Susceptibility of Stenotrophomonas maltophilia Clinical Isolates to Antibiotics and Contact Lens Multipurpose Disinfecting Solutions

Abstract

Purpose.: To determine the susceptibility of Stenotrophomonas maltophilia to various antibiotics and contact lens multipurpose disinfecting solutions.

Methods.: Forty S. maltophilia strains from contact lens cases, contact lenses, or eye swabs of contact lens wearers including 27 asymptomatic wearers and 13 keratitis patients were examined for their susceptibility to different antibiotics, using a disc diffusion assay, and to multipurpose disinfecting solutions using a broth microdilution method.

Results.: Certain strains were resistant to aztreonum (15%), imipenem (93%), chroramphenicol (13%), and cefepime (8%). Two of those strains were multidrug resistant. All strains were sensitive to trimethoprim-sulfamethoxazole, tigecycline, ceftazidime, and fluoroquinolones. Overall, the minimum inhibitory concentration (MIC) for all strains was significantly higher (P < 0.05) for AQuify (50% dilution) and OPTI-FREE RepleniSH (25%) than all other multipurpose contact lens disinfecting solutions (MPDS) (3%–14%, except RepleniSH versus MeniCare Soft [14%]). AQuify, OPTI-FREE RepleniSH, and MeniCare Soft had significantly higher minimum bactericidal concentrations (undiluted MPDS) than other disinfecting solutions (P < 0.05).

Conclusions.: The Australian ocular isolates of S. maltophilia remain susceptible to trimethoprim-sulfamethozole, tigecycline, and most fluoroquinolones. However, the isolates showed resistance to certain multipurpose disinfecting solutions.

Introduction

Stenotrophomonas maltophilia, previously referred to as Pseudomonas maltophilia¹ and Xanthomonas maltophilia,² is an emerging multidrug-resistant opportunistic pathogen.^3–6 S. maltophilia is able to persist in nutrient-poor aqueous environments, and can be isolated from water, soil, and various plants and animals. S. maltophilia is associated with various systemic infections including cystic fibrosis, bacteremia, endocarditis, meningitis, and respiratory/urinary tract and skin/soft tissue infections in immune-compromised individuals.^7–10 S. maltophilia infection has a high mortality rate,¹¹ probably due to its poor response to antibiotic therapy. Ocular infections caused by S. maltophilia have been reported, including conjunctivitis,¹² keratitis,^13–17 and endophthalmitis.^18–20 Microbial keratitis is one of most important complications for contact lens wearers, with a reported incidence of 2.7 to 4.2 per 10,000 wearers per year.^21,22 

Epidemiological studies have demonstrated that lens case hygiene is significantly associated with the risk of developing microbial keratitis. Poor storage case hygiene, defined as not air-drying contact lens cases when not in use, was associated with a 6.4 times (95% confidence interval [CI], 1.9–21.8) risk of developing infectious keratitis, and infrequent storage case replacement (less frequently than every 3 months) with a 5.4 times (95% CI, 1.5–18.9) risk.²³ Similarly, according to data obtained from clinical trials, the risk of developing a corneal infiltrative event is correlated with the amount of Gram-negative (including S. maltophilia) contamination of contact lens cases.²⁴ The rate of contamination of contact lens cases and types of microbes within the lens cases depend on the type of multipurpose contact lens disinfecting solutions (MPDS) being used, and not the type of lens.²⁵ This implicates resistance to the disinfectants by some microbes. The resistance may manifest during biofilm growth of microbes,²⁶ but may also be due to inherent resistance of the bacteria. Indeed, S. maltophilia, Achromobacter spp., and Delftia acidovorans isolated from lens cases of people who have corneal infiltrates have been shown to be resistant to certain MPDS.²⁷ The current study was designed to determine the susceptibility of S. maltophilia isolates, mostly from contact lens wearers or their paraphernalia, in Australia to antibiotics and multipurpose solutions for contact lenses. 

Materials and Methods

Sample Collection and Bacterial Isolates

S. maltophilia clinical isolates (40 in total, Table 1) were retrieved from our culture collection; they had been isolated over an 18-year period between February 1994 and July 2011. Strains were divided into two groups, 20 strains isolated from February 1994 to April 2001 and 20 strains from December 2005 to July 2011, for comparative analysis. Strains were also divided into those isolated from cases of keratitis or from lens wearers with no adverse events. Isolated colonies of each strain were grown on nutrient agar overnight and then resuspended in 5 mL Mueller-Hinton broth (MHB; Thermo Fisher Scientific Australia Pty Ltd., Adelaide, SA, Australia), and bacterial cells were adjusted to a turbidity of 1 × 10⁸ CFU/mL (optical density [OD]660_nm = 0.1). 

Table 1

 

Strains of S. maltophilia and Their Source

Strain	Date of Isolation	Source	Isolated at Time of Keratitis (Y/N)
Xmal1	4/02/1994	Contact lens	N
Xmal2	4/02/1994	Eye swab	N
Xmal3	4/02/1994	Eye swab	N
Xmal4	4/02/1994	Eye swab	N
Xmal5	4/02/1994	Not known	N
Xmal7	23/09/1994	Eye swab	Y
Xmal8	14/10/1994	Contact lens	N
Xmal9	24/10/1994	Contact lens	N
Xmal10	15/12/1994	Eye Swab	Y
Xmal11	21/09/1995	Contact lens	Y
Xmal12	12/10/1995	Eye swab	Y
Xmal13	15/03/1996	Contact lens	Y
Xmal14	15/11/1996	Contact lens	N
Xmal15	19/03/1998	Contact lens	Y
Xmal16	3/04/1998	Contact lens	Y
Xmal17	24/08/1998	Not known	N
Xmal18	14/12/1998	Contact lens	Y
Xmal19	16/12/1998	Contact lens	N
Xmal20	12/02/1999	Contact lens case	Y
Xmal21	2/04/2001	Contact lens	Y
Smal1	12/12/2005	Cystic fibrosis swab	N
Smal2	27/05/2008	Contact lens case	N
Smal3	12/02/2008	Contact lens case	N
Smal4	3/04/2009	Contact lens case	Y
Smal5	9/04/2009	Contact lens case	N
Smal6	9/09/2010	Contact lens case	Y
Smal7	18/11/2010	Contact lens case	N
Smal8	1/12/2010	Contact lens case	N
Smal9	10/02/2011	Contact lens case	N
Smal10	24/02/2011	Contact lens case	N
Smal11	9/03/2011	Contact lens case	N
Smal12	24/03/2011	Contact lens case	Y
Smal13	8/04/2011	Contact lens case	N
Smal14	15/04/2011	Contact lens case	N
Smal15	17/04/2011	Contact lens case	N
Smal16	27/05/2011	Contact lens case	N
Smal17	1/07/2011	Contact lens case	N
Smal18	1/07/2011	Contact lens case	N
Smal19	8/07/2011	Contact lens case	N
Smal20	15/07/2011	Contact lens case	N

To measure the growth rate of the strains, 1 μL of each strain in MHB was added to 100 μL fresh MHB. The strains were incubated at 37°C for 24 hours, and the absorbance of the media at 660 nm was recorded for 24 hours. Plots of lnOD₆₆₀ versus time were generated, and the mean generation time was calculated from the exponential growth phase as ln2/([lnOD_t − lnOD_t0]/t). 

Antibiotic Sensitivity

The antibiotics used were fluoroquinolones (ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, and ofloxacin), β-lactams (aztreonam, ceftazidime, cefepime, imipenem, and ticarcillin), aminoglycosides (gentamicin and tobramycin), glycycline (tigecycline), SXT (trimethoprim/sulfamethozole), chloramphenicol, and polymyxin B. Bacterial susceptibilities to these antibiotics were determined using the disc diffusion method in accordance with the Kirby-Bauer method as recommended by the Clinical and Laboratory Standards Institute (M100-S21, CLSI, 2011), and the results were interpreted according to the criteria of the CLSI for Pseudomonas spp. (Table 2). The antimicrobial discs were obtained from Thermo Fisher Scientific Australia Pty Ltd. or Becton Dickinson (North Ride, NSW, Australia). 

Table 2

 

Antibiotic Susceptibilities of S. maltophilia

Antibiotic Class	Antibiotics	Disc Content	Number of Strains (%)
			S	I	R
Fluoroquinolones	Ciprofloxacin	5 μg	37 (92.5%)	1 (2.5%)	2 (5%)
	Gatifloxacin	5 μg	40 (100%)	0 (0%)	0 (0%)
	Levofloxacin	5 μg	40 (100%)	0 (0%)	0 (0%)
	Moxifloxacin	5 μg	40 (100%)	0 (0%)	0 (0%)
	Ofloxacin	5 μg	39 (97.5%)	1 (2.5%)	0 (0%)
β-lactams	Aztreonam	30 μg	21 (52.5%)	13 (32.5%)	6 (15%)
	Ceftazidime	30 μg	40 (100%)	0 (0%)	0 (0%)
	Cefepime	30 μg	35 (87.5%)	2 (5%)	3 (7.5%)
	Imipenem	10 μg	3 (7.5%)	0 (0%)	37 (92.5%)
	Ticarcillin	75 μg	39 (97.5%)	0 (0%)	1 (2.5%)
Aminoglycosides	Gentamicin	10 μg	37 (92.5%)	2 (5%)	1 (2.5%)
	Tobramycin	10 μg	38 (95%)	1 (2.5%)	1 (2.5%)
Glycycline	Tigecycline	15 μg	40 (100%)	0 (0%)	0 (0%)
SXT	Trimethoprim/sulfamethozole (cotrimoxazole)	25 μg	40 (100%)	0 (0%)	0 (0%)
Chloramphenicol	Chloramphenicol	10 μg	17 (42.5%)	18 (45%)	5 (12.5%)
Polypeptide	Polymyxin B	300 units	37 (92.5%)	-	3 (7.5%)

 S, sensitive; I, intermediate resistance; R, resistant.

Susceptibility to MPDS

RevitaLens OcuTec (Abbott Medical Optics, Santa Ana, CA, USA), renu fresh and Biotrue (Bausch + Lomb, Rochester, NY, USA), AQuify (Ciba Vision, Atlanta, GA, USA), OPTI-FREE PureMoist and OPTI-FREE RepleniSH (Alcon, Fort Worth, TX, USA), and MeniCare Soft (Menicon, Nagoya, Japan) were used to investigate the susceptibility of S. maltophilia isolates (Table 3). The minimum inhibitory concentration (MIC) for each MPDS was measured using the broth microdilution method in MHB as recommended by the CLSI.²⁸ The MPDS were 2-fold serially diluted in MilliQ water (Millipore Australia Pty Ltd., North Ryde, NSW, Australia) to give final concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. Each 500-μL dilution was inoculated with 50 μL bacterial suspension prepared in MHB (see above). Minimum inhibitory concentrations were measured using a microtiter plate reader (Multiscan spectrum; Thermo Fisher Scientific Australia Pty Ltd.) at 660_nm after incubation for 24 hours at 37°C_. The MIC endpoint was defined as the lowest concentration for which there was an 80% reduction in growth compared to the positive control (MIC₈₀). Minimum bactericidal concentration (MBC) in broth was assessed by plating out the 20-μL broth culture from the wells giving an MIC₈₀ and concentrations above this onto a Dey-Engly agar plate (Becton Dickinson) and incubating at 37°C for 24 hours. The MBC were determined as the concentration at which bacterial growth did not occur after incubation. All isolates were tested in duplicate for both MIC and MBC and repeated three times. 

Table 3

 

MIC and MBC Concentrations of Different MPDS for Strains of S. maltophilia

MPDS	MPDS Ingredients	Median MIC Concentration, %	Median MBC Concentration, %
RevitaLens OcuTec	Polyquaternium-1 (0.0003%), alexidine dihydrochloride (0.00016%), edetate sodium, boric acid, sodium borate, Tetronic 904, sodium citrate, sodium chloride, water	3.13	18.75
renu fresh	Polyaminopropyl biguanide (PHMB; 0.0001%), edetate sodium, poloxamine, hydroxyalkylphosphonate, boric acid, sodium borate, sodium chloride, water	3.13	75
Biotrue	Polyquaternium-1 (0.0001%), polyaminopropyl biguanide (PHMB; 0.00013%), edetate sodium, boric acid, sodium borate, poloxamine, sulfobetaine, hyaluronan, sodium chloride, water	6.25	50
AQuify	Polyaminopropyl biguanide (PHMB; 0.0001%), edetate sodium, Pluronic 127, tromethamine, sorbitol, sodium phosphate dihydrogen, dexpanthenol, water	50	100
OPTI-FREE RepleniSH	Polyquaternium-1 (0.001%), myristamidopropyl dimethylamine (0.0005%), nonanoyl-ethylenediaminetriacetic acid, Tetronic 1304, sodium borate, sodium citrate, sodium chloride, propylene glycol, water	25	100
OPTI-FREE PureMoist	Polyquaternium-1 (0.001%), myristamidopropyl dimethylamine (0.0006%), edetate sodium, Tetronic 1304, poly(ethyleneoxide-butyleneoxide)-41, sodium citrate, sodium chloride, boric acid, sorbitol, aminomethylpropanol, water	5.47	18.75
MeniCare Soft	Polyaminopropyl biguanide (PHMB; 0.0001%), macrogolglycerol hydroxystearate 60, edetate sodium, glycine, glycolic acid, propylene glycol, “isotonic agents,” water	14.06	100

Statistical Analysis

Statistical analysis was performed using SPSS 20.0 (SPSS; IBM, St. Leonards, NSW, Australia). Data were analyzed between factors using a two-sided Pearson χ² or Fisher's exact test as appropriate. P values ≤ 0.05 were considered significant. 

Results

Antibiotic Susceptibility

None of the isolates were resistant to trimethoprim-sulfamethozole, tigecycline, ceftazidime, gatifloxacin, levofloxacin, or moxifloxacin (Table 2). The sensitivity rates (excluding intermediate resistance) of isolates were 7% for imipenem, 85% for aztreonam, 87% for chloramphenicol, and 92% for cefepime (Table 2). The rate of multidrug resistance was 10%. 

Comparing between the two groups of S. maltophilia strains (group 1, isolated 1997–2001; group 2, 2005–2011), group 1 isolates were significantly more sensitive to imipenem (0% vs. 19%; P = 0.001) and significantly less sensitive to ciprofloxacin (90% vs. 29%; P = 0.019). There was no difference in the sensitivity to any antibiotic for strains isolated from lens wearers with keratitis at the time of isolation versus lens wearers with no keratitis at the time of isolation. However, between the two groups, isolates from keratitis in group 1 were more likely to be sensitive to imipenem than isolates from keratitis in group 2 (20% vs. 0%; P = 0.012). 

MPDS Susceptibility

There was no difference between the isolates in their mean generation time in MHB (2.9 ± 0.5 hours). The MIC and MBC of each MPDS are given in Table 3. In this table, MIC and MBC dilutions do not always agree with the dilutions used, as in some cases different repeats resulted in slightly different dilutions for MIC or MBC (usually differing by only one dilution either way). Overall, the MIC for AQuify (50%) and OPTI-FREE RepleniSH (25%) were significantly higher than the MIC of other MPDS (P < 0.05), with the exception of the comparison of OPTI-FREE RepleniSH versus MeniCare Soft (Table 3). There were no significant differences between the MIC for RevitaLens OcuTec, renu fresh, Biotrue, OPTI-FREE PureMoist, and MeniCare Soft (Table 3). The MBC of AQuify, OPTI-FREE RepleniSH, and MeniCare Soft (undiluted MPDS) were significantly higher than those of the other MPDS (range, 18.75% [RevitaLens OcuTec and OPTI-FREE PureMoist] to 75% [renu fresh]). 

There were differences between strains for MIC and MBC of each MPDS, and the number of strains having MIC and MBC in various ranges are given in Table 4. However, there were no differences in the MIC or MBC between strains isolated during different years (1997–2001 vs. 2005–2011) or from subjects with or without keratitis. 

Table 4

 

Number of Strains Within Particular Ranges of MIC and MBC for the Various MPDS

MPDS	MIC/MBC, Number of Strains
	1%–5%	>5%–15%	>15%–30%	>30%–50%	>50%–100%
RevitaLens OcuTec	28/1	13/14	0/25	0/0	0/0
renu fresh	33/1	7/0	0/1	0/14	0/24
Biotrue	9/1	31/0	0/0	0/37	0/2
AQuify	1/1	1/0	1/0	23/0	14/39
OPTI-FREE RepleniSH	0/0	1/1	22/1	12/8	5/30
OPTI-FREE PureMoist	20/1	19/8	1/29	0/2	0/1
MeniCare Soft	3/1	17/0	19/0	0/2	1/37

Discussion

The current study demonstrated that strains of S. maltophilia isolated from contact lens wearers in Australia retain high rates of susceptibility to antibiotics. The high rates of susceptibility to fluoroquinolones (ciprofloxacin, levofloxacin, or moxifloxacin), sulfamethoxazole-trimethoprim, and tigecycline are in agreement with findings from strains isolated in Taiwan from endophthalmitis¹⁸ and from around the world from diverse infections.²⁹ The isolates in the current study had higher rates of sensitivity to ceftazidime or the combination of trimethoprim and sulfamethoxazole than isolates from keratitis or sclerotic lesions in a Taiwanese population.¹⁴ Indeed, all of the isolates from Australia were susceptible to trimethoprim-sulfamethozole and, generally, the fluoroquinolones, antimicrobials often used to treat S. maltophilia infection.⁴ 

The current study did not find any differences in susceptibility of isolates from keratitis and nonkeratitis patients. This is in contrast to reports on isolates from lungs showing that isolates from cases of cystic fibrosis had significantly higher levels of resistance to piperacillin, cefotaxime, cefepime, moxalactam, ciprofloxacin, ofloxacin, sparfloxacin, gatifloxacin, and doxycycline.³⁰ In the case of the cystic fibrosis isolates, the increased rate of resistance is likely due to the frequent exposure of bacteria in the lungs of these patients to antibiotics.³⁰ The current study did show that there were some changes in antibiotic sensitivity patterns over time, with isolates from 1997 to 2001 showing increased sensitivity to imipenem but reduced sensitivity to chloramphenicol. This may reflect changes to antibiotic use within the community over time. 

The present study also examined the rates of resistance of isolates of S. maltophilia to multipurpose disinfecting solutions. These solutions contained a variety of disinfectants (Table 3) and other ingredients. These MPDS showed very different MIC and MBC values. In contrast to the situation with antibiotics, there are no accepted values to label strains resistant or susceptible to these agents; hence we used a broth dilution method to measure MIC/MBC. Many of the S. maltophilia isolates in the current study had been isolated from contact lens disinfecting cases, with many of these cases being used in conjunction with either AQuify or OPTI-FREE RepleniSH.²⁵ Others have shown that strains of this bacterium were resistant to these MPDS.²⁷ It is of interest to compare the resistance to OPTI-FREE RepleniSH and OPTI-FREE PureMoist. These two MPDS from the same manufacturer differ in a slight increase in the concentration of myristamidopropyl dimethylamine (to 0.0006%) in the latter; a change from nonanoyl-ethylenediaminetriacetic acid in RepleniSH to edetate sodium in PureMoist; the addition of poly(ethyleneoxide-butyleneoxide)-41, sorbitol, and aminomethylpropanol to PureMoist; and the omission of propylene glycol from PureMoist. This change in constituents has made a substantial difference in the ability of the MPDS to prevent the growth and to kill strains of S. maltophilia (Tables 3, 4). These results are supported by those of Kilvington et al.,²⁷ who used a slightly different assay to test for susceptibility. These data, as well as the resistance of strains to the MPDS MeniCare Soft, point to the fact that S. maltophilia can be quite intrinsically resistant to the disinfectants polyquaternium-1, myristamidopropyl dimethylamine, and polyhexamethyl biguanide (PHMB), and the importance of other MPDS excipients in controlling growth of this bacterium. It would be interesting to monitor the contamination of contact lens cases used with MPDS to determine whether MIC or MBC is important for control of contamination during use. Overall, the best performing-MPDS for controlling the growth of S. maltophilia were RevitaLens OcuTec and OPTI-FREE PureMoist, which showed the lowest MIC and MBC for the strains. 

The finding that strains of S. maltophilia are resistant to the disinfectant action of MPDS is probably one of the reasons that these bacteria have been cultured from contact lens cases using those MPDS.^25,27 S. maltophilia may be a scaffold bacterium in contact lens case biofilms, promoting the further colonization of other microbial types within the biofilm. It has been postulated that the presence of bacteria such as S. maltophilia in cases could lead to colonization of cases with Acanthamoeba strains, as they can feed on these bacteria.^27,31,32 S. maltophilia can act as endosymbiotes of Acanthamoeba.³³ These protozoa may then go on to cause microbial keratitis, and cases of coculture of Acanthamoeba and S. maltophilia from keratitis have been reported.^16,17 Also, S. maltophilia itself has been isolated from contact lenses at the time of keratitis.³⁴ In the study by Wiley et al.,³⁴ strains of S. maltophilia and other Gram-negative bacteria such as Achromobacter spp. and D. acidovorans were isolated from contact lenses at the time of keratitis, and the level of contact lens case contamination by these bacteria was associated with increased severity of keratitis.³⁴ 

In conclusion, this study has demonstrated that strains of S. maltophilia isolated in Australia from contact lenses, contact lens cases, or ocular swabs over a 17-year period (1994–2011) remained relatively susceptible to antibiotics commonly used to treat infections caused by this bacterium. However, strains were very resistant to the effects of certain MPDS, and this was not solely dependent on the type of disinfectants used in the formulation of the MPDS. 

Acknowledgments

Supported by the Brien Holden Vision Institute. The authors alone are responsible for the content and writing of the paper. 

Disclosure: K. Watanabe, None; H. Zhu, None; M. Willcox, Alcon (R); Bausch + Lomb (R)