Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility
ABSTRACT
Risk assessments of the potential for microbicides to select for reduced bacterial susceptibility have been based largely on data generated through the exposure of bacteria to microbicides in aqueous solution. Since microbicides are normally formulated with multiple excipients, we have investigated the effect of formulation on antimicrobial activity and the induction of bacterial insusceptibility. We tested 8 species of bacteria (7 genera) before and after repeated exposure (14 passages), using a previously validated gradient plating system, for their susceptibilities to the microbicides benzalkonium chloride, benzisothiozolinone, chlorhexidine, didecyldimethyl ammonium chloride, DMDM-hydantoin, polyhexamethylene biguanide, thymol, and triclosan in aqueous solution (nonformulated) and in formulation with excipients often deployed in consumer products. Susceptibilities were also assessed following an additional 14 passages without microbicide to determine the stability of any susceptibility changes. MICs and minimum bactericidal concentrations (MBC) were on average 11-fold lower for formulated microbicides than for nonformulated microbicides. After exposure to the antimicrobial compounds, of 72 combinations of microbicide and bacterium there were 19 ≥4-fold (mean, 8-fold) increases in MIC for nonformulated and 8 ≥4-fold (mean, 2-fold) increases in MIC for formulated microbicides. Furthermore, there were 20 ≥4-fold increases in MBC (mean, 8-fold) for nonformulated and 10 ≥4-fold (mean, 2-fold) increases in MBC for formulated microbicides. Susceptibility decreases fully or partially reverted back to preexposure values for 49% of MICs and 72% of MBCs after further passage. In summary, formulated microbicides exhibited greater antibacterial potency than unformulated actives and susceptibility decreases after repeated exposure were lower in frequency and extent.
INTRODUCTION
Microbicides are broad-spectrum chemical agents that inactivate microorganisms (1–3). They are widely deployed throughout health care (4–6), domestic (7, 8), and industrial (9–11) environments, where their application includes antisepsis (12), hard surface disinfection (13), and pharmaceutical product preservation (14). They may also be incorporated into medical device coatings, for instance in sutures (15), wound dressings (16), and urinary catheters (17) to inhibit bacterial adhesion and subsequent biofilm formation.
It has been hypothesized that the use of microbicides could select for bacterial adaptation, resulting in reduced efficacy of the primary agent, as well as potentially decreasing bacterial susceptibility to chemically unrelated agents such as other microbicides and antibiotics (18). While there have been reports documenting the laboratory selection of bacteria with decreased microbicide sensitivity following repeated exposure to microbicides in highly selective conditions, it remains unclear whether this commonly occurs in the environment (19–24).
The majority of studies reporting reductions in microbicide susceptibility have used the active compound in aqueous solution with or without the addition of cosolvents such as dimethyl sulfoxide (DMSO) (25) or ethanol (26, 27). In real use, however, microbicides are deployed in formulated products with multiple excipients that may enhance potency. The potential effect of the formulation of microbicides on reducing the development of bacterial insusceptibility has received little research attention. Furthermore, despite the research effort that has been directed toward the possible risk of induced microbicide insusceptibility, the stability of such susceptibility changes has been investigated infrequently (24).
With the ultimate aim of developing realism-based approaches to risk assessment, the current investigation evaluates the frequency, magnitude, and reversibility of susceptibility changes that may be induced by the repeated exposure of a range of bacteria to microbicides in aqueous solution or in formulation. The microbicides selected reflect those frequently used in consumer products such as laundry detergents, hard surface disinfectants and personal care products. Planktonic susceptibilities (MIC and minimum bactericidal concentration [MBC]) and minimum biofilm eradication concentrations (MBECs) were determined before and after repeated exposure to sublethal concentrations of the microbicides benzalkonium chloride (BAC), benzisothiozolinone (BIT), chlorhexidine (CHX), didecyldimethyl ammonium chloride (DDAC), glydant (DMDM hydantoin), polyhexamethylene biguanide (PHMB), thymol, and triclosan in aqueous solution and in formulation with commonly used sequestrants and surfactants. Bacteria were also passaged further in the absence of any antimicrobial to determine the stability of any observed change in susceptibility.
MATERIALS AND METHODS
Bacteria.
Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, and Escherichia coli ATCC 25922 were obtained from Oxoid (Basingstoke, United Kingdom). Acinetobacter baumannii MBRG15.1, Pseudomonas putida MBRG15.2, Moraxella osloensis MBRG15.3, Escherichia coli MBRG15.4, and Cronobacter sakazakii MBRG15.5, were isolated from a domestic kitchen drain biofilm. Enterococcus faecalis was provided by Angela Oates, The University of Manchester.
Chemical reagents and growth media.
Bacteriological growth media were purchased from Oxoid (Basingstoke, United Kingdom). All other chemical reagents were purchased from Sigma-Aldrich (Dorset, United Kingdom) unless otherwise stated. Bacterial growth media were sterilized at 121°C and 15 lb/in2 for 15 min prior to use. Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis were cultured on tryptone soy agar and broth. Acinetobacter baumannii, Pseudomonas putida, Moraxella osloensis, and Cronobacter sakazakii were grown on Wilkins Chalgren agar and broth containing 2% sucrose. All bacteria were incubated aerobically at 37°C for 18 h unless stated otherwise.
Antimicrobial compounds.
BAC, CHX, thymol, and triclosan were purchased from Sigma-Aldrich (Dorset, United Kingdom). DDAC (50% [vol/vol]) was purchased from Merck Millipore (Durham, United Kingdom). Vantocil (a 20% [vol/vol] aqueous solution of PHMB) was obtained from Arch Chemicals, Inc. (Manchester, United Kingdom). Glydant (DMDM hydantoin) was obtained from Lonza (Bishop's Stortford, United Kingdom). All microbicides were tested in aqueous solution as previously described (27) or added to microbicide-free formulation chassis containing sequestrants and surfactants, at concentrations reflective of their normal deployment (in consumer products. Specifically, BAC, CHX, DDAC, DMDM hydantoin, PHMB, and thymol were prepared at 1% (vol/vol) in a general purpose cleaner. Triclosan and benzisothiozolinone were formulated into a laundry detergent at 0.0066 and 0.02% (wt/vol), respectively.
Exposure of bacteria to sublethal concentrations of microbicides as active and formulation.
A previously validated system (20, 25) was used to generate reproducible ∼100-fold-concentration gradients of the antimicrobial compounds on tryptone soy agar plates using an automated spiral plater (Don Whitley Scientific, Shipley, United Kingdom). Antimicrobials in aqueous solution or in formulation (50 μl) were deposited on the agar surface. Plates were dried for 1 h at room temperature prior to radial deposition of bacterial pure cultures and then incubated (4 days; 37°C) in an aerobic incubator. After incubation, growth observed at the highest microbicide concentration was aseptically removed and streaked onto a fresh plate containing the same antimicrobial compound concentration gradient. Where growth was observed across the whole antimicrobial gradient, a new plate produced with a 5-fold-higher microbicide concentration was used (25). This process was repeated until 14 passages had occurred (P14). Bacteria that exhibited ≥4-fold changes in MIC, MBC, or MBEC were then passaged a further 14 times in the absence of any antimicrobial compound (X14) to ascertain the stability of adaptation. Bacteria at P0, P14, and X14 were archived for subsequent MIC and MBC testing. Susceptibility testing (MIC, MBC, MBEC) was performed in two separate experiments each with three technical replicates.
Determination of bacterial MICs and MBCs.
MIC values were determined using a microdilution method as described previously (28). Briefly, overnight bacterial cultures were adjusted to an optical density at 600 nm (OD600) of 0.8 and diluted 1 in 100 in tryptone soy both or Wilkins Chalgren broth with 2% sucrose in a 96-well microtiter plate containing doubling dilutions of the relevant microbicide. Plates were incubated at 37°C (24 h) with agitation (100 rpm). The MIC was defined as the lowest concentration for which bacterial growth did not occur. Growth was viewed as turbidity (600 nm) compared to an uninoculated well (negative control) and was detected using a microtiter plate reader (PowerWave XS; BioTek, Bedfordshire, United Kingdom).
MBCs were determined as described previously (25), in brief aliquots (10 μl) from wells exhibiting no turbidity were transferred to sterile tryptone soy agar or Wilkins Chalgren agar prior to 4 days of incubation at 37°C to determine the MBC (25). The MBC was defined as the lowest concentration of microbicide at which no growth occurred after 4 days of incubation.
Determination of MBECs.
Single species biofilms were grown on the pegs of a Calgary biofilm device (CBD) (29). To produce inocula for biofilm susceptibility testing, single colonies of test bacteria were inoculated into 10 ml of sterile tryptone soy broth or Wilkins Chalgren broth with 2% sucrose and incubated at 37°C in a shaking aerobic incubator (100 rpm) for 18 h. Cultures were diluted to an OD600 of 0.8 and then further diluted 1:100 using fresh growth medium. Next, 100 μl of bacterial inoculum was added to each well of the CBD base, and the plates were then incubated at 37°C and 30 rpm for 48 h to allow biofilm formation on the pegs. Doubling dilutions for microbicides (150 μl) were prepared in sterile broth across a 96-well microtiter plate. Biofilms were exposed to antimicrobial compounds and incubated for 24 h at 37°C and 100 rpm. After incubation, the lid was transferred to a 96-well plate containing 200 μl of sterile broth and was incubated for 24 h at 37°C and 100 rpm. The MBECs were determined as the lowest concentration for which bacterial growth did not occur after 18 h of incubation. Growth was viewed as turbidity compared to an uninoculated well (negative control) and was detected using a microtiter plate reader (BioTek).
Statistical analyses.
Variability between replicates in the susceptibility tests were expressed as standard deviations. These were calculated using Microsoft Excel.
RESULTS
Two main variables describe data associated with the selection of decreased susceptibility by exposure to microbicides in the present study: (i) the frequency of susceptibility decreases of >2-fold (25) for multiple test bacteria and microbicides and (ii) the extent of susceptibility changes for each combination of bacterium and microbicide.
Repeated exposure to the microbicide-containing formulations resulted in a lower frequency of susceptibility reductions than did exposure to the same microbicide in aqueous solutions, and where decreases in susceptibility did occur, these were generally smaller for formulated microbicides. All individual values for bacterial susceptibility before, during, and after microbicide exposure have been given in Tables 1 to 8. However, due to the large number of combinations of bacterium and antimicrobial compound that were tested, the extent of susceptibility has also been expressed as mean values in the following section.
After repeated exposure to unformulated microbicides there were 19 ≥4-fold increases in MIC (1 of which fully reverted back to preexposure values after subsequent passage in the absence of microbicide, 13 of which partially reverted, and 5 of which did not revert; the average increase in MIC [P0 to P14] was 11-fold across the test panel of bacteria and microbicides). There were 20 increases in MBC (2 fully revertible, 11 partially revertible, and 7 nonrevertible; average, 8-fold increase) and 17 increases in MBEC (7 fully revertible, 6 partially revertible, and 4 nonrevertible; average, 4-fold increase) after microbicide exposure (Tables 1 to 8). After exposure to microbicide-containing formulations, there were 8 ≥4-fold increases in MIC (2 fully revertible and 6 nonrevertible; average, 2-fold increase), 10 increases in MBC (3 fully revertible, 5 partially revertible, and 2 nonrevertible; average, 2-fold increase) and 16 increases in MBEC (5 fully revertible, 8 partially revertible, and 3 nonrevertible; average, 3-fold increase) (Tables 1 to 8). In terms of antimicrobial potency, when comparing the formulated to nonformulated microbicides across the test panel of bacteria we saw ∼11-fold lower MIC and MBC values and a 3-fold lower MBEC for the unexposed (P0) bacterial isolates. For the P14 isolates, we observed an ∼35-fold lower MIC, an ∼36-fold lower MBC, and an ∼4-fold lower MBEC (Tables 1 to 8).
Benzalkonium chloride.
All test bacteria, with the exception of M. osloensis, C. sakazakii, and the E. coli drain isolate, exhibited a ≥4-fold increase in MIC after exposure to BAC (Table 1). Increases in MBC, while generally smaller than those in MIC, were also observed at ≥4-fold for S. aureus, E. coli, and P. aeruginosa. Furthermore, ≥4-fold increases in MBEC occurred for S. aureus and E. faecalis after BAC exposure. After growth in the absence of BAC, subsequent full or partial reversion in MIC, MBC, or MBEC occurred for all test bacteria with the exception of E. coli and P. aeruginosa (MIC and MBC). In contrast, after exposure to the BAC formulation only S. aureus, E. coli, P. aeruginosa, and A. baumannii showed a ≥4-fold increase in MIC, with S. aureus and E. coli also demonstrating a ≥4-fold increase in MBC. S. aureus, E. faecalis, and P. aeruginosa also exhibited a ≥4-fold increase in MBEC after exposure to BAC formulation. After recovery in the absence of BAC formulation, only S. aureus demonstrated any reversion in susceptibility (MBEC). The MIC for BAC was reduced in M. osloenis at P14 compared to P0 when this bacterium was exposed to BAC in aqueous solution.
TABLE 1
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 0.1 | 3.9 | 2.0 | 0.5 | 2.0 | 2.0 | 2.0 | 15.6 | 7.8 | 2.0 | 7.8 | 7.8 | 2.6 (1) | 31.3 | 15.6 | 3.9 | 125 | 7.8 |
| E. coli† | 4.6 (1) | 31.3 | 31.3 | 3.9 | 31.3 | 31.3 | 7.2 (2) | 41.7 (16) | 62.5 | 7.8 | 31.3 | 62.5 | 31.3 | 31.3 | 62.5 | 31.3 | 62.5 | 62.5 |
| E. faecalis† | 2.0 | 7.8 | 3.9 | 2.0 | 3.9 | 3.9 | 3.3 (1) | 7.8 | 7.8 | 3.9 | 7.8 | 7.8 | 6.5 (1) | 31.3 | 7.8 | 6.7 (2) | 46.9 (17) | 46.9 (17) |
| P. aeruginosa† | 14.3 (2) | 62.5 | 62.5 | 15.6 | 62.5 | 125 | 23.4 (9) | 125 | 125 | 31.3 | 62.5 | 250 | 125 | 250 | 500 | 62.5 | 250 | 500 |
| M. osloensis* | 3.9 | 2.0 | NA | 1.0 | 1.0 | NA | 7.8 | 15.6 | NA | 2.0 | 2.0 | NA | 7.8 | NA | NA | 7.8 | 2.0 | NA |
| A. baumannii* | 2.0 | 62.5 | 31.3 | 3.9 | 31.3 | 31.3 | 93.8 (34) | 250 | 125 | 62.5 | 62.5 | 125 | 125 | 250 | 125 | 125 | 125 | 93.8 (34) |
| P. putida* | 15.6 | 62.5 | 31.3 | 15.6 | 15.6 | NA | 125 | 125 | 62.5 | 62.5 | 31.3 | NA | 125 | NA | 62.5 | 125 | 31.3 | NA |
| C. sakazakii* | 62.5 | 52.1 (16) | NA | 31.3 | 31.3 | NA | 125 | 125 | NA | 31.3 | 31.3 | NA | 31.3 | NA | NA | 31.3 | 62.5 | NA |
| E. coli* | 18.4 (7) | 52.1 (16) | NA | 15.6 | 31.3 | NA | 62.5 | 125 | NA | 31.3 | 31.3 | NA | 62.5 | NA | NA | 62.5 | 62.5 | NA |
a
MBC, minimum bactericidal concentration; MBEC, minimum biofilm eradication concentration. P0, before antimicrobial exposure; P14, during antimicrobial exposure; X14, after passage in the absence of antimicrobial. †, nondrain isolates; *, drain isolates. UF, unformulated (microbicide in aqueous solution); F, formulated (microbicide in formulation). Organisms that underwent a ≥4-fold increase in MIC, MBC, or MBEC (indicated by boldfacing) were passaged a further 14 times in the absence of microbicide. NA, bacteria that did not undergo a ≥4-fold change and were not assessed for reversibility. The data represent the results from six replicates. Where data varied between biological replicates, standard deviations have been given in parentheses. For controls, bacteria were tested against formulations without microbicide; all bacteria were nonsusceptible to in-use concentrations.
Benzisothiozolinone.
No bacterium displayed a substantial change in susceptibility (≥4-fold MIC, MBC, or MBEC) to BIT or to BIT formulation after long-term exposure to the respective agent (Table 2).
TABLE 2
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 7.8 | 15.6 | NA | 1.0 | 2.0 | NA | 31.3 | 62.5 | NA | 15.6 | 15.6 | NA | 62.5 | 62.5 | NA | 31.3 | 62.5 | NA |
| E. coli† | 15.6 | 15.6 | NA | 7.8 | 7.8 | NA | 31.3 | 62.5 | NA | 31.3 | 31.3 | NA | 250 | 187.5 (68) | NA | 125 | 125 | NA |
| E. faecalis† | 7.8 | 15.6 | NA | 0.5 | 1.0 | NA | 7.8 | 7.8 | NA | 0.5 | 1.0 | NA | 250 | 41.7 (16) | NA | 125 | 125 | NA |
| P. aeruginosa† | 125 | 250 | NA | 15.6 | 31.3 | NA | 250 | 500 | NA | 62.5 | 125 | NA | 500 | 500 | NA | >125b | >125b | NA |
| M. osloensis* | 1.0 | 1.0 | NA | 0.5 | 0.5 | NA | 1.0 | 1.0 | NA | 0.5 | 0.5 | NA | 2.0 | 2.0 | NA | 0.5 | 1.0 | NA |
| A. baumannii* | 31.3 | 31.3 | NA | 7.8 | 15.6 | NA | 31.3 | 62.5 | NA | 31.3 | 62.5 | NA | 250 | 250 | NA | 62.5 | 125 | NA |
| P. putida* | 15.6 | 31.3 | NA | 31.3 | 31.3 | NA | 62.5 | 62.5 | NA | 31.3 | 62.5 | NA | 250 | 250 | NA | 62.5 | 125 | NA |
| C. sakazakii* | 7.8 | 7.8 | NA | 7.8 | 7.8 | NA | 31.3 | 31.3 | NA | 31.3 | 31.3 | NA | 250 | 500 | NA | 62.5 | 125 | NA |
| E. coli* | 15.6 | 31.3 | NA | 15.6 | 15.6 | NA | 62.5 | 62.5 | NA | 15.6 | 31.3 | NA | 250 | 187.5 | NA | 125 | 125 | NA |
a
See footnote in Table 1.
b
Benzisothiozolinone was formulated into a laundry detergent at 0.02%. Therefore, the highest test concentration for the formulation was 125 mg/liter.
Chlorhexidine.
After repeated exposure to chlorhexidine both S. aureus and E. coli showed ≥4-fold increases in MIC and MBC, which partially reverted in the absence of the microbicide (Table 3). P. aeruginosa demonstrated a ≥4-fold increase in MIC which did not revert after regrowth in a chlorhexidine free environment. E. faecalis and M. osloensis exhibited ≥4-fold increases in MBEC, which partially and fully reverted in the absence of chlorhexidine, respectively. In contrast, after exposure to chlorhexidine formulation no bacterium exhibited a ≥4-fold decrease in susceptibility at the MIC, MBC, or MBEC level.
TABLE 3
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 1.7 (1) | 7.8 | 3.9 | 2.0 | 2.0 | NA | 5.2 (2) | 46.9 (17) | 31.3 | 7.8 | 7.8 | NA | 13 (4) | 31.3 | 31.3 | 7.8 | 15.6 | NA |
| E. coli† | 2.4 (1) | 11.7 (4) | 7.9 | 2.0 | 3.9 | NA | 9.8 (5) | 62.5 | 31.3 | 15.6 | 31.3 | NA | 52.1 (16) | 62.5 | 31.3 | 62.5 | 31.3 | NA |
| E. faecalis† | 3.9 | 7.8 | 15.6 | 3.9 | 7.8 | NA | 14.3 (3) | 31.3 | 31.3 | 7.8 | 15.6 | NA | 31.3 | 125 | 62.5 | 31.3 | 62.5 | NA |
| P. aeruginosa† | 7.8 | 31.3 | 31.3 | 7.8 | 15.6 | NA | 68.8 (34) | 250 | 125 | 125 | 125 | NA | 250 | 125 | 125 | 250 | 125 | NA |
| M. osloensis* | 3.9 | 2.0 | 2.0 | 1.0 | 1.0 | NA | 31.3 | 15.6 | 3.9 | 1.0 | 1.0 | NA | 31.3 | 125 | 15.6 | 15.6 | 31.3 | NA |
| A. baumannii* | 7.8 | 7.8 | NA | 3.9 | 7.8 | NA | 125 | 62.5 | NA | 15.6 | 31.3 | NA | 125 | 125 | NA | 125 | 31.3 | NA |
| P. putida* | 7.8 | 7.8 | NA | 4.6 (2) | 3.9 | NA | 93.8 (34) | 62.5 | NA | 7.8 | 7.8 | NA | 62.5 | 125 | NA | 62.5 | 62.5 | NA |
| C. sakazakii* | 7.8 | 7.8 | NA | 3.9 | 3.9 | NA | 62.5 | 125 | NA | 7.8 | 15.6 | NA | 62.5 | 125 | NA | 31.3 | 10.4 (4) | NA |
| E. coli* | 7.8 | 10.4 (4) | 15.6 | 3.9 | 3.9 | NA | 46.8 (17) | 125 | 125 | 7.8 | 15.6 | NA | 125 | 125 | 125 | 62.5 | 23.4 (9) | NA |
a
See footnote in Table 1.
Didecyldimethyl ammonium chloride.
After repeated DDAC exposure P. aeruginosa, A. baumannii, and the E. coli drain isolate exhibited a ≥4-fold increase in MBC, out of which P. aeruginosa fully reverted, while A. baumannii and E. coli partially reverted following repeated growth the absence of DDAC. S. aureus, E. coli, E. faecalis, and the E. coli drain isolate all exhibited a ≥4-fold increase in MBEC, out of which E. faecalis and the E. coli drain isolate partially reverted, E. coli fully reverted, and S. aureus did not revert back to preexposure values after growth in the absence of the microbicide (Table 4). After exposure to the DDAC-containing formulation, P. aeruginosa and the E. coli drain isolate exhibited a ≥4-fold increase in MBC, out of which E. coli partially reverted and P. aeruginosa fully reverted after passage in an antimicrobial free environment. In agreement with the changes in MBEC observed after exposure to DDAC active, S. aureus, E. coli, E. faecalis, and the E. coli drain isolate also showed a ≥4-fold increase in MBEC after exposure to DDAC formulation. MBEC values partially reverted for both E. coli isolates and for E. faecalis but did not revert for S. aureus after recovery in the absence of DDAC.
TABLE 4
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 0.5 | 1.0 | 1.0 | 0.5 | 0.5 | 0.5 | 2.0 | 3.9 | 3.9 | 2.0 | 0.5 | 0.5 | 3.9 | 31.3 | 31.3 | 3.9 | 62.5 | 62.5 |
| E. coli† | 7.8 | 11.7 (4) | 7.8 | 3.9 | 7.8 | 3.9 | 3.9 | 11.7 (4) | 15.6 | 3.9 | 7.8 | 3.9 | 31.3 | 125 | 15.6 | 7.8 | 36.5 (13) | 15.6 |
| E. faecalis† | 1.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 1.0 | 2.0 | 2.0 | 2.0 | 3.9 | 3.9 | 2.0 | 125 | 31.3 | 2.0 | 104.2 (32) | 62.5 |
| P. aeruginosa† | 14.3 (2) | 31.3 | 15.6 | 15.6 | 31.3 | 15.6 | 31.3 | 125 | 31.3 | 31.3 | 125 | 31.3 | 125 | 125 | 250 | 62.5 | 125 | 62.5 |
| M. osloensis* | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | NA | 1.4 (0.5) | 3.9 | 2.0 | 2.0 | 2 | NA | 2.0 | 3.9 | 3.9 | 2.0 | 2.0 | NA |
| A. baumannii* | 15.6 | 31.3 | 15.6 | 3.9 | 7.8 | NA | 15.6 | 62.5 | 31.3 | 62.5 | 62.5 | NA | 62.5 | 125 | 31.3 | 62.5 | 62.5 | NA |
| P. putida* | 47.4 (17) | 31.3 | NA | 4.6(1) | 3.9 | NA | 62.5 | 41.7 (17) | NA | 31.3 | 62.5 | NA | 62.5 | 62.5 | NA | 62.5 | 62.5 | NA |
| C. sakazakii* | 7.2 (2) | 15.6 | 15.6 | 7.8 | 15.6 | NA | 15.6 | 31.3 | 31.3 | 7.8 | 15.6 | NA | 31.3 | 62.5 | 62.5 | 15.6 | 31.3 | NA |
| E. coli* | 4.6 (2) | 15.6 | 15.6 | 3.9 | 7.8 | 3.9 | 10.4 (4) | 41.7 (17) | 31.3 | 3.9 | 15.6 | 7.8 | 15.6 | 62.5 | 31.3 | 15.6 | 62.5 | 23.5 (9) |
a
See footnote in Table 1.
Glydant (DMDM hydantoin).
The E. coli drain isolate exhibited a ≥4-fold increase in MBC after repeated exposure to DMDM hydantoin; this susceptibility decrease fully reverted in the absence of the microbicide (Table 5). Comparatively, after exposure to DMDM hydantoin formulation both E. coli isolates and C. sakazakii showed a ≥4-fold increase in MBEC, all of which fully reverted in an antimicrobial-free environment.
TABLE 5
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 187.5 | 187.5 | NA | 187.5 | 187.5 | NA | 375 | 482 (183) | NA | 375 | 375 | NA | 3,000 | 3,000 | NA | 1,500 | 3,000 | NA |
| E. coli† | 375 | 375 | NA | 375 | 375 | 375 | 1,500 | 1,500 | NA | 375 | 750 | 375 | 6,000 | 6,000 | NA | 1,500 | 6,000 | 1,500 |
| E. faecalis† | 187.5 | 187.5 | NA | 187.5 | 187.5 | NA | 1,500 | 1,500 | NA | 1,500 | 750 | NA | 3,000 | 3,000 | NA | 3,000 | 6,000 | NA |
| P. aeruginosa† | 187.5 | 187.5 | NA | 187.5 | 187.5 | NA | 6,000 | 6,000 | NA | 1,500 | 1,500 | NA | 6,000 | 6,000 | NA | 6,000 | 12,000 | NA |
| M. osloensis* | 375 | 375 | NA | 46.9 | 62.5 | NA | 325 | 375 | NA | 187.5 | 187.5 | NA | 750 | 1,500 | NA | 750 | 1,500 | NA |
| A. baumannii* | 375 | 325 | NA | 187.5 | 187.5 | NA | 750 | 750 | NA | 375 | 375 | NA | 6,000 | 6,000 | NA | 6,000 | 6,000 | NA |
| P. putida* | 375 | 375 | NA | 375 | 375 | NA | 750 | 750 | NA | 750 | 375 | NA | 6,000 | 6,000 | NA | 3,000 | 6,000 | NA |
| C. sakazakii* | 375 | 375 | NA | 187.5 | 187.5 | 375 | 3,000 | 3,000 | NA | 375 | 750 | 375 | 6,000 | 6,000 | NA | 1,500 | 6,000 | 1,500 |
| E. coli* | 187.5 | 466 (219) | 187.5 | 187.5 | 375 | 187.5 | 375 | 1,500 | 375 | 375 | 750 | 375 | 6,000 | 6,000 | 6,000 | 1,500 | 12,000 | 1,500 |
a
See footnote in Table 1.
Polyhexamethylene biguanide.
S. aureus, E. faecalis M. osloensis and A. baumannii exhibited a ≥4-fold increase in MIC after PHMB exposure out of which M. osloensis and A. baumannii fully reverted and S. aureus and E. faecalis partially reverted after growth in the absence of PHMB (Table 6). S. aureus, E. coli, P. aeruginosa, E. faecalis, and the E. coli drain isolate demonstrated a ≥4-fold increase in MBC, out of which S. aureus, E. faecalis, and the E. coli drain isolate showed partial reversion, and E. coli and P. aeruginosa showed no reversion to preexposure values in the absence of PHMB. After PHMB exposure, S. aureus, E. faecalis, A. baumannii, C. sakazakii, and the E. coli drain isolate also displayed a ≥4-fold increase in MBEC, which fully reverted for S. aureus, A. baumannii, and E. coli drain isolate and partially reverted for E. faecalis and C. sakazakii after regrowth in the absence of PHMB. After exposure to PHMB formulation S. aureus, E. faecalis, and P. aeruginosa showed substantial changes in their PHMB susceptibility displaying ≥4-fold increases in MBC, all of which fully or partially reverted in the absence of the antimicrobial formulation. S. aureus and E. faecalis also exhibited a ≥4-fold increase in MBEC after exposure to PHMB formulation, all of which partially reverted back to preexposure values after regrowth in the absence of the formulation.
TABLE 6
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 3.9 | 23.5 (9) | 15.6 | 3.9 | 3.9 | 3.9 | 3.9 | 125 | 15.6 | 3.9 | 15.6 | 7.8 | 15.6 | 125 | 15.6 | 15.6 | 125 | 31.3 |
| E. coli† | 15 (10) | 31.3 | 15.6 | 7.8 | 15.6 | NA | 15 (10) | 62.5 | 62.5 | 15.6 | 31.3 | NA | 62.5 | 62.5 | 62.5 | 62.5 | 31.3 | NA |
| E. faecalis† | 7.8 | 31.3 | 15.6 | 5.9(1) | 15.6 | 7.8 | 7.8 | 125 | 15.6 | 7.8 | 31.3 | 7.8 | 14.3 (3) | 125 | 31.3 | 15.6 | 125 | 31.3 |
| P. aeruginosa† | 22.8 (15) | 31.3 | 62.5 | 15.6 | 15.6 | 15.6 | 22.8 (15) | 125 | 125 | 31.3 | 125 | 31.3 | 250 | 250 | 250 | 250 | 62.5 | 62.5 |
| M. osloensis* | 7.8 | 31.3 | 3.9 | 1.0 | 1.0 | NA | 62.5 | 31.3 | 31.3 | 7.8 | 7.8 | NA | 62.5 | 62.5 | 31.3 | 31.3 | 62.5 | NA |
| A. baumannii* | 7.8 | 31.3 | 7.8 | 9.1 (3) | 15.6 | NA | 62.5 | 125 | 62.5 | 31.3 | 62.5 | NA | 62.5 | 250 | 62.5 | 62.5 | 125 | NA |
| P. putida* | 28.9 (8) | 31.3 | NA | 15.6 | 15.6 | NA | 62.5 | 62.5 | NA | 31.3 | 62.5 | NA | 125 | 125 | NA | 125 | 125 | NA |
| C. sakazakii* | 7.8 | 15.6 | 15.6 | 31.2 | 15.6 | NA | 104 (32) | 125 | 125 | 15.6 | 31.3 | NA | 62.5 | 250 | 125 | 62.5 | 125 | NA |
| E. coli* | 7.8 | 7.8 | 31.3 | 7.8 | 15.6 | NA | 15.6 | 250 | 31.3 | 15.6 | 31.3 | NA | 62.5 | 250 | 31.3 | 62.5 | 31.3 | NA |
a
See footnote in Table 1.
Thymol.
After long-term thymol exposure, none of the bacterial isolates showed a ≥4-fold decrease in thymol susceptibility at MIC, MBC, or MBEC level (Table 7). After exposure to the thymol-containing formulation, E. coli and A. baumannii both underwent ≥4-fold increases in MBC, while P. putida demonstrated a ≥4-fold increase in MIC and MBC, all of which partially reverted in the absence of thymol formulation. Furthermore, both E. coli isolates showed a ≥4-fold increase in MBEC, which partially reverted after growth in the absence of thymol formulation. The MBC for thymol was reduced in P. aeruginosa at P14 compared to P0 when this bacterium was exposed to thymol in aqueous solution.
TABLE 7
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 187.5 | 187.5 | NA | 187.5 | 187.5 | NA | 375 | 375 | NA | 375 | 750 | NA | 416 (160) | 375 | NA | 375 | 750 | NA |
| E. coli† | 1,500 | 1,500 | NA | 187.5 | 375 | 375 | 1,500 | 1,500 | NA | 375 | 1,500 | 750 | 1,500 | 1,500 | NA | 375 | 3,000 | 1,500 |
| E. faecalis† | 375 | 750 | NA | 187.5 | 375 | NA | 750 | 750 | NA | 375 | 750 | NA | 750 | 750 | NA | 750 | 1,500 | NA |
| P. aeruginosa† | 3,000 | 3,000 | NA | 1,500 | 3,000 | NA | 6,000 | 3,000 | NA | 3,000 | 6,000 | NA | 6,000 | 6,000 | NA | 6,000 | 12,000 | NA |
| M. osloensis* | 750 | 750 | NA | 187.5 | 375 | NA | 750 | 750 | NA | 187.5 | 375 | NA | 3,000 | 1,500 | NA | 3,000 | 375 | NA |
| A. baumannii* | 750 | 750 | NA | 375 | 375 | 375 | 1,500 | 3,000 | NA | 750 | 6,000 | 3,000 | 6,000 | 6,000 | NA | 6,000 | 6,000 | 6,000 |
| P. putida* | 750 | 750 | NA | 375 | 3,000 | 375 | 1,500 | 3,000 | NA | 1,500 | 6,000 | 3,000 | 6,000 | 6,000 | NA | 6,000 | 6,000 | 12,000 |
| C. sakazakii* | 750 | 750 | NA | 375 | 375 | NA | 2,250 (822) | 3,000 | NA | 375 | 750 | NA | 6,000 | 6,000 | NA | 3,000 | 750 | NA |
| E. coli* | 665 (190) | 750 | NA | 187.5 | 375 | NA | 3,000 | 3,000 | NA | 375 | 750 | NA | 6,000 | 6,000 | NA | 750 | 3,000 | 1,500 |
a
See footnote in Table 1.
Triclosan.
All bacterial isolates, with the exception of E. faecalis, A. baumannii and P. aeruginosa, which is nonsusceptible to triclosan, demonstrated an increase in MIC after repeated triclosan exposure, none of which fully reverted back to preexposure levels after regrowth in the absence of triclosan (Table 8). All isolates apart from P. aeruginosa, A. baumannii, and P. putida showed a ≥4-fold increase in MBC out of which C. sakazakii and the E. coli drain isolate showed partial reversion, while the others showed no reversion after passage in the absence of triclosan. Both E. coli isolates in addition to C. sakazakii, E. faecalis, and A. baumannii showed ≥4-fold increase in MBEC after repeated triclosan exposure, out of which C. sakazakii and E. faecalis did not revert and both E. coli isolates completely reverted in the absence of the microbicide. In comparison, after exposure to triclosan formulation only the E. coli isolates and P. aeruginosa showed ≥4-fold increase in MIC, which fully reverted for P. aeruginosa but did not revert for either E. coli strain in the absence of triclosan formulation. The MBECs increased ≥4-fold for S. aureus and E. faecalis but fully reverted for both bacteria after regrowth in the absence of triclosan formulation.
TABLE 8
| Bacterium | Concn (mg/liter) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MBEC | ||||||||||||||||
| UF | F | UF | F | UF | F | |||||||||||||
| P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | P0 | P14 | X14 | |
| S. aureus† | 0.2 | 62.5 | 31.3 | 0.1 | 0.1 | 0.1 | 3.9 | 62.5 | 62.5 | 0.1 | 0.1 | 0.1 | 65.1 | 125 | 125 | 2.0 | 7.8 | 2.0 |
| E. coli† | 2.0 | 62.5 | 62.5 | 0.1 | 2.0 | 3.9 | 2.0 | 125 | 125 | 7.8 | 7.8 | 3.9 | 125 | 500 | 125 | 62.5 | 15.6 | 15.6 |
| E. faecalis† | 62.5 | 62.5 | 62.5 | 0.1 | 0.1 | 0.1 | 62.5 | 125 | 125 | 0.1 | 0.1 | 0.1 | 15.6 | 125 | 125 | 2.0 | 7.8 | 2.0 |
| P. aeruginosa† | NS | NS | NS | 7.8 | 62.5 | 7.8 | NS | NS | NS | 62.5 | 62.5 | 7.8 | NS | NS | NS | 62.5 | 62.5 | 7.8 |
| M. osloensis* | 1.0 | 15.6 | 7.8 | 1.0 | 1.0 | NA | 7.8 | 31.3 | 31.3 | 3.9 | 3.9 | NA | 125 | 125 | 125 | 3.9 | 3.9 | NA |
| A. baumannii* | 125 | 125 | 125 | 2.0 | 2.0 | NA | 125 | 250 | 125 | 31.6 | 15.6 | NA | 125 | 250 | 125 | 62.5 | 15.6 | NA |
| P. putida* | 15.6 | 62.5 | 62.5 | 1.0 | 2.0 | NA | 62.5 | 125 | 125 | 15.6 | 15.6 | NA | 125 | 250 | 500 | 62.5 | 15.6 | NA |
| C. sakazakii* | 7.8 | 500 | 188 | 2.0 | 2.0 | NA | 7.8 | 1000 | 250 | 31.3 | 31.3 | NA | 1.3 (0.5) | 125 | 125 | 62.5 | 31.3 | NA |
| E. coli* | 1.0 | 125 | 62.5 | 0.1 | 2.0 | 3.9 | 2.0 | 250 | 125 | 15.6 | 15.6 | 15.6 | 125 | 500 | 125 | 62.5 | 15.6 | 15.6 |
a
See footnote in Table 1. NS, not susceptible (MBC/MIC/MBEC > 1,000 mg/liter).
DISCUSSION
The majority of investigations into the potential of microbicides to select for changes in bacterial susceptibility have been conducted by exposing pure cultures of bacteria to microbicides as pure actives in aqueous solution or in simple formulations (aqueous solutions containing the active and in some studies, cosolvents such as DMSO [25] or ethanol [27]). It has been hypothesized that formulated products may interact with bacteria in a manner that is distinct from aqueous solutions (28, 30) potentially reducing the frequency and extent of susceptibility changes. Although numerous studies have evaluated the antimicrobial potency of formulated microbicides (3, 31, 32), to our knowledge there are no studies in the literature that have compared the effects of repeated bacterial exposure to microbicides in aqueous solution and in complex formulation for a range of bacteria and microbicides. In the present investigation therefore, we evaluated the effect of the formulation of microbicides on antimicrobial potency and on the mitigation of bacterial insusceptibility for a selection of bacterial isolates and microbicides encompassing biguanides, quaternary ammonium compounds, phenolics, isothiazolinones, formaldehyde releasers, and essential oils. Microbicides were tested as aqueous solutions of the active compounds and in complex formulations with sequestrants and ionic/nonionic surfactants to mimic their real-world use as hard-surface disinfectants (for BAC, chlorhexidine, DDAC, DMDM hydantoin, PHMB, and thymol), and laundry detergents (for BIT and triclosan). The reversibility of any induced susceptibility changes was also investigated to ascertain the stability of adaptation.
Reductions in bacterial susceptibility to an antimicrobial compound can be influenced by several factors related to the antimicrobial or the microorganism. Bacterial susceptibility may be affected by the structural integrity of the bacterial cell envelope and its ability to function as an effective permeability barrier (33, 34, 35). Innate bacterial nonsusceptibility toward an antimicrobial agent may occur due to effective barrier components of the bacterial cell, such as an outer membrane in Gram-negative bacteria (36) or the spore coat in bacterial endospores (37). Changes in cell envelope permeability may therefore affect bacterial susceptibility, which can include alterations in lipopolysaccharide expression and structure (33), reduction in the number of outer membrane porins (23), and alterations in membrane fatty acid composition (38). The expression of efflux pumps has also been linked to decreases in microbicide susceptibility in bacteria, particularly toward membrane-active compounds such as biguanides (39) (CHX and PHMB) and quaternary ammonium compounds (40) (BAC and DDAC in the present investigation). The increased expression of efflux pumps may therefore also potentially account for some of the susceptibility changes observed in many of our bacterial isolates. Reversible susceptibility changes to microbicides may result from temporary phenotypic adaptations in bacteria, such as the induction of stress responses that revert once the bacteria recover in an antimicrobial-free environment (41, 42). Equally, the development of microbicide insusceptibility may be attributable to the selection of insusceptible mutants, for instance mutations in FabI are reportedly render some bacteria insusceptible to triclosan (43, 44). However, the inherent stability of a particular mutation largely depends upon the overall fitness cost that it exerts on the host microorganism versus the competitive advantage that it provides in a particular environment (45). Hence, any mutation that renders a bacterium less susceptible toward an antimicrobial compound may eventually be lost once the selective pressure is removed if the mutation results in a biologically significant reduction in the fitness of the microorganism (46).
While previous studies have reported the induction of microbicide insusceptibility in bacteria, it should be noted that adapted bacterial isolates often remain susceptible to the microbicide at concentrations used in consumer products and that true microbicide resistance is likely to be uncommon (25). In the present investigation, the only test bacterium that was refractory to a microbicide was P. aeruginosa to triclosan. This was apparent before microbicide exposure and has previously been attributed to the physiology of this bacterium, including expression of efflux pumps (47). Interestingly, P. aeruginosa was comparatively susceptible to the triclosan formulation, illustrating marked differences in potency for the microbicide in aqueous solution compared to the formulated product.
Of all the microbicides in unformulated form, BAC and triclosan induced the highest frequency of ≥4-fold increases in MIC with 6/9 bacterial isolates showing a reduction in susceptibility to both antimicrobials at this level. This was followed by PHMB (4 isolates) and CHX (3 isolates). Triclosan exposure resulted in the highest frequency of ≥4-fold increases in MBC (6 isolates), followed by PHMB (5 isolates), DDAC and BAC (3 isolates) and then CHX (2 isolates) and DMDM hydantoin (1 isolate). In terms of the susceptibility of bacteria when grown as biofilms, PHMB adaptation resulted in the highest number of isolates showing ≥4-fold increases in MBEC (5 isolates), followed by triclosan and DDAC (4 isolates each) and then BAC and CHX (2 isolates).
With respect to the formulated microbicides, BAC induced the highest number of ≥4-fold increases in MIC (4 isolates), followed by triclosan (3 isolates) and thymol (1 isolate). DMDM hydantoin-, thymol-, and PHMB-containing formulations induced the largest number of ≥4-fold increases in MBC (3 isolates each), followed by BAC and DDAC (2 isolates each). Exposure to the DDAC-containing formulations resulted in the highest numbers of bacterial isolates exhibiting a ≥4-fold increase in MBEC (4 isolates), followed by BAC and DMDM hydantoin (3 isolates) and then PHMB, thymol, and triclosan formulations (2 isolates).
Although the current investigation demonstrates that induced reductions in susceptibility toward both microbicides and microbicide-containing formulations may occur, a substantially higher number of bacterial isolates underwent ≥4-fold increases in MIC, MBC, or MBEC when exposed to microbicides in aqueous solution, in comparison to those in formulation. The only exception to this was thymol, for which changes in susceptibility were more frequent in bacteria exposed to the compound in formulation. Thymol is poorly soluble in water and formulation may therefore have substantially improved solubility, increasing bacterial exposure and thus selectivity. Furthermore, since incorporating microbicides into formulations frequently enhanced antimicrobial potency, the formulated microbicides often maintained higher antimicrobial activity in comparison to microbicides in aqueous solution, even after repeated exposure. The incorporation of nonionic surfactants and sequestrants into microbicide-containing formulations therefore appears to increase antimicrobial potency, as well as mitigating the development of antimicrobial insusceptibility both in terms of frequency and magnitude of susceptibility change. Since excipients can interact with different cellular targets to the accompanying microbicide, formulations may have a cumulative antimicrobial effect that would require multiple further physiological adaptations to render the microorganism insusceptible.
Alcohol ethoxylates are a major class of nonionic surfactants which are often used in household detergents, cleaners and personal care products and have previously shown bacteriostatic effects due to their direct impact on the bacterial cell membrane leading to the leakage of cytoplasmic components, indicating an increase in membrane permeability (48). An increase in membrane permeability would allow microbicides to more readily transverse the cytoplasmic membrane increasing their access to intracellular target sites. Therefore, combining microbicides and alcohol ethoxylates in formulation may enhance overall antimicrobial potency compared to the pure active. Sodium tripolyphosphate, a chelating agent commonly used in domestic detergents, has previously shown antibacterial activity against several bacteria often found as food contaminants (49). Since sodium tripolyphosphate is a chelating agent, it is plausible, as with other chelators such as EDTA, that this antibacterial activity occurs by disruption of the bacterial cell envelope through the sequestration of stabilizing divalent cations. Such cations normally link bacterial lipopolysaccharides to the outer membrane and interference with this process can destabilize the outer membrane in Gram-negative bacteria, impairing barrier function (50, 51, 52). Furthermore, strong chelating agents may inhibit bacterial growth by sequestering trace minerals required for bacterial metabolism (51, 53).
Essential oils such as thymol are often incorporated into antimicrobial formulation due to their inhibitory effects on bacterial growth. The antimicrobial activity of essential oils reportedly occurs through interaction with the bacterial cytoplasmic membrane, resulting in increased cell permeability and the disruption of energy generation (54, 55). Compensatory adaptations may occur, but whether these would result in outcome-changing effects during deployment depends on the extent of any susceptibility decreases, the concentration used in the product and the antimicrobial potency of the formulation (i.e., the active compound and excipients in combination).
In a small number of cases bacterial susceptibility was increased following repeated exposure to microbicides. This could be due to knock-on effects of cellular damage caused by microbicide exposure.
Conclusion.
With the ultimate aim of developing realistic approaches to risk assessment, we observed that repeated exposure of 9 bacteria to 8 microbicides in aqueous solution or within complex formulations with sequestrants and ionic/nonionic surfactants, induced reductions in bacterial susceptibility in a highly selective laboratory exposure system. Susceptibility changes varied in reversibility, possibly reflecting a range of underlying mechanisms, including temporary phenotypic adaptation, such as the induction of stress responses or the selection of stable mutations. Importantly, the formulation of microbicides markedly increased overall antimicrobial potency for the test microbicides against the majority of the bacteria, as well as reducing the frequency and magnitude of susceptibility changes. Although it remains unclear how observations based on the in vitro exposure of bacteria to microbicides can be extrapolated to their use in the real world, understanding the potential selectivity of microbicide-containing formulations is likely to better served by testing formulations, as well as active aqueous solutions. This highlights the need to conduct risk assessments of induced microbicide susceptibility changes using conditions that more accurately reflect their deployment.
ACKNOWLEDGMENTS
We thank Joanne O'Keeffe and Andrew Jamieson from Unilever R&D, Port Sunlight, for their advice regarding the selection of microbicides and formulations.
This project was funded by Unilever's Safety and Environmental Assurance Centre (SEAC).
Alejandro Amézquita is an employee of Unilever. Peter McClure was an employee of Unilever when this project was initiated.
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Information & Contributors
Information
Published In
Applied and Environmental Microbiology
Volume 81 • Number 20 • 15 October 2015
Pages: 7330 - 7338
Editor: H. L. Drake
PubMed: 26253662
Copyright
© 2015 American Society for Microbiology.
History
Received: 19 June 2015
Accepted: 5 August 2015
Published online: 22 September 2015
Contributors
Editor
H. L. Drake
Editor
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Citations
Citation
2015.
Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility. Appl Environ Microbiol
81:.
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