Journal of Antimicrobial Chemotherapy (2006) 58, 193–197doi:10.1093/jac/dkl206Advance Access publication 2 June 2006
Antimicrobial and toxicological profile of the new biocide
Astrid Buxbaum, Christina Kratzer, Wolfgang Graninger and Apostolos Georgopoulos*
Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy,
Medical University of Vienna, Vienna, Austria
Received 1 December 2005; returned 16 January 2006; revised 18 April 2006; accepted 27 April 2006
Objectives: Akacid plusÒ is a new member of the polymeric guanidine family of disinfectants. It wasespecially developed to enhance the antimicrobial activity of this class with significantly less toxicity.
The in vitro activity of Akacid plusÒ compared with chlorhexidine digluconate and mupirocin was testedagainst a total of 369 recent clinical isolates.
Methods: The organisms tested by CLSI reference methods included the following: Staphylococcusaureus (98), Staphylococcus epidermidis (9), Bacillus spp. (2), Enterococcus faecalis (32), Klebsiellaspp. (45), Enterobacter spp. (20), Escherichia coli (65), Salmonella spp. (6), Shigella spp. (2), Yersiniaenterocolitica (1), Acinetobacter spp. (4), Proteus spp. (7), Pseudomonas aeruginosa (59), Stenotrophomo-nas maltophilia (4), Candida spp. (10) and Aspergillus spp. (7). In vitro selection of resistance to AkacidplusÒ was carried out on 24 strains. Toxicological analyses were also performed.
Results: All tested agents were more effective against Staphylococcus spp. and Bacillus spp. than againstE. faecalis and Gram-negative bacteria. The MIC90s of chlorhexidine and mupirocin showed a 4-fold and32-fold increase for methicillin-resistant S. aureus in comparison with methicillin-susceptible strains,while MIC values of Akacid plusÒ were similar for antibiotic-susceptible and multiresistant strains.
Bactericidal action of Akacid plusÒ was observed at 1–2· MIC. The in vitro selection of resistance testshowed no increase in MIC values of Akacid plusÒ for any isolate after 30 passages. In addition, AkacidplusÒ showed low oral and dermal toxicity.
Conclusions: These preliminary results demonstrate the broad antimicrobial properties of AkacidplusÒ, which makes it a promising tool for topical application in the prophylaxis and treatment ofbacterial and fungal infections.
Keywords: bactericidal, resistance, toxicity
of multiresistant infectious-disease organisms that are difficultand, sometimes, impossible to treat, the search for new agents
The discovery and application of antimicrobial chemotherapy and
that do not select for resistant clones becomes ever more
the use of biocides in the form of antiseptics and disinfectants,
particularly in the latter half of the twentieth century, allowed
However, this issue has been further complicated by the find-
control over most infectious diseases. The emergence of bacterial
ing that, as for antibiotics, intensive exposure of hospital patho-
resistance to antimicrobial agents began shortly after their intro-
gens to biocides may result in the emergence of resistance to
duction to clinical practice and has developed rapidly and
these agents. Evidence for reduced susceptibility to biocides from
exposure to these agents has been both laboratory based3 and
Biocides are clearly different from antibiotics in their mode of
action, in their condition of use and in their respective acquired
Akacid plusÒ is a new member of the polymeric guanidine
and intrinsic mechanisms by which bacteria resist their toxic
family of disinfectants. It was especially developed to enhance
effects, and they often display non-specific killing. In the face
the antimicrobial activity of this class with significantly less
*Corresponding author. Tel: +43-1-40400/5139; Fax: +43-1-40400/5200; E-mail: [email protected]
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toxicity. This paper evaluates the antimicrobial profile of Akacid
plusÒ in comparison with chlorhexidine digluconate (due to its
The toxicological studies were performed at the Toxicology Depart-
widespread use) and mupirocin [due to its topical use in the
ment of ARC Seibersdorf Research GmbH (Seibersdorf, Austria).
hospital setting against methicillin-resistant Staphylococcus aur-
The approval numbers for the animal experiments are LF1-TVG-
eus (MRSA)], its toxicity and the potential for induction of
The acute toxic effects of Akacid plusÒ after a single peroral
administration to rats were determined according to EU method
B.1.10 Initially the study was carried out with one group consistingof three female animals given a dose of 200 mg of active ingredient
per kg of body weight. Based on these observation results the dosewas increased to 2000 mg/kg of body weight. All rats were killed by
A total of 369 recent clinical isolates were tested from patients
with documented infections in hospitals located in Austria. The
2 on day 14 and subjected to a gross necropsy
distribution of species and strain counts was as follows: methicillin-
The acute toxic effects of Akacid plusÒ after a single dermal
susceptible S. aureus (MSSA) (36); MRSA (62); methicillin-resistant
administration to rats were investigated according to EU method
Staphylococcus epidermidis (MRSE) (9); vancomycin-susceptible
B.3.11 Akacid plusÒ at a dose of 2000 mg/kg of body weight was
Enterococcus faecalis (27); vancomycin-resistant E. faecalis (VRE)
(5); Klebsiella spp. (45, 15.5% ESBLs); Enterobacter spp. (20);
thoracal region of five male and five female CRL:CD(SD) BR
Escherichia coli (65, 13.8% ESBLs) Salmonella spp. (6), Shigella
Sprague Dawley rats from Charles River Wiga (Germany) and the
spp. (2); Yersinia enterocolitica (1); Acinetobacter spp. (4); Proteus
duration of the exposure was 24 h. They were killed by inhalation of
spp. (7); Pseudomonas aeruginosa (59, 28.8% ESBLs); Stenotro-
phomonas maltophilia (4); Candida spp. (10); Aspergillus spp. (7).
2 after 14 days and subjected to a necropsy including a gross
Identifications were performed using the API system. In addition,Bacillus subtilis (spore suspension for the inhibitor test, Merck)
To examine a possible irritation or corrosion by Akacid plusÒ
and Bacillus anthracis CH10 (anthrax spores Merck reg. no.
following a single application to the intact skin of rabbits the EU
method B.412 was performed. The test substance (1.5 g) was spreadon cellulose patches in a size of about 2.5 · 2.5 cm and was appliedto the intact skin of each of three female New Zealand White rabbits
from Charles River Wiga. At the end of the exposure period (4 h) the
A stock solution of Akacid plusÒ, a 3:1 mixture of poly-(hexam-
dressings and the patches were removed. The skin was examined for
ethylen-guanidinium-chloride) and poly-[2-(2-ethoxy)-ethoxyethyl)-
erythema/eschar and oedema as well as for other local alterations 1,
guanidinium-chloride] (Ch. 1007, POC), as 25% aqueous solution
24, 48 and 72 h after patch removal.
was used and diluted with sterile distilled water to the desired con-centrations. Chlorhexidine digluconate 20% (Sigma, St Louis, MO,USA) and mupirocin powder (Smith Kline Beecham, London, UK)
were selected as reference substances.
Table 1 illustrates the activity of Akacid plusÒ in comparisonwith chlorhexidine digluconate and mupirocin against ATCC
To assess the antimicrobial activity of Akacid plusÒ in comparison
strains and clinical bacterial and fungal isolates. MIC values
with chlorhexidine and mupirocin, MICs were determined using the
of chlorhexidine digluconate and mupirocin were comparable
CLSI broth microdilution method with Mueller–Hinton broth.5 Forfungal testing 3-(N-morpholino)propanesulfonic acid-buffered RPMI
to the results obtained by other studies.4 Akacid plusÒ showed
1640 medium was used.6,7 MIC endpoints were read as the lowest
good activity against staphylococci with MICs of 0.06–0.5 mg/L,
concentration of antimicrobial that totally inhibited macroscopically
regardless of their susceptibility to oxacillin. The MIC90s of
visible growth of the inoculum. Quality control was provided by the
chlorhexidine and mupirocin showed a 4-fold (0.5 to 2 mg/L)
concurrent testing of ATCC strains. MBCs of Akacid plusÒ were
and 32-fold (0.25 to 8 mg/L) increase for MRSA in comparison
determined by methods published by the CLSI.8 All susceptibility
with methicillin-susceptible strains. All tested agents achieved
lesser activity against E. faecalis (2–128 mg/L), but no difference
Killing curves for Akacid plusÒ were carried out on S. aureus
in the MIC values was detected for vancomycin-susceptible
ATCC 29213 and E. coli ATCC 35218. Concentrations of Akacid
E. faecalis and VRE. Potent activity was also observed regarding
plusÒ at 0.5·, 1·, 2· and 4· MIC were used and monitored at time
inhibition of spore germination of B. subtilis and B. anthracis. All
point 0 and at 5 min, 30 min, 2 h, 6 h and 24 h. Three independent
tested substances were less active against Gram-negative bacteria.
experiments were performed per strain.
The testing of clinically relevant fungal species of Candida andAspergillus furthermore proved the antifungal efficacy of Akacid
plusÒ and confirmed that of chlorhexidine.
In vitro selection of resistance to Akacid plusÒ was carried out on 24
Ten strains, including CLSI quality control strains and clinical
strains: MSSA (1), MRSA (2), MRSE (4), VRE (5), Klebsiella spp.
isolates of S. aureus, E. faecalis, S. pneumoniae, E. coli,
(2), E. coli (4, 50% ESBLs), P. aeruginosa (4, 50% ESBLs) and
K. pneumoniae and P. aeruginosa were tested to compare Akacid
Acinetobacter spp. (2 strains). The broth selection method described
plusÒ MIC and MBC results. MBC values of Akacid plusÒ were
by Markopoulos et al.9 was used for the experiments. Thirty passages
observed at 1–2· MIC. Killing curves were also carried out using
of each test isolate were performed. All tests were performed in
Akacid plusÒ concentrations at 0.5·, 1·, 2· and 4· the measured
triplicate for each isolate. If the three replicates differed at the
organism MIC. Killing curves for S. aureus ATCC 29213 and
end of all cycles, the highest MIC was taken as the result.
E. coli ATCC 35218 (inoculum 106 cfu/mL) are given in Figure 1
Table 1. MICs of Akacid plusÒ (AP), chlorhexidine digluconate
(CHG) and mupirocin (MUP) for clinical strains of bacteria (352),
The oral and dermal LD50 of Akacid plusÒ in rats was found to
be above 2000 mg of active ingredient/kg of body weight. After asingle oral administration of Akacid plusÒ at a dose of 200 mg/kg
of body weight to female rats, all animals survived and no abnor-malities in life were revealed from day 1 until the end of the
observation period on day 14. One female and one male rat died
on account of the treatment with 2000 mg/kg. The necropsyrevealed no pathological abnormalities with exception of animals
no. 4 and no. 8. These rats showed light lungs, a flat liver and
spleen, and light mucous membranes. After a single dermal
administration of Akacid plusÒ at a dose of 2000 mg/kg of
body weight all animals survived until the scheduled termination
of the study and no toxic effects of the test substance were noted
in life. Body weights and body weight gain were inconspicuous
during the whole study in all rats, and all animals were normal at
In the acute dermal irritation/corrosion study with rabbits, no
general toxic effects of Akacid plusÒ were observed and all
exposed skin sites were normal at each examination term.
The present study demonstrates the broad antimicrobial profile of
Akacid plusÒ in comparison with chlorhexidine, another of the
family of cationic antimicrobials, and mupirocin, an antibiotic
with high activity against Gram-positive pathogens. MIC values
of chlorhexidine digluconate and mupirocin were comparable to
the results obtained by other studies.4 Previous studies by Irizarry
et al.13 and Suller and Russell.14 detected MRSA strains to be less
susceptible than MSSA strains to chlorhexidine, triclosan and
quaternary ammonium compounds. Likewise, Kresken et al.15
observed mupirocin resistance almost exclusively in methicillin-
resistant strains of Staphylococcus spp. In the present work
the MIC90s of chlorhexidine and mupirocin showed a 4-fold and
32-fold increase for MRSA in comparison with methicillin-
susceptible strains, while MIC values for Akacid plusÒ were
similar for both MRSA and MSSA. Recently, we have evaluated
bactericidal activity of Akacid plusÒ 0.1% after exposure for
5 min in basic quantitative suspension tests against quality
control strains of S. aureus, Enterococcus hirae, E. coli and
P. aeruginosa.16 Additionally, we have shown potent activity
of nebulized Akacid plusÒ 0.5% for eradication of antibiotic-
Includes Klebsiella pneumoniae, Klebsiella oxytoca.
susceptible and multiresistant S. aureus, P. aeruginosa and
Includes Enterobacter aerogenes, Enterobacter cloacae.
cIncludes Proteus mirabilis, Proteus vulgaris.
E. coli on hard surfaces.17 In the absence of neutralizing solution
dIncludes Salmonella enteritidis, Salmonella typhimurium.
and presence of Akacid plusÒ bacterial cells of S. aureus ATCC
eIncludes Shigella sonnei, Shigella flexneri.
29213 and E. coli 35218 were eliminated at 1· MIC within
Includes Acinetobacter baumannii, Acinetobacter lwoffii.
<5 h. A multiple of the MIC of Akacid plusÒ accelerated the
Includes Candida albicans, Candida glabrata, Candida krusei, Candida trop-
eradication of the exposed bacteria.
hIncludes Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus.
The increasing use of biocides has also raised concerns about
the development of biocide resistance. In the present study we
(lower detection limit 5 · 101 cfu/mL). Akacid plusÒ at ‡2· MIC
were not able to induce bacterial resistance to Akacid plusÒ.
and 1· MIC eradicated S. aureus and E. coli within 2 and 5 h.
Exposure of subinhibitory concentrations did not result inreduced susceptibility of Staphylococcus spp., Klebsiella spp.,E. coli, P. aeruginosa and Acinetobacter spp. In contrast, Marko-
poulos et al.9 showed significant increases in MICs of teicoplanin
For this test not only susceptible ATCC strains but also multires-
for S. epidermidis after broth and agar selection methods.
istant clinical isolates of Gram-positive and Gram-negative
Up to now it was a well-accepted fact that biocidal activity
organisms were used. There was no increase in MIC values of
comes at a price; that is to say that high activity equals high
Akacid plusÒ for any isolate after 30 passages.
toxicity. Chlorhexidine, which is registered as a disinfectant and
Figure 1. Time–killing curves for Akacid plusÒ versus S. aureus ATCC 29213 (MIC, 0.5 mg/L) and E. coli ATCC 35218 (MIC, 2 mg/L). Mean viable bacterial count(cfu/mL) of S. aureus (a) and E. coli (b) was evaluated in the presence and absence of Akacid plusÒ at 0.5·, 1·, 2· and 4· MIC at 5 min, 30 min, 2 h, 6 h and 24 h.
is used as a preservative in cosmetics and as a surgical rub, is
promising tool for topical application in the prophylaxis and
irritating to the eyes. According to the results obtained in the
treatment of bacterial and fungal infections. No difference in
toxicological studies, Akacid plusÒ showed a low acute oral and
the MIC values between MSSA and MRSA was detected.
dermal toxicity with an LD50 > 2000 mg/kg of body weight (a
Since the exact mechanism of action of Akacid plusÒ is not
concentration high above the therapeutic dose) and was not irrit-
fully understood yet, further tests are underway to study the
ating to the skin. Further toxicity studies including acute eye
mode of action and full range of activity of this promising
toxicity, skin sensitization, mutagenicity and chronic exposure
are needed to determine the complete toxicity profile of AkacidplusÒ.
The preliminary results of the present study demonstrate
the broad antimicrobial properties, also against MRSA and
We thank W. Schmidt, K. Stich and H. Sigmund for excellent
ESBL-producing Gram-negatives, which make Akacid plusÒ a
9. Markopoulos E, Graninger W, Georgopoulos A. In-vitro selection
of resistance to vancomycin and teicoplanin in Enterococcus faecium
and Enterococcus faecalis compared with Staphylococcus epidermidis.
J Antimicrob Chemother 1998; 41: 43–7.
10. European Commission. EU Method B.1 tris Acute oral
toxicity—acute toxic class method. Dir. 2004/73/EC; O.J. L 152,
1. Percival A. Increasing resistance to antibiotics—public health
crisis. Hosp Pharmacol 1997; 4: 193–6.
11. European Commission. EU Method B.3 Acute dermal toxicity.
2. Levy SB. Antibiotic and antiseptic resistance: impact on public
health. Ped Infect Dis 2000; 19: 120–2.
3. Walsh SE, Maillard JY, Russell AD et al. Development of bacterial
12. European Commission. EU Method B.4 Acute toxicity: dermal
resistance to several biocides and effects on antibiotic susceptibility.
irritation/corrosion. Dir. 2004/73/EC; O.J. L 152, 2004.
13. Irizarry L, Merlin T, Rupp J et al. Reduced susceptibility of
4. Block C, Furman M. Association between intensity of chlorhexid-
methicillin-resistant Staphylococcus aureus to cetylpyridinium chloride
ine use and micro-organisms of reduced susceptibility in a hospital
and chlorhexidine. Chemotherapy 1996; 42: 248–52.
environment. J Hosp Infect 2002; 51: 201–6.
14. Suller MT, Russell AD. In-vitro selection of resistance to
5. National Committee for Clinical Laboratory Standards. Methods
vancomycin and teicoplanin in Enterococcus faecium and Enterococcus
for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
faecalis compared with Staphylococcus epidermidis. J Hosp Infect
Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne,
15. Kresken M, Hafner D, Schmitz FJ et al. Prevalence of mupirocin
6. National Committee for Clinical Laboratory Standards. Reference
resistance in clinical isolates of Staphylococcus aureus and Staphylo-
coccus epidermidis: results of the antimicrobial resistance surveillance
study of the Paul-Ehrlich-Society for Chemotherapy. Int J Antimicrob
7. National Committee for Clinical Laboratory Standards. Reference
16. Kratzer C, Tobudic S, Graninger W et al. In vitro antimicrobial
Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-
activity of the novel polymeric guanidine Akacid plus. J Hosp Infect
Forming Filamentous Fungi: Proposed Standard M38-A. NCCLS,
8. National Committee for Clinical Laboratory Standards. Methods
17. Kratzer C, Tobudic S, Assadian O et al. Validation of Akacid plus
for Determining Bactericidal Activity of Antimicrobial Agents: Approved
as a room disinfectant in the hospital setting. Appl Environ Microbiol
Guideline M26-A. NCCLS, Wayne, PA, USA, 1999.
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