocin resistance were positive for the mupA gene.⁴ This controversy finding was justified with the chromosomal location of the gene.⁵

Considering the emergence of mupirocin resistance among MRSA strains, an increase in the frequent use of other drugs effective against MRSA is observed. The resistance profiles of the isolates show that iMLS_B (HLMUPR: 83.3% vs. LLMUPR: 16.7%) and cMLS_B (HLMUPR: 28.6% vs. LLMUPR: 71.4%) phenotypes are distributed among MUPR-MRSA strains. This result is consistent with a previous report, where a high rate of aminoglycoside and macrolide resistance was reported.^5,29 High prevalence of resistance among HLMUPR-MRSA strains may be attributed to a mupA gene located on a plasmid that typically carries resistance determinants to other antimicrobial agents, including macrolides, gentamicin, tetracycline, and trimethoprim.⁴

The HLMUPR-MRSA strains were grouped in four different clones by a combination of genotyping methods. The predominant clone was ST8-SCCmec IV/t064 (19.6%) followed by ST5-SCCmec IV/t002 (9.8%), ST8-SCCmec IV/t008 (7.8%), and ST239-SCCmec III/t631 (7.8%). In line with our previous survey that reported mupirocin resistance in ST8-SCCmec IV/t064 clone,³⁰ Udo and colleagues investigated clonal composition and distribution of MRSA isolates in Kuwait's hospitals between 1992 and 2010, in 13 public hospitals, and identified 31 MRSA clones. They reported ST8-SCCmec IV/t064 clone with the HLMUPR pattern that was Panton–Valentine leukocidin (PVL) and ACME negative, but carried different enterotoxin and resistance genes.³¹ With regard, similar findings of this clone were reported from Nigeria³² and Ireland.³⁰ It appears that the ST8-SCCmec IV/t064 clone circulating in Iranian hospitals might be imported from neighboring countries.

Another clone found among HLMUPR-MRSA strains was ST5-SCCmec IV/t002. Regarding the literature, ST5 belonged to clonal complex 5 (CC5) and can distribute in communities and hospitals.³¹ This clone was previously detected in many countries such as Australia, Ireland, and the United Arab Emirates and that is the predominant clone in South Korea and Japan.^30,31 PVL-positive CC5-MRSA-V was reported sporadically from Germany and the United Arab Emirates.³⁰ Based on our results, none of these strains carried pvl, eta, etb, and tst genes and they were resistant to most of the tested antibiotics. González-Domínguez et al. in a study in Spain to identify the prevalence, clonal lineages, and virulence genes of HLMUPR-MRSA clinical isolates showed ST125 and ST5 in 97.5% and 2.5% of tested isolates, respectively. They showed that all strains carried SCCmec type IVc and agr type II and none of the virulence genes studied was identified among HLMUPR-MRSA isolates.⁶

Song et al. in a study in China showed that there was a high prevalence of ST5-spa t002 among their clinical S. aureus isolates.³³ In contrast to our study, they showed that most of the ST5 isolates were susceptible to trimethoprim–sulfamethoxazole and rifampicin. Resistance to mupirocin in ST5-spa t002 strains was earlier reported by several researchers.^30,34 In contrast to previously published data, which reported ST5-SCCmec IV/t002 isolate as a mupA-negative strain,³⁴ in this study, we found ST5-SCCmec IV/t002 isolates with a high level of resistance to mupirocin that was positive for the mupA gene.

As our results showed, the third most common HLMUPR-MRSA clone detected in this study was ST8-SCCmecIV/t008 [PVL+], which resembled the USA300. Interestingly, the emergence of USA300 clone MRSA isolates harboring mupA was reported in the United States of America.³⁵ USA300 was infrequently reported from Switzerland, Japan, Hong Kong, Australia, Spain, Kuwait, and the United Emirates Arab.^30,31 As stated in the literature, resistance to mupirocin and other non-β-lactams is emerging among USA300 isolates. Although drug resistance patterns in ST8-SCCmecIV/t008 isolates may vary, trimethoprim–sulfamethoxazole resistance in ST8 isolates is reported by several investigators,³⁵ which is in accordance with this study. In line with previous published data from Iran, which reported ST8-t008 isolate as a vancomycin-intermediate S. aureus strain,³⁶ in this study, we found ST8-SCCmecIV/t008 isolate [vanA⁻] with vancomycin MIC of 8 μg/mL. The absence of vanA gene among our tested strains was in agreement with Tiwari and Sen's study in 2006 in India.³⁷ To the best of our knowledge, this finding may be the first report of ST8 with resistance to mupirocin and heterogeneous to vancomycin simultaneously from Tehran, Iran, and even Asia. This may soon become a global problem because USA300 isolates are virulent and readily transmissible, and are likely to spread to other areas. This emergence of ST8-SCCmecIV/t008 multidrug resistance may be attributed to the building of the selective pressure of drugs, especially mupirocin and vancomycin as the main antimicrobial agent available to treat life-threatening infections with MRSA.

Another clone detected was ST239-SCCmec III/t631 among HLMUPR-MRSA strains (7.8%) harboring eta and etb genes. It is thought that ST239 has evolved from ST 8 (the USA300 clone lineage).³⁰ This clone was initially described as the main clone in South America, Europe, Saudi Arabia, Kuwait, and most Asian countries.^30,31 In this study, it was shown that all the ST239-SCCmec III/t631 strains harbored mupA gene, which is consistent with the studies of Abimanyu et al. and Boswihi et al. who reported high-level mupirocin resistance in MRSA ST239 clone.^29,31 According to some reports, MRSA ST239 strains from Asian countries are resistant to additional antibiotics compared with their counterparts in the Western countries.²⁹ The AST results of HLMUPR-MRSA ST239-SCCmec III/t631 strains revealed two isolates with both mupirocin and inducible clindamycin resistance. In this connection, Udo and colleagues reported multiresistant ST239-SCCmec III clone as a predominant species in Kuwait.³¹ Goudarzi et al. identified genetic diversity of MRSA strains isolated from burn patients in Tehran's hospitals. They found that HLMUPR-MRSA strains belonged to ST15-SCCmec IV/t084 (40%), ST22-SCCmec IV/t790 (23.3%), ST239-SCCmec III/t631 (20%), and ST239-SCCmec III/t030 (16.7%) clones.¹⁰ These data support the idea that this strain is actively circulating in our region.

This study revealed that LLMUPR-MRSA isolates belonged to three major clones: ST22-SCCmec IV/t790 (21.6%), ST239-SCCmec III/t860 (17.7%), and ST15-SCCmec IV/t084 (15.7%). As previously highlighted, ST22-SCCmec IV/t790 was the most-common LLMUPR-MRSA clone identified in this research (39.8%). ST22-MRSA-IV is a pandemic CC22-MRSA strain spread widely in many countries and can occur in hospitals and outpatients.³⁰ Variable virulence markers in ST22-MRSA-IV have been reported by investigators.^9,30 In our study, among ST22-SCCmec IV/t790 strains, two (18.2%) and one isolate (9.1%) were positive for pvl and tst genes, respectively. S. aureus ST22 harboring pvl gene was detected in Iran,¹ Saudi Arabia,³⁸ Kuwait,³⁹ and England.⁴⁰ As summarized in Table 2, the ST22-SCCmec IV/t790 strains were positive for many antibiotic resistance genes, including mupA (18.2%), erm(A) (90.9%) erm(B) (81.8%), erm(C) (81.8%), msr(A) (100%), msr(B) (54.5%), tet(M) (63.6%), ant (4′)-Ia (100%), aac (6′)-Ie/aph (2′′) (81.8%), aph (3′)-IIIa(90.9%). This finding is consistent with Shore and colleagues³⁰ study in Ireland. They showed the presence of erm(C), lnu(A), aacA-aphD, aadD, and mupA in the majority of their cases.⁴¹ As mentioned earlier, two strains belonging to ST22-SCCmec IV/t790 clone, despite carrying the mupA gene, demonstrated LLMUPR phenotype. This result is attributed to a frameshift mutation in the mupA gene that inactivates the gene product.⁴ These data support the idea that the high prevalence of ST22 among the clinical LLMUPR-MRSA strains in this study could result from their transfer from the community to hospitals.

In this study, 17.7% of LLMUPR-MRSA strains belonged to ST239-SCCmec III/t860 clone. It is remarkable that almost half of LLMUPR-MRSA strains belong to the identified clone harbor eta gene. Although there are many reports of mupirocin resistance among ST239 strains, this study might be the first report of ST239-SCCmec III/t860 as an LLMUPR-MRSA clone in Iran. The previously published data from Kuwait also showed a high prevalence of ST239-SCCmec III/t860 clone with the multiresistant pattern, but mupirocin resistance was not detected.³¹ This result suggests dissemination of ST239-SCCmec III/t860 clone among clinical MRSA strains in our region.

Other LLMUPR-MRSA clones found in this study were from ST15-SCCmec IV/t084. In this study, except these two isolates, the other that belonged to ST15 was pvl positive. Several studies reported the various prevalence of ST15. A recent multicenter study found ST15 as the second most frequent clone of samples collected in 25 European countries,⁴² while in another study, this clone was detected in 11 European countries, although at a low level.⁴³ The results of our previous study to identify molecular types of MRSA obtained from burn patients in Tehran, Iran, showed the most common mupirocin-resistant MRSA isolates belonged to ST15-SCCmec IV/t084, which is consistent with our results.¹⁰

Conclusions

Our study provided an estimate of the prevalence of high-level and low-level mupirocin resistance among MRSA isolates. Strains of HLMUPR-MRSA belonged to CC5 and CC8, while strains of LLMUPR-MRSA were assigned to CC22 and CC8. To the best of our knowledge, this study presents the first report of MUPR-MRSA clonal dissemination in Tehran's hospitals. Although the prevalence of resistance to mupirocin in our region was relatively low, surveys such as identification and effective control of hospital clones and diligence in continued surveillance might be necessary to prevent its spread.

Footnotes

Acknowledgments

The authors thank the Research Deputy of Shahid Beheshti University of Medical Sciences for funding of this study (grant number. 9294). The funder had no role in study design, data collection, analysis, and decision to publish or preparation of the article. We appreciate Dr. Edet E. Udo and Dr. Agnes Marie Sá Figueiredo for providing references strains.

Disclosure Statement

No competing financial interests exist.

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