Effects of sarA inactivation on the intrinsic multidrug resistance mechanism of Staphylococcus aureus

FEMS Microbiology Letters 237 (2004) 297–302 www.fems-microbiology.org

Effects of sarA inactivation on the intrinsic multidrug resistance mechanism of Staphylococcus aureus Jessica O. OÕLeary a, Mark J. Langevin a, Christopher T.D. Price b, Jon S. Blevins c, Mark S. Smeltzer c, John E. Gustafson a,* a

c

Microbiology Group, Department of Biology, New Mexico State University, Las Cruces, NM 88003-8001, USA b School of Biomedical Sciences, Curtin University of Technology, Perth, WA 6845, Australia Departments of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Received 18 May 2004; received in revised form 27 June 2004; accepted 28 June 2004 First published online 6 July 2004

Abstract The sarA locus of Staphylococccus aureus regulates the synthesis of over 100 genes on the S. aureus chromosome. We now report the effects of sarA inactivation on intrinsic multidrug resistance expression by S. aureus. In a strain-dependent fashion, sarA::kan mutants of three unrelated strains of S. aureus demonstrated significantly increased susceptibility to five or more of the following substances: the antibiotics ciprofloxacin, fusidic acid, and vancomycin; the DNA-intercalating agent ethidium; and four common household cleaner formulations. In addition, all three sarA::kan mutants demonstrated significantly increased accumulation of ciprofloxacin and one sarA::kan mutant demonstrated increased ethidium accumulation. Our data therefore indicate that sarA plays a role in the intrinsic multidrug resistance mechanism expressed by S. aureus, in part by regulating drug accumulation.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Staphylococcus aureus; Multidrug resistance; sarA

1. Introduction The synthesis of Staphylococcus aureus virulence factors is in part coordinately controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) (for review see [6,15]). The agr operon encodes for two divergent transcripts designated RNAII and RNAIII which are controlled by two promoters (P2 and P3). RNAII is translated into the polypeptides, AgrB, AgrD, AgrC and AgrA, all making up a quorom sensing system that is required for the activation of RNAII and RNAIII [15]. RNAIII encodes a small hemolysin and acts as a regulatory RNA, repressing surface *

Corresponding author. Tel.: +1-505-646-5660; fax: +1-505-6465665. E-mail address: [email protected] (J.E. Gustafson).

protein production and concomitantly increasing the production of exotoxins [14,17,19]. sarA mutants demonstrate diminished production of RNAII and RNAIII [5], and physical binding of SarA to a sequence between the agr P2 and P3 promoters has been demonstrated [4]. The sarA locus harbors three promoters which produce three overlapping transcripts, all having a common 3 0 end and encoding SarA [1]. In addition to coordinating the synthesis of virulence factors, sarA influences the expression of over 100 genes located on the S. aureus chromosome, revealing its global regulatory nature [8]. Recent evidence has demonstrated that SarA may be physically binding to many of the promoters of genes that sarA has been shown to regulate [26]. Besides its role in virulence factor production, sarA is also required for the full expression of intrinsic

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2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.06.047

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resistance to b-lactam antibiotics [9,21]. agr has also been implicated to play a role in the expression of vancomycin-intermediate resistance expression [24]. These reports demonstrate a role for sarA and agr in the intrinsic response mechanism of S. aureus to cell wall active antibiotics. We now provide direct evidence demonstrating a role for sarA in the intrinsic multidrug resistance mechanism of S. aureus.

2. Materials and methods 2.1. Bacterial strains, strain maintenance and chemicals The strains used in this study were previously described by Blevins et al. [3]. The collection investigated included strains Newman, RN6390, S6C and their isogenic sarA::kan mutants, Newman sarA::kan, RN6390 sarA::kan and S6C sarA::kan. All experiments were performed at 37 C and liquid cultures were maintained at 200 rpm and initiated with 1% (v/v) inoculums from overnight cultures (18 h). All parent strain working stocks were maintained on DifcoTM Luria broth base (Becton Dickinson and Company, Sparks MD, USA) agar (LBA) at 4 C or stored following growth in Luria broth (LB) and glycerol addition (20% v/v final concentration) at
20 or
80 C. sarA::kan mutant working stocks were maintained on LBA containing kanamycin (50 mg l
1) and stored as the parent strains following growth in LB containing kanamycin.

Stocks of ciprofloxacin (Bayer Corporation, Morristown, NJ, USA), fusidic acid (Leo Pharmaceuticals, Ballerup, Denmark), vancomycin, oxacillin, and ethidium bromide (Sigma Aldrich, St. Louis, MO, USA) were all made up in ddH2O and stored at
20 C until required. The house cleaners used were Pine-Sol (PS) (Clorox Company, Oakland, CA, USA ) Orange Clean Super Concentrate (OC) (Orange Glo International Inc., Littleton, CO, USA) Simple Green (SG) (Sunshine Makers Inc., Huntington Harbor, CA, USA) and Parsons Lemon Ammonia (PA) (Procter & Gamble, Cincinnati, OH, USA) which were stored in their original containers at 25 C. Single lots of each cleaner formulation were used for all experimentation. 2.2. Drug gradient plate analysis and antibiotic resistance population analysis Gradient plates were prepared in square plates (90 mm · 90 mm) with LBA. Initially 40 ml of molten LBA (50 C) was poured into a square plate raised at one edge by a 1 ml glass pipette (6 mm) and allowed to solidify overnight. The next day, 40 ml of molten LBA containing one of antibacterials being analyzed (see Table 1 for gradient concentrations) was poured into a plate containing the first slanted layer of LBA lying flat, and allowed to solidify. All plates containing the continuous drug gradients were then dried open face down at 37 C for 1–2 h before being inoculated. Overnight LB cultures inoculated with single colonies were diluted to an OD625 of 0.1 with LB and streaked three

Table 1 Gradient plate analyses

Strain

0 fi 0.5 (mg l
1) or 0 fi 2.0 (mg l
1)a Cip

0 fi 0.2 (mg l
1) or 0 fi 0.3 (mg l
1)a Fus

0 fi 1.5 (mg l
1) or 0 fi 1.2 (mg l
1)a Van

0 fi 20 (mg l
1) or 0 fi 30 (mg l
1)a EtBr

Newman Newman sarA::kan RN6390 RN6390 sarA::kan S6C S6C sarA::kan

21 ± 2.0 11 ± 4.0b 21 ± 0 14 ± 1.5b 29 ± 1.0 23 ± 2.0b

23 ± 3.0 16 ± 1.0b 25 ± 2.0 19 ± 3.5 34 ± 1.5 24 ± 4.5b

27 ± 2.5 14 ± 2.0b 13 ± 2.0 9 ± 2.0 15 ± 0.5 9 ± 1.5b

28 ± 1.2 18 ± 0.6b 21 ± 0.6 10 ± 0.6b >90 28 ± 0.6b

0 fi 1.0% (v/v) or 0 fi 0.6% (v/v)a PS

0 fi 0.15% (v/v)

0 fi 0.45% (v/v)

OC

0 fi 13% (v/v) or 0 fi 14% (v/v)a SG

PA

36 ± 1.0 0b 17 ± 0.5 10 ± 2.5b 13 ± 1.5 1 ± 2.0b

37 ± 7.3 0b 31 ± 1.2 9 ± 1.2b 15 ± 0.6 9 ± 1.5b

>90 6 ± 3.2b 18 ± 1.0 16 ± 1.2 30 ± 0.6 15 ± 1.7b

37 ± 11.6 15 ± 0.5 58 ± 0.6 30 ± 0.6b 21 ± 1.0 10 ± 0.6b

Drug gradients

Newman Newman sarA::kan RN6390 RN6390 sarA::kan S6C S6C sarA::kan

Numbers represent mm grown on 90 mm gradient plates and standard deviations (n = 3). Abbreviations: Cip, ciprofloxacin; Fus, fusidic acid; Van, vancomycin; EtBr, ethidium bromide; PS, Pine Sol; OC, Orange Clean Super Concentrate; SG, Simple Green; and PA, ParsonÕs Lemon Ammonia. a Gradients used for strains S6C and S6C sarA::kan. b Represents significant decrease in resistance in comparison to parent strain (p-value <0.05).

J.O. OÕLeary et al. / FEMS Microbiology Letters 237 (2004) 297–302

2.3. Ciprofloxacin and ethidium accumulation assays Fluorometric ciprofloxacin (kexcite, 277 nm; kemit, 447 nm) and ethidium (kexcite, 530 nm; kemit, 600 nm) accumulation assays were carried out as previously described [22] using a luminescence spectrometer (Cary Eclipse).

8

log 10 CFUs ml-1

7 6 5 4 3 2 1 0 0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Ciprofloxacin mg 1-1

(a) 8 7

log 10 CFUs ml-1

times onto freshly prepared gradient plates with sterile cotton swabs. Inoculated plates were incubated at 37 C and read following 48 h incubation. The point at which confluent bacterial growth halted was reported as mm grown on the gradient. Vancomycin and ciprofloxacin resistance population analyses were performed using the microdilution method of Pfeltz et al. [20] with LBA plates containing different concentrations of vancomycin or ciprofloxacin. All plates were spot-inoculated with 10 ll aliquots of 10
1–10
8 dilutions of an overnight LB culture previously diluted to an OD625 of 1.0. The spots were then allowed to soak into the agar surface and the plates were incubated 48 h before colony forming units (CFUs) within the spots were scored.

299

6 5 4 3 2 1 0 0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Ciprofloxacin mg 1-1

(b) 8

3. Results

Distances grown by the strains on the drug gradients investigated demonstrated that: S6C expressed the greatest resistance to ciprofloxacin, fusidic acid and ethidium; Newman expressed the greatest resistance to vancomycin, PS and SG; and RN6390 demonstrated the greatest resistance to PA and nearly equaled Newman in expressing the highest level of OC resistance (Table 1). On ciprofloxacin gradient plates all three sarA::kan mutants demonstrated reductions in distances grown compared to parent strains Newman, RN6390, and S6C (Table 1). On fusidic acid and vancomycin gradients, reductions in distances grown were observed for Newman sarA::kan and S6CsarA::kan compared to their respective parent strains. Compared to RN6390, RN6390 sarA::kan did not demonstrate significant reductions in distances grown on the vancomycin and fusidic acid gradients (Table 1). On ethidium gradient plates all three sarA::kan mutants demonstrated reductions in distances grown compared to parent strains (Table 1). On PS, OC, SG and PA gradient plates, significant reductions in distances grown were observed for all sarA::kan mutants compared to their respective parent strains, except for Newman sarA::kan on PA gradients, and RN6390 sarA::kan on SG gradients (Table 1).

log 10 CFUs ml-1

3.1. Effects of sarA inactivation on intrinsic multidrug resistance

7 6 5 4 3 2 1 0 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

(c)

Ciprofloxacin mg 1-1

Fig. 1. Ciprofloxacin resistance population analyses: (a) Newman, open circle (s), Newman sarA::kan, closed circle (d); (b) RN6390, open square (h), RN6390 sarA::kan, closed square (j); and (c) S6C, open diamond (e), S6C sarA::kan closed diamond (r).

3.2. Effects of sarA ciprofloxacin- and vancomycinresistance population analysis All sarA::kan mutants demonstrated reduced survival on increasing ciprofloxacin concentrations compared to their respective parent strains in ciprofloxacin resistance population analyses (Fig. 1). The number of Newman CFUs surviving on 0.05, 0.1 and 0.15 mg l
1 ciprofloxacin, were one log greater than Newman sarA::kan. Neither Newman nor Newman sarA::kan survived on concentrations over 0.25 mg l
1 (Fig. 1(a)). RN6390 and RN6390 sarA::kan compare in CFU survival up

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to 0.20 mg l
1 ciprofloxacin. On ciprofloxacin concentrations above 0.20 mg l
1 RN6390 sarA::kan did not survive, while RN6390 CFUs continued to arise up to 0.35 mg l
1 (Fig. 1(b)). S6C CFUs appeared on all ciprofloxacin concentrations investigated, whereas S6C sarA::kan did not survive past a concentration of 2.0 mg l
1 (Fig. 1(c)). Compared to the other strain sets investigated, S6C and S6C sarA::kan survived on the high-

Table 2 Drug accumulation Strain

Ciprofloxacina

Ethidiumb

Newman NewmansarA::kan

116 ± 2.9 125 ± 3.4c

266 ± 30 249 ± 15

RN6390 RN6390sarA::kan

130 ± 2.5 154 ± 6.4c

298 ± 10 325 ± 18

S6C S6CsarA::kan

88 ± 5.2 114 ± 1.7c

266 ± 16 321 ± 4.7c

ng Ciprofloxacin mg
1 dry cell weight. Arbitrary units. c Represents significant increase in accumulation in comparison parent strain (n = 3, p-value <0.05). a

b

9 8

log 10 CFUs ml-1

7 6 5 4 3 2 1 0 0.0

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Vancomycin mg 1-1

(a) 8 7

log 10 CFUs ml-1

6 5 4 3 2 1 0 0.0

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Vancomycin mg 1-1

(b)

est concentrations of ciprofloxacin investigated (Fig. 1), which concurs with ciprofloxacin gradient plate data. Varied results were obtained when vancomycin resistance population analyses were performed with sarA::kan mutants and parent strains. Both Newman sarA::kan and S6C sarA::kan demonstrated reduced survival on increasing concentrations of vancomycin compared to parent strains (Fig. 2(a) and (c)). Compared to Newman sarA::kan, Newman demonstrated greater survival on all vancomycin concentrations examined except 0.8 mg l
1 (Fig. 2(a)). Compared to the other strain sets investigated, Newman and Newman sarA::kan survived on the highest vancomycin concentrations investigated (Fig. 2), which concurs with vancomycin gradient plate data (Table 1). Interestingly, greater numbers of RN6390 sarA::kan CFUs appeared on 0.3–0.9 mg l
1 vancomycin, compared to RN6390, yet both strains did not survive past 0.9 mg l
1 vancomycin (Fig. 2(b)). On vancomycin gradient plates, no significant difference in distances grown was observed between RN6390 and RN6390 sarA::kan (Table 1). S6C sarA::kan did not survive past 0.4 mg l
1 vancomycin, while S6C CFUs survived up to 0.7 mg l
1 (Fig. 2(c)).

8

3.3. Ciprofloxacin and ethidium accumulation assays

7

log10 CFUs ml-1

6

All three sarA::kan mutants demonstrated greater ciprofloxacin accumulation compared to parent strains in ciprofloxacin accumulation assays (Table 2). Only S6C sarA::kan demonstrated a significant increase in ethidum accumulation compared to S6C (Table 2).

5 4 3 2 1

4. Discussion 0 0.00

(c)

0.20

0.30

0.40

0.50

0.55

0.60

0.70

0.80

Vancomycin mg 1-1

Fig. 2. Vancomycin resistance population analyses: (a) Newman, open circle (s), Newman sarA::kan, closed circle (d); (b) RN6390, open square (h)), RN6390 sarA::kan, closed square (j); and (c) S6C, open diamond (e), S6C sarA::kan closed diamond (r).

In a strain-dependent fashion, sarA::kan mutants of three unrelated strains of S. aureus demonstrated significantly increased susceptibility to at least five or more of the antibacterials investigated. The antibacterials investigated included: a DNA synthesis inhibitor

J.O. OÕLeary et al. / FEMS Microbiology Letters 237 (2004) 297–302

(ciprofloxacin), a protein synthesis inhibitor (fusidic acid); a peptidoglycan synthesis inhibitor (vancomycin); a DNA-intercalating agent (ethidium); and four common household cleaner formulations which probably target the cell membrane at least. In addition, all three sarA::kan mutants demonstrated significantly increased accumulation of ciprofloxacin and one sarA::kan mutant demonstrated increased ethidium accumulation. Besides representing unique genetic lineages, the strains utilized in this study also exhibited unique multidrug resistance phenotypes (Table 1, Figs. 1 and 2). Inactivation of sarA in RN6390 did not lead to significant decreases in vancomycin, fusidic acid and SG resistance levels. In fact, vancomycin resistance population analysis demonstrated increased survival of RN6390 sarA::kan compared to RN6390, but both strains did die off at the same vancomycin concentration. RN6390 is an 8325-4 derivative, which is known to harbor a mutation in the rsbU gene of the alternative sigma factor (sigB) operon [12]. Since RsbU is required for the activation of SigB, 8325-4 derivatives express phenotypes comparable to sigB deletion mutants [12]. SigB inactivation has been reported to lead to a reduction in sarA expression [2,11] and SigB is required for the activation of one of the sarA promoters (P3) in vitro [7]. Indeed, RN6390 previously was shown to produce reduced amounts of the P3 SigB-dependent sarA transcript [3]. SigB is also required for the full expression of resistance to vancomycin and PS [23,25], as well as the response of this organism to other stressing environments [10]. Therefore the lack of active SigB in RN6390 could lead to reduced SarA production and perhaps inactivation of sarA in this strain does not impact multidrug resistance expression as much as the other strains investigated. The ethidium level (>30 mg l
1) that S6C grew up to may indicate that it carries one of the qac genes which encode efflux pumps that are known to mediate resistance to ethidium [27]. Only S6C demonstrated significantly increased ethidium accumulation following sarA inactivation. Perhaps an ethidium efflux mechanism in S6C requires sarA to function appropriately. In addition, the level of ciprofloxacin resistance expressed by S6C on gradient plate and population analysis probably indicates this strain to be a first step fluoroquinolone-resistant topisomerase IV mutant as well [18]. Regardless of strain background, inactivation of sarA led to a reduction in the resistance to at least five antibacterial agents, clearly defining a role for sarA in the intrinsic multidrug resistance mechanism of S. aureus. Since sarA controls over 100 genes on the S. aureus chromosome, defining the exact role for sarA in the response to multiple antibacterials becomes complicated. However, recent reports from other laboratories offer tantalizing connections between sarA and intrinsic multidrug resistance expression. The norR gene (also

301

known as rat or mgr [13,16]), of S. aureus produces a gene regulator that affects intrinsic resistance to fluoroquinolones and ethidium, is a member of the SarA family of proteins, and acts by upregulating the expression of norA, which encodes a multidrug efflux pump [28]. The overexpression of norR in sarA mutants does not result in overexpression of norA, demonstrating that the effect of norR on norA expression is sarA-dependent [28]. Furthermore, sarA also upregulates the synthesis of another putative NorA paralogue of S. aureus [8]. We hypothesis that sarA inactivation leads to the reduced synthesis or activity of a putative multidrug efflux pump(s) and disallows norR from upregulating NorA, thereby leading to increased drug accumulation and susceptibility to multiple antibacterials. The exact role that SarA plays in the response of S. aureus to multiple antibacterials deserves further attention. The potential role of additional sarA paralogues [6] in the multidrug resistance mechanism of S. aureus should also be investigated.

Acknowledgement This work was supported by NIH R15 grant AI054382-01 (JEG), MBRS-NIH-RISE GM612222 (NMSU) and NIH-MARC GMO-7666726 (NMSU).

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