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JAC Advance Access originally published online on November 30, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):104-109; doi:10.1093/jac/dki428
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vitro activity of a novel compound, the metal ion chelating agent AQ+, against clinical isolates of Staphylococcus aureus

Benjamin R. D. Short, Miguel A. Vargas, Jonathan C. Thomas, Simon O'Hanlon and Mark C. Enright*

Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College London, Old Medical School Building, St Mary's Hospital, Norfolk Place, London W2 1PG, UK

* Corresponding author. Tel: +44-207-5943417; Fax: +44-207-5943693; E-mail: m.c.enright{at}imperial.ac.uk

Received 8 September 2005; returned 5 October 2005; revised 6 October 2005; accepted 1 November 2005


   Abstract
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Objectives: To determine the efficacy of a novel antimicrobial compound, AQ+, against a genetically heterogeneous collection comprising 213 Staphylococcus aureus isolates from global sources. AQ+ is an aqueous preparation containing 0.5% 8-hydroxyquinoline.

Methods: MICs were found for all the isolates tested using the BSAC microdilution method. Time–kill studies were performed according to NCCLS guidelines. Transmission electron microscopy (TEM) was used to view the ultrastructural effects of AQ+.

Results: AQ+ was shown to strongly inhibit the growth of all isolates with a median MIC of 0.25% at a pH optimum of 9.2. Lowering the pH to 7.5 gave an ~4-fold reduction in efficacy and at pH 5.5 there was an ~8-fold reduction in efficacy. Methicillin-resistant S. aureus (MRSA) as well as vancomycin-intermediate S. aureus were shown to be as equally susceptible to AQ+ as methicillin-susceptible S. aureus. Time–kill curves for AQ+ were similar to those for gentamicin. TEM showed that AQ+ actively disrupts the cell wall of S. aureus leading to cell lysis.

Conclusions: These results suggest that AQ+ has strong antimicrobial activity and may be useful in preparations to reduce nasal and skin carriage of MRSA.

Keywords: 8-hydroxyquinoline , S. aureus , MRSA , time–kill , antimicrobial susceptibility


   Introduction
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The Gram-positive bacterial pathogen Staphylococcus aureus is responsible for widespread community-acquired and nosocomial infections worldwide.1 This versatile pathogen can cause a wide range of disease in humans from boils and furuncles to more serious conditions such as endocarditis, septicaemia and pneumonia.2

S. aureus has a proven ability to adapt to different selection pressures and is a particularly acute problem in the hospital environment owing to its ability to rapidly acquire resistance to antibiotics and disinfectants.3 Resistant isolates have emerged following the introduction of all antibiotic classes. This was the case following the introduction of methicillin (a semi-synthetic penicillin) in 1959 with the first isolation of methicillin-resistant S. aureus (MRSA) in 1961.4 This example has been repeated for various antibiotics and now the emergence of MRSA strains with decreased susceptibility to the antibiotic of last resort, vancomycin, has been reported.5 The prevalence of antibiotic-resistant S. aureus strains has risen dramatically in the last decade and in the UK MRSA bacteraemias (as a percentage of S. aureus bacteraemias) rose from <2% in 1990 to 42% in 2000.6 Coupled to its predominance as a nosocomial infection MRSA is also emerging as a community-acquired pathogen in several countries.7

Effective hospital infection control practices including stringent isolation and disinfection guidelines are important in controlling the spread of antibiotic-resistant pathogens in hospitals.8 Regular handwashing in healthcare workers is often cited as being critical in reducing direct transmission of S. aureus and there is some evidence for this.9 Unfortunately, increasing resistance to commonly used disinfectants reduces the efficacy of regular handwashing10–12 and dermatological side effects associated with some compounds may lead to poor adherence to handwashing guidelines.13,14

AQ+ is a novel antimicrobial compound based on an aqueous formulation of 8-hydroxyquinoline. 8-Hydroxyquinoline is a potent lipophilic metal chelator whose 8-hydroxyquinoline copper chelate has been used as a fungicide in agriculture and for the preservation of textiles, wood and paper. We have determined MICs of AQ+ for 213 diverse isolates of S. aureus including representatives of all major MRSA clones.3 Time–kill studies were performed with AQ+, gentamicin, oxacillin and vancomycin to determine the comparative killing effect of the compound. Transmission electron microscopy (TEM) was used to evaluate the effect of AQ+ on cellular ultrastructure.


   Materials and methods
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Microorganisms

We examined a collection of 213 genotypically and phenotypically diverse S. aureus isolates including 147 isolates from pandemic MRSA lineages3 and 66 isolates representing community-acquired MRSA and methicillin-susceptible S. aureus (MSSA) lineages. This collection contained 110 MSSA and 103 MRSA representing all major MRSA lineages and included six isolates with reduced susceptibility to vancomycin [vancomycin-intermediate S. aureus (VISA)]. NCTC strain 6571 was used as a control isolate according to British Society for Antimicrobial Therapy (BSAC) guidelines.15 Isolates were stored at –80°C in a glycerol broth solution. Fresh subcultures were used for each experiment.

AQ+

AQ+ is the trademark name of a patented compound whose active ingredient is 8-hydroxyquinoline. This compound is buffered in a solution containing water, propyl glycol, Synperionic 91/6 and tetra-sodium EDTA. AQ+ is manufactured by AQ+ Plc, Angus, UK. It is not possible to test the active ingredient 8-hydroxyquinoline separately from its buffering solution owing to the instability of the compound in a non-aqueous phase. The AQ+ formulation tested contained 0.5% 8-hydroxyquinoline and MICs were found as the percentage of the complete formulation required to inhibit the visible growth of each strain. For ease of understanding MICs are referred to as %AQ+ in this study—as an example, an MIC value of 0.5% AQ+ would represent 25 ppm of the active compound 8-hydroxyquinoline.

Susceptibility testing

Susceptibility to different antibiotics and AQ+ was assessed using broth microdilution according to the BSAC guidelines for the determination of MICs.15 Iso-Sensitest broth (ISB; Oxoid, Basingstoke, UK) was used for all AQ+ susceptibility testing.

The susceptibility of isolates to oxacillin, gentamicin, vancomycin, cefoxitin, ciprofloxacin, clindamycin, erythromycin, linezolid, tetracycline, fusidic acid and penicillin was also measured using the agar dilution method outlined in the BSAC guidelines.15 These susceptibility data were corroborated using Etest strips (AB Biodisk, Sweden). Iso-Sensitest agar (Oxoid, UK) was also used for all plate dilution susceptibility testing.

Susceptibility of S. aureus to AQ+ was investigated at the optimum pH of 9.2. The effect of lowering the pH to 7.5 and 5.5 on the efficacy of AQ+ was also examined on a random subset of 50 isolates. The Mann–Whitney U-test was used to test for a significant difference in MIC between pH treatments.

Antimicrobial activity of buffer solution

Owing to the nature of 8-hydroxyquinoline it is very difficult to produce in a non-aqueous phase. Thus, all testing was performed on the complete formulation known by its trademark name of AQ+. This formulation contains two other compounds known to have antimicrobial activity; EDTA and Synperionic 91/6. Both of these compounds are present in very low concentrations but in order to be certain that the antimicrobial activity of AQ+ is mainly owing to the active compound (8-hydroxyquinoline) the buffer alone was tested against a representative subset of isolates.

Time–kill studies

Time–kill studies were performed on a subset of the strain collection comprising an epidemic MRSA isolate of clone UK EMRSA-7, a carried isolate from Oxford, D137, a laboratory control strain NCTC 6571 and the homo-VISA (homogeneous-VISA) isolate Mu50. Time–kill experiments were performed according to NCCLS guidelines16 using a protocol based upon a published study that examined the killing effect of tea tree oils.17 Isolates were grown overnight in ISB at 37°C with shaking. Overnight cultures were diluted to a final concentration of between 1 x 105 and 5 x 105 cfu/mL. Flasks were then shaken at 150 rpm for 90 min at 37°C so that the cultures were in logarithmic growth phase.

Each isolate was inoculated into two flasks, containing ISB and AQ+, oxacillin, gentamicin or vancomycin and a negative growth control of broth only. After 90 min of incubation at 37°C, 100 µL of the broth was taken to determine the initial inoculum by serial dilution. AQ+ or an antibiotic was then added to one flask and the buffer solution to the control flask. The final concentration of each antibiotic used was one doubling dilution above the MIC for each particular isolate. AQ+ was added in various concentrations above the MIC in order to determine how the killing rate of AQ+ varied with concentration. The MICs for each isolate are shown in Table 1. Each time–kill experiment was performed in triplicate.


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  		Table 1.. MICs for four S. aureus isolates examined using time–kill studies

 
Colony counts were performed by making appropriate dilutions in sterile distilled water then plating each onto blood agar (TSA, Oxoid) supplemented with 5% defibrinated sheep blood (TCS, Botolph Claydon, UK). Plates were incubated for 18 h at 37°C and viable counts were calculated to give cfu/mL.

Electron microscopy

TEM was performed on the laboratory strain 6571, and a homo-VISA isolate Mu50. These two isolates were chosen to reflect the diversity of the strain collection. The 6571 strain is an MSSA strain from the 1960s and Mu50 is a multidrug-resistant strain from the late 1990s. Samples were grown overnight in TSB, then inoculated into flasks containing either TSB or TSB supplemented with AQ+. Final concentrations of bacteria were 1–5 x 108 cfu/mL with AQ+ concentrations of 0.25% in treated flasks. Samples were then incubated for 90 min with shaking prior to centrifugation at 10 000 g for 10 min to pellet the cells. The supernatant was discarded and the cells were washed three times with PBS (Sigma, UK) prior to treatment for electron microscopy.

To fix the cells 2% gluteraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate was added and samples were incubated overnight at 4°C. To post-fix the cells 1% osmium tetraoxide was added and the samples were incubated for 1 h. The samples were then dehydrated in graded acetone dilutions of 30, 50, 70, 90 and 100% with three washes at each stage. Infiltration was achieved with various ratios of propylene oxide and Spurr's resin prior to an overnight incubation in 100% Spurr's resin and polymerization in 100% Spurr's resin at 70°C for 8 h.

Sample blocks were then trimmed and ultrathin-sectioned using a Reichert Ultracut Ultramicrotome to produce sections ~100 nm thick. These sections were then viewed on a JEOL1200 TEM and images were taken with a 35 mm camera.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
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Susceptibility testing

All isolates were inhibited by 10% AQ+, or lower, at an optimum pH of 9.2. The median MIC at this pH was 0.25%. MSSA, MRSA and VISA isolates were shown to be equally susceptible to AQ+. The susceptibility of the strain collection to AQ+ is illustrated in Table 2. The isolates were shown to have a considerable range of antibiotic resistance and this is summarized in Table 3. The susceptibility of S. aureus to AQ+ was not apparently influenced by the presence or absence of other antibiotic resistance determinants.


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  		Table 2.. Summary table showing the MICs of AQ+ for 213 global isolates of S. aureus at pH 9.2

 

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  		Table 3.. MIC data for various antibiotics against the strain collection

 
It was found that the buffer without 8-hydroxyquinoline was only inhibitory at concentrations of 20% or above. The median MIC for the strain collection was 0.25%, representing an ~80-fold higher antimicrobial activity with the main active ingredient than without.

The effect of pH on the efficacy of AQ+ was investigated in a subset of 50 isolates from the collection. At pH 7.5 the median MIC was significantly increased to 1% (P = 0.0000) and at pH 5.5 it was again significantly increased to 2.5% (P = 0.0004). Figure 1 shows the effect of changing pH on the efficacy of AQ+.



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  		Figure 1.. Effect of pH on the efficacy of AQ+ at inhibiting the growth of isolates of S. aureus.

 
Time–kill studies

Measurements of killing for AQ+, gentamicin, vancomycin and oxacillin were made for each isolate. Antimicrobials were added at a concentration of one doubling dilution above the MIC for each particular isolate unless otherwise stated. Each experiment was performed in triplicate and in all cases the buffer solution-containing controls showed at least a 103 increase in cfu/mL by the end of the 6 h test period.

AQ+ was shown to kill 99.9% of all cells from test isolates within the 6 h test period. The control MSSA strain 6571 was killed within 1 h, and Mu50 within 2 h. It took 6 h for a 99.9% kill of the MRSA strain EMRSA-7 and an MSSA strain D137 (Figure 2).



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  		Figure 2.. Killing of S. aureus by AQ+. Circles, epidemic MRSA strain EMRSA-7; crosses, nasally-carried MSSA strain D137; diamonds, VISA strain Mu50; and triangles, control MSSA strain 6571.

 
The time–kill curve for AQ+ against 6571 was similar in appearance to that of gentamicin. Oxacillin and vancomycin did not reach 99.9% kills over the 6 h study period (Figure 3).



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  		Figure 3.. Killing of MSSA strain 6571 by AQ+, oxacillin, vancomycin and gentamicin. Filled circles, AQ+; open circles, gentamicin; crosses, oxacillin; and filled triangles, vancomycin.

 
Isolates were killed more rapidly at higher concentrations of AQ+ (Figure 4). It took 2 h for a 99.9% kill of 6571 at a concentration one dilution above the MIC for that isolate. Increasing the concentration 10-fold to 5% reduced the time required to reach a 99.9% kill to 1 h. The latter concentration is much closer to those in AQ+-containing products.



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  		Figure 4.. Killing of laboratory MSSA strain 6571 by different concentrations of AQ+. Circles, 0.5%; crosses, 5%.

 
Electron microscopy

TEM was used to investigate the nature of the interaction between AQ+ and S. aureus. Two exponentially growing isolates of S. aureus were treated with AQ+ at concentrations equal to their MIC (Table 1) before they were fixed, ultrathin-sectioned and examined using TEM. We examined the laboratory MSSA strain 6571 and the homo-VISA strain Mu50.

The images of cells treated with AQ+ were greatly different from those left untreated. Untreated cells were uniform in structure with intact cell walls, whereas many of the treated cells had very diffuse cell walls and appeared to have shed cell wall material (Figure 5). In addition, many lysed cells and large amounts of cell debris were present with only empty wall structures remaining. These results suggest that AQ+ induces lysis and subsequent cell death in S. aureus.



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  		Figure 5.. TEM images taken of S. aureus 6571 and Mu50 cells treated with AQ+ at concentrations equal to their MIC. (a and c) Untreated controls. The cell walls of treated cells are darker indicating an increase in electron density, they are also more diffuse and ejection of wall material appears common (b). Ghost cells were also seen in treated samples together with large amounts of cell debris (d and e).

 

   Discussion
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 Materials and methods
 Results
 Discussion
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The number of infections caused by multidrug-resistant bacteria is increasing and this causes major problems in healthcare settings. With increasing antibiotic and disinfectant resistance, effective infection control becomes increasingly important. Our in vitro results suggest that AQ+ might be a useful topical agent for the eradication of S. aureus including multidrug-resistant MRSA.

AQ+ is the trademark name of an aqueous solution containing the lipophilic metal chelator 8-hydroxyquinoline. Here, we demonstrated the bactericidal activity of this novel compound against a collection of MSSA, MRSA and VISA including representatives of all major nosocomial MRSA clones. The median MIC of 0.25% equates to a very low concentration of active ingredient (12.5 ppm) indicating the extremely active nature of this compound.

The median MIC was increased significantly by lowering the pH from 9.2 to 7.5 and further increased at pH 5.5. However, all isolates were inhibited even at these lower pHs and the median MIC at pH 7.5 was 1%, which equates to a much lower concentration of active ingredient than is found in product formulations.

Time–kill studies show that increasing the AQ+ concentration from 0.5% (one doubling dilution above the MIC of 0.25%) to 5% decreases the 99.9% kill from 2 h down to 1 h. Killing curves also demonstrate the ability of AQ+ to kill all bacterial populations of MSSA, MRSA and homo-VISA within 6 h.

Cells treated with AQ+ show altered cell wall structure and were also more electron dense than untreated controls. The presence of ghost cells was common in treated samples indicating cell death and subsequent lysis.

MRSA is preferentially carried in the anterior nares and mupirocin-containing nasal ointment is widely used to eradicate nasal carriage; however, resistance to this antibiotic, although not yet widespread, is increasing.18 Our results suggest that AQ+ could be a viable alternative to mupirocin in eradicating nasal carriage of MRSA although the activity of the compound against mupirocin-resistant isolates was not tested here.

MRSA and other drug-resistant pathogens are difficult to eradicate from the skin and direct contact is known to be an important route of transmission.19 Various studies have highlighted the importance of adhering to a strict hand disinfection regimen20 with the use of alcohol gels promoted by various bodies.21,22 However, regular use of alcohol gels may promote dermatological side effects.23 These may lead healthcare personnel to reduce their compliance with handwashing regimens recommended by Infection Control specialists. The rapid time–kill of AQ+ against MRSA indicates that it could be an effective active ingredient as part of a hand cleaning product. No dermatological side effects have been observed with heavy use (more than 30 washes per day) of this compound (B. R. D. Short, unpublished results).

We conclude that AQ+ could be a valuable antimicrobial agent for use in formulations to eradicate carriage and help reduce MRSA transmission.


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MCE is a paid consultant of AQ+ PLC. The funding source had no role in study design, data collection, analysis, interpretation or writing of the report.


   Acknowledgements
 
We kindly thank Ursula Potter for the TEM work and Laura Piddock for a critical review of the manuscript prior to submission. This work was funded by a research grant from AQ+ PLC.


   References
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 Materials and methods
 Results
 Discussion
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 References
 
1. Diekema DJ, Pfaller MA, Schmitz FJ et al. Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis 2001; 32 Suppl 2: S114–32.

2. Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998; 339: 520–32.[Free Full Text]

3. Robinson DA, Enright MC. Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2003; 47: 3926–34.[Abstract/Free Full Text]

4. Jevons M. Celbenin-resistant Staphylococci. Br Med J 1961; i: 124–5.

5. Chang S, Sievert DM, Hageman JC et al. Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene. N Engl J Med 2003; 348: 1342–7.[Free Full Text]

6. Reacher MH, Shah A, Livermore DM et al. Bacteraemia and antibiotic resistance of its pathogens reported in England and Wales between 1990 and 1998: trend analysis. Br Med J 2000; 320: 213–6.[Abstract/Free Full Text]

7. Chambers HF. Community-associated MRSA-resistance and virulence converge. N Engl J Med 2005; 352: 1485–7.[Free Full Text]

8. Cooper BS, Stone SP, Kibbler CC et al. Systematic review of isolation policies in the hospital management of methicillin-resistant Staphylococcus aureus: a review of the literature with epidemiological and economic modelling. Health Technol Assess 2003; 7: 1–194.[Medline]

9. Larson EL. APIC guideline for handwashing and hand antisepsis in health care settings. Am J Infect Control 1995; 23: 251–69.[CrossRef][ISI][Medline]

10. Irizarry L, Merlin T, Rupp J et al. Reduced susceptibility of methicillin-resistant Staphylococcus aureus to cetylpyridinium chloride and chlorhexidine. Chemotherapy 1996; 42: 248–52.[ISI][Medline]

11. Kampf G, Jarosch R, Ruden H. Limited effectiveness of chlorhexidine based hand disinfectants against methicillin-resistant Staphylococcus aureus (MRSA). J Hosp Infect 1998; 38: 297–303.[CrossRef][ISI][Medline]

12. Bamber AI, Neal TJ. An assessment of triclosan susceptibility in methicillin-resistant and methicillin-sensitive Staphylococcus aureus. J Hosp Infect 1999; 41: 107–9.[CrossRef][ISI][Medline]

13. Kampf G, Muscatiello M, Hantschel D et al. Dermal tolerance and effect on skin hydration of a new ethanol-based hand gel. J Hosp Infect 2002; 52: 297–301.[CrossRef][ISI][Medline]

14. Wilkinson SM. Effects of infection control measures on skin of health care workers. Commun Dis Public Health 2000; 3: 305–6.[Medline]

15. Working Party on Susceptibility Testing of the British Society for Antimicrobial Chemotherapy. Antimicrobial Susceptibility Testing: BSAC Working Party Report. J Antimicrob Chemother 2001; 48 Suppl S1.

16. National Committee for Clinical and Laboratory Standards. Methods for Determining Bactericidal Activity of Antimicrobial Agents: Tentative Guideline M26-T. NCCLS, Villanova, PA, 1992.

17. May J, Chan CH, King A et al. Time–kill studies of tea tree oils on clinical isolates. J Antimicrob Chemother 2000; 45: 639–43.[Abstract/Free Full Text]

18. Irish D, Eltringham I, Teall A et al. Control of an outbreak of an epidemic methicillin-resistant Staphylococcus aureus also resistant to mupirocin. J Hosp Infect 1998; 39: 19–26.[CrossRef][ISI][Medline]

19. Reybrouck G. Handwashing and hand disinfection. J Hosp Infect 1986; 8: 5–23.[CrossRef][ISI][Medline]

20. MacDonald A, Dinah F, MacKenzie D et al. Performance feedback of hand hygiene, using alcohol gel as the skin decontaminant, reduces the number of inpatients newly affected by MRSA and antibiotic costs. J Hosp Infect 2004; 56: 56–63.[CrossRef][ISI][Medline]

21. Boyce JM, Pittet D. Guideline for Hand Hygiene in Health-Care Settings: recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Infect Control Hosp Epidemiol 2002; 23: S3–40.[CrossRef][ISI][Medline]

22. Healthcare Infection Control Practices Advisory Committee and Hand-Hygiene Task Force; Society for Healthcare Epidemiology of America; Association for Professionals in Infection Control and Epidemiology; Infection Diseases Society of America. Guideline for hand hygiene in healthcare settings. J Am Coll Surg 2004; 198: 121–7.[CrossRef][ISI][Medline]

23. Forrester BG, Roth VS. Hand dermatitis in intensive care units. J Occup Environ Med 1998; 40: 881–5.[CrossRef][ISI][Medline]


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