Emergence of high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus in Western Australia

Journal

of Hospital

Infection

(1994)

26, 157-l

65

Emergence of high-level mupirocin methicillin-resistant Staphylococcus Western Australia E. E. Udo,

J. W. Pearman*

resistance

in

aureus

in

and W. B. Grubb

School of Biomedical Sciences, Curtin Uniz~ersity of Technology, GPO Box U 1987, Perth 6001, Western Australia and Department of L21icrobiology, Royal Perth Hospital, Perth, Westevn Australia Accepted for publication

20 September 199.3

Summary:

Six mupirocin-resistant Staphylococcus arcreus were isolated from patients living in the northern part of Western Australia (\VA). The? were all resistant to methicillin, tetracycline, trimethoprim and cadmium and harboured similar 41.4 kb plasmids. Transfer and curing experiments \\ith one of the isolates, WBG7569, demonstrated that the 41.4 kb plasmid encoded resistance to mupirocin, tetracycline, trimethoprim and cadmium. The isolates were compared by pulsed-field gel electrophoresis lvith methicillin-resistant S. awew (URSA) nreviouslr isolated from the Kimberlcl region in the northern-most part* of LVA -(\\;A RIRSA). The mupirocinresistant isolates were found to be closely related to M:X RlRSX suggesting that they were WA nlRSA which had acquired a new multiple-resistance plasmid encoding high-level mupirocin resistance. Keywords: hIRSX, electrophoresis.

mupirocin

resistance,

plasmid

analysis,

pulsed-field

gel

Introduction

Mupirocin (pseudomonic acid A) is a naturally occurring antibiotic produced by Pseudomonasfkovescens which acts by binding reversibly and specifically to bacterial isoleucyl transfer-RN.4 synthetase, thereby preventing protein synthesis.’ It is only used as a topical agent because, systemically, it is rapid11 metabolized. Mupirocin is activre against including methicillin-resistant StaphJjlococcus aweus staphylococci (MRSA) and other Gram-positive cocci.’ It is highly effective in clearing nasal carriage of S. aweus from both hospital staff and patients” ’ and has been used successfully to treat Gram-positive infections of ulcers, wounds, burns’aa and eczema.’ Mupirocin resistance has been observed in methicillin-susceptible and methicillin-resistant staphylococci. I,ovv level resistance (MIC 12-50 mg l- ‘) was obtained by repeated subculture of staphylococci in the presence of the antibiotic.’ Subsequently, high levrel resistance (MIC > 512 mg 1~ ‘) has

158

E. E. Udo et al.

been reported in many laboratories. lo-l5 The determinants for mupirocin resistance have been located on the chromosome and on a variety of conjugative and non-conjugative plasmids.“,” Dyke et al.‘* have cloned and sequenced the mupirocin resistance determinant of a 23 kb plasmid and demonstrated some identity between the derived amino acid sequences of the mupirocin resistance gene and the sequence of isoleucyl tRNA synthetase. A recent study l9 has demonstrated the presence of two distinct isoleucyl-tRNA synthetases in highly resistant staphylococci. In 1982 an outbreak of MRSA occurred in Royal Perth Hospital (RPH) following the admission of an infected patient from a hospital in another part of Australia. 2o After this outbreak it was decided that all patients being admitted to a hospital in Western Australia (WA) who had been in a hospital outside WA in the previous 12 months should be screened for MRSA. This policy has been highly successful in preventing MRSA from becoming established in any hospital in WA. Approximately seven years ago a new type of MRSA (WA MRSA) some communities in the Kimberley region in the appeared in northern-most part of WA.2’ This new type of MRSA has been isolated from a number of patients from the Kimberley who have been admitted to Perth metropolitan hospitals.2’ Molecular an al y sis of some of these isolates2’ has revealed that they are different from MRSA isolated from hospitals in eastern Australia and those which caused an outbreak in RPH in 1982.22 Six mupirocin-resistant S. aweus were isolated at RPH between August 1992 and January 1993. These isolates were all obtained from patients living in the northern part of WA. This study investigated the genetic nature of the mupirocin resistance determinants in these isolates and compared them, using pulsed-field gel electrophoresis and plasmid typing, with the WA MRSA. Materials Bacterial strains The strains studied

and their

and methods

properties

are listed

in Table

I.

Media and reagents Brain Heart Infusion Agar (BHIA), Brain Heart Infusion Broth, Trypticase Soy Broth and Mueller Hinton Agar were purchased from Gibco Diagnostics (Madison, Wi, USA). Lysostaphin, ribonuclease A, and proteinase K were purchased from Sigma Chemical Co. (St Louis, MO, USA). Susceptibility testing Susceptibility to antimicrobial agents was tested and by the replica plating methods.25 Susceptibility

by the disc diffusion,23v2” to mupirocin was tested

Mupirocin-resistant Table

I. Staphylococcus

MRSA aureus

strains

159 studied.

Isolates

Resistance

Plasmids*

Reference

WBG541 WBG1876 WBG7565 WBG7569 WBG7570 WBG7573 WBG7667 WBG7719 WBG7583t WBG7590t WBG75971 WBG7605t

Fa,Rf Fa.Rf Md,Pc,Tc,Tp,Cd,Mup Mc,Pc,Tc,Tp,Cd,Mup Mc,Pc,Tc,Tp,Cd,Mup Mc,Pc,Tc,Tp,Cd,Mup Mc,Pc,Tc,Tp,Cd,Mup Mc,Pc,Tc,Tp,Cd,Mup Mc,Pc,Tc,Cd Mc,Pc,Km,Nm,Sm,Em,Lm Mc,Pc,Tc,Cd Mc,Pc,Tc,Cd,Gm,Km,Nm. Em,Lm,Cm,Tp,Pi,Eb

None None 11.4 41-k 41.4 41.4 41.4 41.4 30.9 30.0 30~0 > 30,30,2.8 1.8

22 22 ‘I-his This ‘I’his This ‘I’his This 21 21 21 21

XLIc, methicillin; PC, benz;-I penicillin; Tc, tetracycline; ‘l‘p, trimrthoprim; hlup, gentamicin; Km, kanamycm; Nm, neomycin; Sm, streptomycin; Cm, chloramphenicol; mycin; Lm, lincomycin; Fa, fusidic acid; Rf, rifwnpicin; Cd, cadmium; Pi, propamldine ethidium bromide. *, sizes of plasmids in kilobases; t. \VX 51RS.A.

Lvith discs containing 5 pg of antibiotic. High-level with discs containing 200 I-18of mupirocin.

studv study stud!stud! stud\ stud!

mupirocin; Gm, Em, rrythroisethionate; Eh,

resistance ~vas tested

Cultures Lvere gro\vn on BHIA at 43°C for 24 h, plated onto BHI.4 and incubated at 37°C o\.ernight. Single colonies \yere screened for loss of resistance to antimicrobial agents by replica plating. Disc diffusion tests \vere performed on strains which appeared to ha\.e lost resistance.

Isolation

and anal>qsis of plasmid

DNA

Plasmids were isolated using the cetyltrimethylammonium bromide method’” and their molecular jveight determined by comparing their migration with plasmids in 1VBGM83 by horizontal-gel electrophoresis using 0.6% \v/v agarose (Type 1 A-6013, Sigma Chemical Co, St I,ouis, WIo, US,4). Plasmids \vere digested \yith EroRI restriction endonuclease according to the manufacturer’s instructions and the fragments separated on 1.0% (y/\r) agarose. Phage 1,ambda DNA4 (‘l’oyobo Co. Ltd, Osaka, Japan) digested with HilzdlI I (‘l‘oyobo Co. I,td., Osaka, Japan) \vas used as a size standard.

Plasmid

tvansfe~

l!Iixed-culture transfer (nIC’l’) and conjugation experiments using strains \YBC;1876 and \VHGSql as recipients respecti\rel!, \\‘ere performed as described previously.” Selections Lvere made on BHIA containing (mg 1 ‘) fusidic acid (5) and rifampicin (2.5) and one of the follo\ving: tctrac!.cline

160

E. E. Udo et al.

(5); trimethoprim (2.5); mupirocin (10) and cadmium acetate (135). Transcipients were screened for plasmids by agarose-gel electrophoresis. Pulsed-field gel electrophoresis of chromosomal DNA This was performed by the contour-clamped homogeneous electric field (CHEF) method. Cells were treated as reported previously2*x2” and digested with SmaI (Promega Corporation, Madison, Wi, USA) according to the manufacturer’s instructions. Electrophoresis was performed in 1% (w/v) agarose (molecular biology grade, BioRad Laboratories, Richmond, Ca, USA) using the CHEF-DR II system (BioRad Laboratories) at 4°C for 42 h in 0.5 x TBE buffer (45 mM Tris, 45 mM Boric acid, 1 mM EDTA, pH 8.0) at 120 V. The pulse times were 10 s initial and 80 s final. Results

Six mupirocin-resistant S. aweus isolates WBG7565, WBG7569, WBG7570, WBG7573, WBG7667 and WBG7719 were studied. They were all resistant to high-level mupirocin, methicillin, benzyl penicillin, tetracycline, trimethoprim, and cadmium (high level) and contained a single 41.4 kb plasmid (Table I). Since they all had similar resistance and plasmid patterns, one of the isolates, WBG7569, was selected and used in curing and transfer experiments. In a curing experiment 39 of 222 colonies screened were found to have lost concomitantly resistance to mupirocin, tetracycline, trimethoprim and the 41.4 kb plasmid. They also had a reduced level of resistance to cadmium. Transfer of mupirocin resistance As mupirocin resistance has been reported on conjugative plasmids,” WBG7569 was used as donor in conjugation experiments with strain WBG541. Selection was made on separate plates containing mupirocin, tetracycline, trimethoprim and cadmium. No transconjugants were obtained on any of the selection plates. Conjugation was repeated with isolates WBG7565, WBG7670, WBG7573, WBG7667 and WBG7719 also without success. WBG7569 was then tested in MCT experiments. Transfer was obtained on mupirocin, tetracycline and trimethoprim but not on cadmium selections. Examination of the transcipients revealed that resistance to mupirocin, trimethoprim, tetracycline and cadmium were co-transferred concomitantly with the 41.4 kb plasmid (Table II). Two of six transcipients from the tetracycline selection were resistant to tetracycline alone and contained no plasmids (not shown). These results demonstrated that the 41.4 kb plasmid, designated pWBG715, codes for resistance to mupirocin, trimethoprim, tetracycline and cadmium. Restriction enzyme analysis The EcoRI digestion fragments of the plasmids from all six isolates were

Mupirocin-resistant Table

I I. Mired-culture

transfer

MRSA

of mupirocin

resistarzce

161 to strain

WBGlX

76

Donor

Selection

R-transferred

Plasmid

\I’BG7569

hIup,Fa,Rf Tp,Fa,Rf Tc,Fa,Rf Tc,Fa,Rf Cd,Fa,Rf

Mup,Tc,Tp,Cd Mup,Tc,Tp,Cd Mup,Tc,Tp,Cd Tc i\‘one

41.1 41.4 -cl .4 sane ,sone

lib. kilobase.

Abbreviations

arc BS in Table

(lib)

I

1234567

23.13 9.42 6.68 4.36

2.32 2.02

Figure 1. EcoRI restriction analysis of plasmids in mupirocin resistant isolates. IJane 1, XIolecular size standards, phage lambda DNA digested with HindIII. Sizes are in kh. The 4.36 kb fragment is not visible on this gel. Lane 2, iVBG7565; Lane 3, \VBG7569 (pWBG715); Lane 4, WBG7570; Lane 5, \VBG7573; Lane 6, \VBG7667; 1,ane 7, \VBG7719.

compared with those of pWBG715. Figure 1 shows that they all had identical EcoRI restriction patterns. Their fragment sizes were 14.3, 11.5, 3.7, 3.0, 2.6, 2.3, 2.1, and 1.9 kilobases which were the same as the EcoRI fragments of pWBG715 (not shown). DNA Pulsed-$eld gel analysis of chromosomal Chromosomal DNA of the six isolates was digested with SmaI, separated b> pulsed-field gel electrophoresis and their restriction patterns compared with similarly digested chromosomal DNA of representative WA MRSA. Except for WBG7719, all the mupirocin-resistant isolates had the same SmaI patterns. This pattern was identical to one of the patterns found amongst the WA MRSA (tract 8, Figure 2). The pattern of WBG7719 \vas

162

E. E. Udo et al. 1

Lanes l-6, WBG7570; representatives WBG7597;

2

3

4

5

6

7

8

9

10

Figure 2. Pulsed field analysis of SmaI digested MRSA isolates. Mupirocin-resistant isolates. Lane 1, WBG7565; Lane 2, WBG7569; Lane 3, Lane 4; WBG7573; Lane 5, WBG7.557; Lane 6, WBG7719. Lanes 7-10, of the WA MRSA. Lane 7, WBG7583; Lane 8, WBG7590; Lane 9, Lane 10, WBG7605.

very similar to the others and was identical to the most common SmaI pattern found in the WA MRSA (tract 7, Figure 2). Discussion

This is the first report of high-level resistance to mupirocin in S. aureu~ in Western Australia (WA). It is interesting that, although isolated at a metropolitan hospital, these isolates were from patients living in the far north of WA where new strains of MRSA have recently been reported.21 This study was conducted to determine if the mupirocin-resistant isolates were related to the newly described WA MRSA. Transfer and curing experiments with WBG7569 demonstrated that mupirocin resistance is located on a 41.4 kb plasmid, pWBG715, which also encodes resistance to tetracycline, trimethoprim and cadmium. Although the plasmid did not transfer in conjugation experiments it did transfer by MCT (phage-mediated conjugation).27 EcoRI restriction analysis demonstrated that all the isolates contained a 41.4 kb plasmid with the same restriction fragments as pWBG715. It also indicated that pWBG715 was not related to any of the mupirocin-resistance plasmids reported by Rahman et a1.16identifying it as a new multi-resistance plasmid in S. aureus. The determinants for cadmium resistance appear to be borne on both plasmid pWBG715 and on the chromosome in WBG7569. Colonies of WBG7569 which had lost pWBG715 still expressed low-level resistance to

Mupirocin-resistant

MRSA

163

cadmium. The difference in the level of the resistance may be due to the presence of two determinants corresponding to cadA (high-level resistance) and cadB (low-level resistance),33 Lvith cadA carried on pWBG71.5 and cadB on the chromosome. Pulsed-field gel electrophoresis is a powerful molecular typing tool and has been used extensively to study the epidemiology of staphylococcal infections,2x.29.3”“” Its use in this study made it possible to establish the relatedness of the six mupirocin-resistant isolates to the WA KIRSX. It demonstrated that WBG7719 had an identical pulsed-field pattern to WBG7583, the most common type of WA MRSA and that the other ti\,e mupirocin-resistant isolates had pulsed-field patterns identical to WBG7590, a WA MRSA closely related to WBG7583. The implication of this finding is that some of the WA4 MRSA ha1.e acquired mupirocin resistance. In a previous report 21 it was cautioned that although the M’X MRSA were susceptible to most antistaphylococcal antibiotics, some lvere multiply-resistant indicating that the strains were able to acquire and transfer resistance determinants. The acquisition of mupirocin resistance by the WA MRSA confirms the fears of the authors.” Of greater concern is their acquisition of the new multi-resistance plasmid pWBG715 which not only carries resistance to mupirocin but also resistance to tetracycline, Th e carriage trimethoprim and cadmium. of these determinants on a plasmid means that the\- can be transferred more easily to other strains. Although it is not yet known how pWBG715 has evolved, its appearance in these isolates is of concern. Although (3-lactamase and resistance to cadmium, tetracycline and trimethoprim have previously been detected in to cadmium ~~1s found to be the WA MRSA, only resistance plasmid-borne.” This suggests that the cadmium-resistance plasmid could have acquired resistance to mupirocin, tetracycline and trimethoprim at the expense of penicillinase determinants. Tetracycline and trimethoprim could have been acquired from the chromosome of the \%‘A MRSA. ‘I’he fact that in two cases tetracycline resistant transcipients were obtained from the isolates without the transfer of a plasmid may provide clues to ho\v this multiple-resistance plasmid has evol\red. HoLvever there is no indication as to bvhere or how the mupirocin resistance determinants have emerged. The six mupirocin-resistant isolates lvere cultured from patients from northern WA on admission to a metropolitan hospital. Holyever, the true incidence of mupirocin resistance amongst MRSA in the communities in northern WA is not known. There is an urgent need to determine the incidence of mupirocin resistance in 5’. auveus and ,1IRSX in these communities. Guidelines for the use of mupirocin in the communit!. and hospitals in \VA are urgently needed. It is knoxvn that topical mupirocin (2% ointment) is frequently used in the north of L1TX to treat skin lesions and burns infected with S. aureus. Holvever, it is not possible at this time to accurately correlate the appearance of mupirocin resistance with the amount of mupirocin use in these communities. Experience else\vhere suggests that

164

E. E. Udo et al.

resistance to mupirocin mupirocin.30-32

can develop

during

prolonged

treatment

with

We thank Mrs P. I,. Perry, Scientist-in-Charge, Infection Control Laboratory, Royal Perth Hospital, for assistance. This work was supported by grants to WBG from the National Health and Medical Research Council and by a grant from the Royal Perth Hospital Medical Research Foundation to the authors.

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Mupirocin-resistant 20.

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22.

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2-C.

25.

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32. 33. 33. 35.

36.

MRSA

165

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