1040-5488/14/9103-0272/0 VOL. 91, NO. 3, PP. 272Y277 OPTOMETRY AND VISION SCIENCE Copyright * 2014 American Academy of Optometry
ORIGINAL ARTICLE
Effect of Multipurpose Solutions against Acinetobacter Carrying QAC Genes Maureen V. Boost*, Jessica Chan†, Guang-sen Shi‡, and Pauline Cho§
ABSTRACT Purpose. Acinetobacter has low virulence but causes infections in subjects with reduced immunity. It has been reported in ocular infections including those of patients using contact lenses. Treatment is difficult because Acinetobacter is frequently multidrug resistant. Antibiotic-resistant strains frequently also harbor genes for antiseptic resistance (quaternary ammonium compound [QAC]) genes. Because Acinetobacter is part of the normal flora, it may contaminate contact lens and accessories. This study aims to investigate carriage rates of QAC genes in household and clinical isolates of Acinetobacter and to determine the effectiveness of two multipurpose solutions (MPSs) for soft lenses against organisms carrying QAC genes. Methods. DNA was extracted from 11 bathroom isolates and 15 clinical isolates and amplified by polymerase chain reaction to determine the presence of qacE$1. Gene-positive and gene-negative control strains were used to challenge the two MPSs, and minimum inhibitory concentrations (MICs) of these organisms to benzalkonium chloride and chlorhexidine gluconate were determined. Results. More than 90% of isolates carried qacE$1. The MICs of clinical isolates were higher than those of isolates of bathrooms. Both MPSs were able to produce a 3-log reduction in the numbers of all isolates. Conclusions. Although most isolates carried qacE$1 and elevated MICs to benzalkonium chloride and chlorhexidine gluconate were observed, all were susceptible to both MPSs tested. However, if there were to be poor compliance with care procedures, it is probable that such organisms could survive in the presence of diluted or expired solutions. (Optom Vis Sci 2014;91:272Y277) Key Words: Acinetobacter, antiseptic resistance, contact lens, MPS, noncompliance
A
cinetobacter are gram-negative bacteria isolated from a wide range of sources, including human skin.1 They are the only gram-negative organisms regularly found as members of the human skin flora and are usually regarded as commensal, being present in 25 to 40% of the population.1 They are more likely to be isolated in the summer months because warmth and humidity favor their growth. Although they have a low virulence, infections caused by these organisms have been increasingly reported, in particular, in patients with decreased resistance or who have undergone medical procedures, such as instrumentation or surgery.1,2 They have been recognized as an important cause of nosocomial infections.1,3
*DPhil, FIBMS † BSc, MLT ‡ MD § PhD, FAAO Department of Health Technology and Informatics (MVB, JC), and School of Optometry (G-sS, PC), The Hong Kong Polytechnic University, Hong Kong SAR, China.
They are able to adhere well to a wide range of materials, including human epithelial cells, red blood cells, plastic, and glass surfaces.4,5 Their nutritional requirements are simple, and they are able to persist in harsh and dry conditions that may allow their survival in the environment.1 This combination of long survival and ability to adhere to a wide range of materials allows Acinetobacter to contaminate contact lenses and accessories. After adherence, Acinetobacter are able to form biofilms that increase their ability to survive on inanimate materials.5,6 The ability of Acinetobacter to adhere to human epithelial cells is an essential step toward colonization and invasion. Although their ability to invade epithelial cells is lower than other gram-negative pathogens such as Pseudomonas aeruginosa and Escherichia coli, it has been suggested that the physiological changes in the eyes of contact lens users may make these subjects more susceptible to infection with opportunistic pathogens including Acinetobacter.4 Although eye infection with Acinetobacter is rare, it is listed as a causative organism of microbial keratitis in several surveys7Y10 and case reports.2,11
Optometry and Vision Science, Vol. 91, No. 3, March 2014
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Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al.
Treatment of Acinetobacter infection is difficult because it is frequently multidrug resistant, having both intrinsic and acquired resistance mechanisms.12 Multidrug resistance in Acinetobacter has been attributed to its ability to accept foreign DNA, including genetic determinants for antimicrobial resistance.13 In addition to resistance to antibiotics, Acinetobacter have also been shown to carry determinants for antiseptic resistance that may be coselected with those for antibiotic resistance.13 Conversely, selection for antiseptic resistance may coselect for antibiotic resistance.14 Antiseptic resistance has been reported for several agents including biguanides (e.g., chlorhexidine gluconate [CHX]), quaternary ammonium compounds (QACs) (e.g., benzalkonium chloride [BAK] and PHMB [polyhexamethylene biguanide]), and diamidines (e.g., propamidine).14 This resistance is attributable to production of efflux proteins encoded by plasmid-mediated genes identified in both gram-positive and gram-negative organisms. These proteins belong to two families, the major facilitator super family and the small multidrug resistance family. The major facilitator super family is only found in gram-positive organisms, but genes for two members of the small multidrug resistance family, qacE and qacE$1, have been identified in a range of gram-negative organisms, including Acinetobacter.15,16 QacE is a multidrug efflux protein that confers resistance to a variety of QACs,15,16 whereas QacE$1 is a defective form of QacE. High levels of qacE$1 carriage have been reported in clinical isolates of Acinetobacter, and it has been suggested that the increase in both antibiotic and antiseptic resistance in this organism is related to the presence of this gene.17 The presence of residues of disinfectant or use of disinfectant at subinhibitory concentrations has been suggested to select for strains carrying QAC genes.18 Multipurpose solutions (MPSs) are widely used for contact lens disinfection and include QACs and biguanides as disinfecting agents. Optifree Replenish uses a polymeric form of QAC, POLYQUAD, as its active disinfecting ingredient. Several other MPSs including Renu Fresh, Complete, and Solocare use polymeric forms of biguanide compounds such as polyhexamethylene biguanide. These compounds are used at low concentrations. If there is poor compliance with the use of MPSs such as topping up or reuse of solutions in the lens case, this may lead to organisms being exposed to subinhibitory levels of disinfecting agents and may lead to selection of strains carrying antiseptic resistance genes. The presence of Acinetobacter from contact lens cases of patients reporting poor compliance with care routines has been reported.13,19Y22 This study aimed to determine the effectiveness of two MPSs against strains of Acinetobacter carrying antiseptic resistance genes in isolates retrieved from the environment of contact lens users.
METHODS Volunteer contact lens users were recruited by means of a poster displayed in the University’s Student Union. A 10-cm2 area adjacent to the bathroom sink of each of the 30 contact lens users were sampled using a sterile swab (Transwabs; Medical Wire & Equipment, UK) moistened with normal saline. The swab was then placed into transport medium and sent to the laboratory for processing within 24 hours. Each sample was plated onto CHROMagar Acinetobacter plate (CHROMagar, France) for iso lation of Acinetobacter. Plates were incubated at 37-C aerobically and examined after 24
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and 48 hours. If red colonies were observed, they were subcultured to tryptone soya agar ([TSA] Oxoid, Basingstoke, UK) and incubated for 18 to 24 hours at 37-C before subsequent identification using a range of biochemical tests.23 A further 15 previously confirmed isolates of Acinetobacter from human infections were obtained from a district hospital in Hong Kong and were cultured on TSA. Susceptibility to two commonly used antiseptics, CHX and BAK (Sigma-Aldrich USA) was determined by broth dilution minimum inhibitory concentration (MIC) method by microdilution in 96-well microtiter plates (BD Falcon USA) for both bathroom and clinical isolates. Each organism was suspended in sterile saline at a concentration equivalent to 0.5 McFarland standard. Twofold serial dilutions of CHX and BAK were prepared in 100-KL aliquots of Mueller-Hinton broth (Oxoid) in a microtiter plate to give a range of dilutions from 1- to 128-mg/L concentrations for each of the antiseptics. Fifty microliters of each bacterial suspension was added to a row on the microtiter plate, and the plate was incubated overnight at 37-C. The microtiter plate was examined using a plate reader (Benchmark Plus Microplate spectrophotometer; BioRad, Hercules, CA). The lowest concentration showing no visible growth of a particular Acinetobacter strain was considered as the MIC. DNA was extracted from each of the isolate using five to 10 colonies taken from a TSA plate and inoculated into 200 KL lysis buffer containing 184 KL Milli-Q water, 2 KL, 1 M Tris, 4 KL 0.5 M EDTA, and 10 KL lysozyme. The mixtures were incubated with agitation (Stuart Orbital Incubator S1500; Bibby Scientific Limited, Stone, UK) for 40 minutes at 37-C, followed by boiling in a water bath for 10 minutes and transferred to an ice bath for a further 10 minutes. After centrifugation at 18,000 rpm for 5 minutes, 100 KL of the supernatant was transferred to a clean Eppendorf tube for measurement of DNA concentration (NanoDrop Spectrometer ND-1000; Thermo Scientific, Waltham, MA). The DNA concentration was adjusted to 50 ng/KL by adding Milli-Q water, and the samples were stored at j30-C before performing polymerase chain reaction (PCR). Primers for qacE$1 (Invitrogen, Carlsbad, CA) were used to amplify the gene in the sample. Primers used were forward primer (P1), 5¶- TAG CGA GGG CTT TAC TAA GC-3¶; reverse primer (P2), 5¶-ATT CAG AAT GCC GAA CAC CG-3¶ based on gene sequence published in GenBank.15 The concentration of each primer was adjusted to 10 KM and was stored at -30-C. For PCR, a master mix consisting of 1 KL DNA sample, 0.5 KM primers (P1 and P2), 200 KM dNTPs, 1.5 mM MgCl2, 1 U Dream Taq DNA polymerase, 1 GoTaq buffer, and Milli-Q water to a total volume of 20 KL was used. Polymerase chain reaction was performed using a thermal cycler (GeneAmp PCR System 9700; Life Technologies, Carlsbad, CA) with initial denaturation at 93-C for 3 minutes, followed by 35 cycles of denaturation at 93-C for 3 seconds, annealing at 55-C for 30 seconds, and DNA extension at 72-C for 1 minute, and final extension at 72-C for 5 minutes. Samples were placed at 4-C after amplification. Water was used as a negative control. The PCR products were detected by electrophoresis on a 1.2% agarose gel at 80 V for 1.5 hours, stained using ethidium bromide and visualized using a UV transilluminator. The PCR product length was 300 bp on comparison with a 1-kb ladder. Eight isolates were selected based on the results of the PCR analyses, and inocula were prepared and used to test against two
Optometry and Vision Science, Vol. 91, No. 3, March 2014
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274 Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al. TABLE 1.
Information of multipurpose solutions used in the study Multipurpose solution ReNu Fresh
OPTI-FREE RepleniSH
Ingredients
MMRDT
Manufacturer
0.0001% polyaminopropyl biguanide, hydroxyalkylphosphonate, boric acid, edetate disodium, poloxamine, sodium borate, sodium chloride 0.001% polyquaternium-1, 0.0005% myristamidopropyl dimethylamine, sodium citrate, sodium chloride, sodium borate, propylene glycol, Tearglyde
4h
Bausch+Lomb Rochester, NY
6h
Alcon Laboratories Fort Worth, TX
MMRDT, manufacturer’s minimum recommended disinfection time.
popular MPS (ReNu Fresh and Optifree RepleniSH). The ingredients of these solutions are shown in Table 1. For each of the selected strains, 0.2 mL of an inoculum with a concentration of 106 CFU/mL was added to 1.8 mL of MPS in a sterile polypropylene test tube (BD Biosciences, San Jose, CA) to give a final concentration of 105 CFU/mL. These mixtures were incubated at room temperature for the manufacturer’s minimum recommended disinfection time as shown in Table 1. Each strain was tested in duplicate for each MPS. At the end of the disinfection time, 1 mL of each of the mixtures was transferred to 9 mL of Dey-Engley broth (BD Biosciences) at room temperature for 10 minutes to neutralize the effect of the disinfectant. After this, 0.1 mL of Dey-Engley broth was spread evenly on nutrient agar in duplicate, and the plates were incubated at 37-C for 18 hours. As a control, phosphate buffered saline was used in place of the MPS and the incubation in parallel for each strain used. Viable counts were performed on the nutrient agars using an automated colony counter (aCOLyte SuperCount; Symbiosis, Frederick, MD). The final CFU/mL was calculated from the viable counts and used to determine the log10 reduction, which is equivalent to the surviving organisms after exposure. Mann-Whitney U tests were used to examine the differences in MICs between solutions. Wilcoxon signed rank tests were used to test for differences in effects of exposure.
RESULTS A total of 11 strains of Acinetobacter were isolated from nine of the 30 household samples. Three samples yielded two distinguishable Acinetobacter strains on CHROMagar Acinetobacter. The results of MIC to CHX and BAK are shown in Table 2. The majority of isolates from the bathroom gave lower MICs than hospital isolates. qacE$1 was present in 24 strains, with 14 of 15 clinical isolates and 10 of 11 bathroom isolates being positive for the gene. For testing effectiveness of MPS, three clinical (A6, A7, A15) and three bathroom (B8b, B12b, B28b) isolates harboring qacE$1 and two strains (A12 and B25) that were negative for the gene were used. Samples A15 displayed the highest MIC to BAK. Both MPSs were effective against all Acinetobacter strains, achieving at least a 3-log reduction, as suggested by the US Food and Drug Administration guidelines (Table 3). Statistical analyses revealed that MICs of clinical strains to both CHX and BAK were significantly higher than bathroom isolates.
There were no statistically significant differences in efficacy of either MPS against Acinetobacter from the two sources nor between the MPS against all strains as log reductions were similar for all isolates.
DISCUSSION In parallel to the rise in users of contact lenses,24,25 there has been an increase in numbers of antiseptic-resistant microorganisms14,26,27
TABLE 2.
Summary of PCR results for qacE$1 gene Minimum inhibitory concentrations, mg/L Isolates* Chlorhexidine Benzalkonium chloride qacE$1 gene A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 B7 B8a B8b B12a B12b B18 B21 B25 B27 B28a B28b
16 16 8 16 16 16 16 16 16 16 8 16 16 16 16 2 4 2 8 8 8 4 16 4 4 4
16 16 16 16 16 16 16 16 16 16 16 16 16 16 32 8 8 4 8 8 8 8 16 4 8 8
* A, clinical strains; B, bathroom strains. + positive.
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+ + + + + + + + + + + j + + + + + + + + + + j + + +
Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al. TABLE 3.
Efficacy of multipurpose solutions at manufacturer’s minimum recommended disinfection time against eight representative Acinetobacter isolates Mean log10 reduction Isolates
ReNu Fresh
OPTI-FREE RepleniSH
A6 A7 A12 A15 B8b B12b B25 B28b Mean T SD
4.75 5.49 5.83 6.05 5.39 5.99 5.54 5.47 5.56 T 0.41
5.83 5.57 5.62 5.55 5.51 6.49 5.34 4.90 5.60 T 0.45
which has led to concern about efficacy of contact lens care products. The US Food and Drug Administration stand-alone test for contact lens solutions contains a limited number of organisms. Clearly, such a test cannot include all potential ocular pathogens, but a good disinfection capacity is essential to minimize the possibility of eye infection in contact lens users. Acinetobacter is ubiquitous in the environment and a colonizer of human skin. It is an unusual cause of microbial keratitis, but it seems to be becoming more common.10,28 It is a common cause of hospital-acquired infections and is difficult to treat because it is frequently multiresistant to antibiotics. In addition to antibiotic resistance, this organism is also reported to display reduced susceptibility to antiseptics. This study investigated the susceptibility of strains from households and hospitals to two popular contact lens solutions. Acinetobacter was isolated from the bathrooms of nine of 30 households of contact lens users, implying that the bathroom is a potential source of Acinetobacter and that cross-contamination between the hands of users and the contact lenses may occur if the user handles the lenses in the bathroom. Three samples yielded more than one Acinetobacter strain, suggesting that the environment favors the growth of this organism. As it was considered possible that hospital isolates would be more resistant than those from the community, 15 hospital isolates were also included in this study. Investigation of the strains for the presence of qacE$1 revealed that almost all strains from both sources harbored this gene, showing that there has been widespread dissemination of this resistance determinant in the population. Other workers have reported high levels of this gene in hospital isolates,29Y31 but this seems to be the first report of the presence of antiseptic resistance genes from community strains of Acinetobacter. Several studies have suggested that residual levels of disinfectants may select for strains harboring antiseptic resistance genes and that inappropriate use of antiseptics may increase selection of such strains. The high rate of antiseptic genes in hospital isolates is thought to be attributable to frequent use of disinfectants in these environments. In Hong Kong, use of disinfectants in the community increased dramatically after the outbreaks of SARS (severe acute respiratory syndrome) and bird flu.32,33 This may be a contributing factor
275
to the high rates of antiseptic resistance genes observed in community isolates. Determination of the MIC revealed that higher MICs to both CHX and BAK were observed in hospital isolates than those in the community. The levels observed for CHX were similar to those reported in a study of Acinetobacter isolates from Japan in 200834 that reported MIC50 of 10 mg/L to CHX and 5 mg/L to BAK but were higher than levels reported in US isolates.35 Both of these studies were conducted using broth dilutions as in the current study, but there is some evidence that higher MICs to BAK but not CHX are obtained if the estimation is performed by agar dilution.13 There is currently no standard method recommended for antiseptic MIC determination, making it difficult to compare results between studies. The MICs obtained showed that all the clinical and bathroom isolates remained susceptible to CHX and BAK at their in-use concentrations, which are 5000 and 2000 mg/L, respectively.13 However, concentrations of QACs and biguanide derivatives used in contact lens solutions are considerably lower than those recommended for general antiseptic use. It was therefore important to determine if the Acinetobacter strains could be effectively killed by routine care procedures with contact lens solutions. Testing of the two chosen MPSs revealed at least a 3-log reduction in viable cells was observed for all strains. This is reassuring, showing that if disinfection is correctly performed, Acinetobacter should be eliminated and would be unlikely to cause infection. However, as have been reported in numerous studies, many patients failed to comply with care procedures.13,19Y22 Such practices as reuse of MPS, topping up of lens cases without pouring away old solution, and use of expired solution may lead to a less than adequate concentration of disinfectant in the lens cases. Reduced concentrations may select for strains with antiseptic-resistant genes. This applies not only to Acinetobacter but also to other organisms including Staphylococcus aureus and Pseudomonas aeruginosa, which are also known to carry antiseptic-resistant genes.15,16,27 Repeated exposures of these strains to a low level of disinfectant can increase their nonsusceptibility to disinfectants. This may lead to development of strains that are resistant to the standard disinfecting concentration. In addition, as it has been shown that there is coselection of antibiotic resistance together with antiseptic resistance,14,35 poor compliance may also lead to the presence of antibiotic-resistant organisms on lenses and accessories. There have been increasing reports of ocular infections with antibiotic-resistant organisms.36,37 Thus, it is important for practitioners to educate their patients in the correct care procedures to minimize the risk of ocular infections.
CONCLUSIONS This study has shown that Acinetobacter is a likely contaminant of contact lens and storage cases. Despite carriage of antiseptic resistance gene qacE$1, it remains susceptible to two popular MPSs when used at the correct concentrations. However, because contact lens wear is associated with an increased risk for microbial keratitis, which can include pathogens with multiple drug resistance, it is important to consider compliance with care routine especially in immunocompromised patients
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276 Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al.
ACKNOWLEDGMENTS The authors thank Ms. Zhang Ming for technical assistance and Dr. Dominic Tsang (Consultant Pathologist, Queen Elizabeth Hospital) for the clinical isolates. Received August 14, 2013; accepted November 27, 2013.
REFERENCES 1. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538Y82. 2. Marcovich A, Levartovsky S. Acinetobacter exposure keratitis. Br J Ophthalmol 1994;78:489Y90. 3. Hsueh PR, Teng LJ, Chen CY, Chen WH, Yu CJ, Ho SW, Luh KT. Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerg Infect Dis 2002; 8:827Y32. 4. Choi CH, Lee JS, Lee YC, Park TI, Lee JC. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiol 2008;8:216. 5. Gaddy JA, Actis LA. Regulation of Acinetobacter baumannii biofilm formation. Future Microbiol 2009;4:273Y8. 6. Wroblewska MM, Sawicka-Grzelak A, Marchel H, Luczak M, Sivan A. Biofilm production by clinical strains of Acinetobacter baumannii isolated from patients hospitalized in two tertiary care hospitals. FEMS Immunol Med Microbiol 2008;53:140Y4. 7. Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PG, Geerards AJ, Kijlstra A. Incidence of contact lensYassociated microbial keratitis and its related morbidity. Lancet 1999;354:181Y5. 8. Goldstein MH, Kowalski RP, Gordon YJ. Emerging fluoroquinolone resistance in bacterial keratitis: a 5-year review. Ophthalmology 1999;106:1313Y8. 9. Alexandrakis G, Alfonso EC, Miller D. Shifting trends in bacterial keratitis in South Florida and emerging resistance to fluoroquinolones. Ophthalmology 2000;107:1497Y502. 10. Fong CF, Tseng CH, Hu FR, Wang IJ, Chen WL, Hou YC. Clinical characteristics of microbial keratitis in a university hospital in Taiwan. Am J Ophthalmol 2004;137:329Y36. 11. Lim L, Loughnan MS, Sullivan LJ. Microbial keratitis associated with extended wear of silicone hydrogel contact lenses. Br J Ophthalmol 2002;86:355Y7. 12. Henwood CJ, Gatward T, Warner M, James D, Stockdale MW, Spence RP, Towner KJ, Livermore DM, Woodford N. Aantibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936). J Antimicrob Chemother 2002;49:479Y87. 13. Kawamura-Sato K, Wachino J, Kondo T, Ito H, Arakawa Y. Correlation between reduced susceptibility to disinfectants and multidrug resistance among clinical isolates of Acinetobacter species. J Antimicrob Chemother 2010;65:1975Y83. 14. Smith K, Gemmell CG, Hunter IS. The association between biocide tolerance and the presence or absence of qac genes among hospitalacquired and community-acquired MRSA isolates. J Antimicrob Chemother 2008;61:78Y84. 15. Kazama H, Hamashima H, Sasatsu M, Arai T. Distribution of the antiseptic-resistance genes qacE and qacE delta 1 in gram-negative bacteria. FEMS Microbiol Lett 1998;159:173Y8. 16. Ku¨cken D, Feucht H, Kaulfers P. Association of qacE and qacEDelta1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria. FEMS Microbiol Lett 2000;183:95Y8.
17. Wang C, Zhan Q, Mi Z, Huang Z, Chen G. Distribution of the antiseptic-resistance gene qacEDelta1 in 283 clinical isolates of gramnegative bacteria in China. J Hosp Infect 2008;69:394Y6. 18. Sasatsu M, Shibata Y, Noguchi N, Kono M. Substrates and inhibitors of antiseptic resistance in Staphylococcus aureus. Biol Pharm Bull 1994;17:163Y5. 19. Willcox MD, Harmis N, Cowell GM, Williams T, Holden DN. Bacterial interactions with contact lenses: effects of lens material, lens wear and microbial physiology. Biomaterials 2001;22:3235Y47. 20. Corrigan KM, Harmis NY, Willcox MD. Association of Acinetobacter species with contact lens-induced adverse responses. Cornea 2001; 20:463Y6. 21. Boost MV, Cho P. Microbial flora of tears of orthokeratology patients, and microbial contamination of contact lenses and contact lens accessories. Optom Vis Sci 2005;82:451Y8. 22. Yung MS, Boost M, Cho P, Yap M. Microbial contamination of contact lenses and lens care accessories of soft contact lens wearers (university students) in Hong Kong. Ophthalmic Physiol Opt 2007; 27:11Y21. 23. Schreckenberger PC, Daneshvar MI, Hollis DG. Acinetobacter, Achromobacter, Chryseobacterium, Moraxella, and other nonfermentative gram-negative rods. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of Clinical Microbiology, 9th ed. Washington, DC: ASM Press; 2007:770Y802. 24. Charm J, Cheung SW, Cho P. Practitioners’ analysis of contact lens practice in Hong Kong. Cont Lens Anterior Eye 2010;33:104Y11. 25. Morgan PB, Woods CA, Knajian R, Jones D, Efron N, Tan KO, Pesinova A, Grei, HJ, Marx J, Santodomingo S, Runberg SE, Tranoudis IG, Chandrinos A, Itoi M, Bendoriene J, van der Worp E, Helland M, Phillips G, Gonza´lez-Me´ijome JM, Belousov V, Mack, CJ. International contact lens prescribing in 2007. Contact Lens Spectrum 2008;23(1):36Y41. 26. Wang JT, Sheng WH, Wang JL, Chen D, Chen ML, Chen YC, Chang SC. Longitudinal analysis of chlorhexidine susceptibilities of nosocomial methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Taiwan. J Antimicrob Chemother 2008;62:514Y7. 27. Zhang M, O’Donoghue MM, Ito T, Hiramatsu K, Boost MV. Prevalence of antiseptic-resistance genes in Staphylococcus aureus and coagulase-negative staphylococci colonising nurses and the general population in Hong Kong. J Hosp Infect 2011;78:113Y7. 28. Kaye S, Tuft S, Neal T, Tole D, Leeming J, Figueiredo F, Armstrong M, McDonnell P, Tullo A, Parry C. Bacterial susceptibility to topical antimicrobials and clinical outcome in bacterial keratitis. Invest Ophthalmol Vis Sci 2010;51:362Y8. 29. Zhang J, Zhu ZH, Su GL, Ping J. Detection of qacE$1-sul1 in Acinetobacter baumannii isolated from Xinjiang and its clinical significance. Chin J Nosocomiol 2009-11. 30. Rajamohan G, Srinivasan VB, Gebreyes WA. Novel role of Acinetobacter baumannii RND efflux transporters in mediating decreased susceptibility to biocides. J Antimicrob Chemother 2010; 65:228Y32. 31. Yavari SA, Rota S, Caglar K, Fidan I. Role of Acinetobacter baumannii AdeB, AdeJ and QacE efflux genes in mediating decreased susceptibility to biocides. Ann Biol Res 2013,4:144Y50. 32. Wong WCW, Lee A, Tsang KK, Wong SYS. How did general practitioners protect themselves, their family, and staff during the SARS epidemic in Hong Kong? J Epidemiol Commun H 2004;58:180Y5. 33. Wong SY, Kung K, Wong MC, Wong C, Tsui W, Chan K, Liang J, Lee NL, Cheung AW, Wong EL. Primary care physicians’ response to pandemic influenza in Hong Kong: a mixed quantitative and qualitative study. Int J Infect Dis 2012;16:687Y91.
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Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al. 34. Kawamura-Sato K, Wachino J, Kondo T, Ito H, Arakawa Y. Reduction of disinfectant bactericidal activities in clinically isolated Acinetobacter species in the presence of organic material. J Antimicrob Chemother 2008;61:568Y76. 35. Vali L, Davies SE, Lai LL, Dave J, Amyes SG. Frequency of biocide resistance genes, antibiotic resistance and the effect of chlorhexidine exposure on clinical methicillin-resistant Staphylococcus aureus isolates. J Antimicrob Chemother 2008;61:524Y32. 36. Pachigolla G, Blomquist P, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities: a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens 2007; 33:45Y9.
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37. McDonald M, Blondeau JM. Emerging antibiotic resistance in ocular infections and the role of fluoroquinolones. J Cataract Refract Surg 2010;36:1588Y98.
Maureen Boost Department of Health Technology and Informatics The Hong Kong Polytechnic University Kowloon, Hong Kong SAR China e-mail: [email protected]
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ORIGINAL ARTICLE
Effect of Multipurpose Solutions against Acinetobacter Carrying QAC Genes Maureen V. Boost*, Jessica Chan†, Guang-sen Shi‡, and Pauline Cho§
ABSTRACT Purpose. Acinetobacter has low virulence but causes infections in subjects with reduced immunity. It has been reported in ocular infections including those of patients using contact lenses. Treatment is difficult because Acinetobacter is frequently multidrug resistant. Antibiotic-resistant strains frequently also harbor genes for antiseptic resistance (quaternary ammonium compound [QAC]) genes. Because Acinetobacter is part of the normal flora, it may contaminate contact lens and accessories. This study aims to investigate carriage rates of QAC genes in household and clinical isolates of Acinetobacter and to determine the effectiveness of two multipurpose solutions (MPSs) for soft lenses against organisms carrying QAC genes. Methods. DNA was extracted from 11 bathroom isolates and 15 clinical isolates and amplified by polymerase chain reaction to determine the presence of qacE$1. Gene-positive and gene-negative control strains were used to challenge the two MPSs, and minimum inhibitory concentrations (MICs) of these organisms to benzalkonium chloride and chlorhexidine gluconate were determined. Results. More than 90% of isolates carried qacE$1. The MICs of clinical isolates were higher than those of isolates of bathrooms. Both MPSs were able to produce a 3-log reduction in the numbers of all isolates. Conclusions. Although most isolates carried qacE$1 and elevated MICs to benzalkonium chloride and chlorhexidine gluconate were observed, all were susceptible to both MPSs tested. However, if there were to be poor compliance with care procedures, it is probable that such organisms could survive in the presence of diluted or expired solutions. (Optom Vis Sci 2014;91:272Y277) Key Words: Acinetobacter, antiseptic resistance, contact lens, MPS, noncompliance
A
cinetobacter are gram-negative bacteria isolated from a wide range of sources, including human skin.1 They are the only gram-negative organisms regularly found as members of the human skin flora and are usually regarded as commensal, being present in 25 to 40% of the population.1 They are more likely to be isolated in the summer months because warmth and humidity favor their growth. Although they have a low virulence, infections caused by these organisms have been increasingly reported, in particular, in patients with decreased resistance or who have undergone medical procedures, such as instrumentation or surgery.1,2 They have been recognized as an important cause of nosocomial infections.1,3
*DPhil, FIBMS † BSc, MLT ‡ MD § PhD, FAAO Department of Health Technology and Informatics (MVB, JC), and School of Optometry (G-sS, PC), The Hong Kong Polytechnic University, Hong Kong SAR, China.
They are able to adhere well to a wide range of materials, including human epithelial cells, red blood cells, plastic, and glass surfaces.4,5 Their nutritional requirements are simple, and they are able to persist in harsh and dry conditions that may allow their survival in the environment.1 This combination of long survival and ability to adhere to a wide range of materials allows Acinetobacter to contaminate contact lenses and accessories. After adherence, Acinetobacter are able to form biofilms that increase their ability to survive on inanimate materials.5,6 The ability of Acinetobacter to adhere to human epithelial cells is an essential step toward colonization and invasion. Although their ability to invade epithelial cells is lower than other gram-negative pathogens such as Pseudomonas aeruginosa and Escherichia coli, it has been suggested that the physiological changes in the eyes of contact lens users may make these subjects more susceptible to infection with opportunistic pathogens including Acinetobacter.4 Although eye infection with Acinetobacter is rare, it is listed as a causative organism of microbial keratitis in several surveys7Y10 and case reports.2,11
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Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al.
Treatment of Acinetobacter infection is difficult because it is frequently multidrug resistant, having both intrinsic and acquired resistance mechanisms.12 Multidrug resistance in Acinetobacter has been attributed to its ability to accept foreign DNA, including genetic determinants for antimicrobial resistance.13 In addition to resistance to antibiotics, Acinetobacter have also been shown to carry determinants for antiseptic resistance that may be coselected with those for antibiotic resistance.13 Conversely, selection for antiseptic resistance may coselect for antibiotic resistance.14 Antiseptic resistance has been reported for several agents including biguanides (e.g., chlorhexidine gluconate [CHX]), quaternary ammonium compounds (QACs) (e.g., benzalkonium chloride [BAK] and PHMB [polyhexamethylene biguanide]), and diamidines (e.g., propamidine).14 This resistance is attributable to production of efflux proteins encoded by plasmid-mediated genes identified in both gram-positive and gram-negative organisms. These proteins belong to two families, the major facilitator super family and the small multidrug resistance family. The major facilitator super family is only found in gram-positive organisms, but genes for two members of the small multidrug resistance family, qacE and qacE$1, have been identified in a range of gram-negative organisms, including Acinetobacter.15,16 QacE is a multidrug efflux protein that confers resistance to a variety of QACs,15,16 whereas QacE$1 is a defective form of QacE. High levels of qacE$1 carriage have been reported in clinical isolates of Acinetobacter, and it has been suggested that the increase in both antibiotic and antiseptic resistance in this organism is related to the presence of this gene.17 The presence of residues of disinfectant or use of disinfectant at subinhibitory concentrations has been suggested to select for strains carrying QAC genes.18 Multipurpose solutions (MPSs) are widely used for contact lens disinfection and include QACs and biguanides as disinfecting agents. Optifree Replenish uses a polymeric form of QAC, POLYQUAD, as its active disinfecting ingredient. Several other MPSs including Renu Fresh, Complete, and Solocare use polymeric forms of biguanide compounds such as polyhexamethylene biguanide. These compounds are used at low concentrations. If there is poor compliance with the use of MPSs such as topping up or reuse of solutions in the lens case, this may lead to organisms being exposed to subinhibitory levels of disinfecting agents and may lead to selection of strains carrying antiseptic resistance genes. The presence of Acinetobacter from contact lens cases of patients reporting poor compliance with care routines has been reported.13,19Y22 This study aimed to determine the effectiveness of two MPSs against strains of Acinetobacter carrying antiseptic resistance genes in isolates retrieved from the environment of contact lens users.
METHODS Volunteer contact lens users were recruited by means of a poster displayed in the University’s Student Union. A 10-cm2 area adjacent to the bathroom sink of each of the 30 contact lens users were sampled using a sterile swab (Transwabs; Medical Wire & Equipment, UK) moistened with normal saline. The swab was then placed into transport medium and sent to the laboratory for processing within 24 hours. Each sample was plated onto CHROMagar Acinetobacter plate (CHROMagar, France) for iso lation of Acinetobacter. Plates were incubated at 37-C aerobically and examined after 24
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and 48 hours. If red colonies were observed, they were subcultured to tryptone soya agar ([TSA] Oxoid, Basingstoke, UK) and incubated for 18 to 24 hours at 37-C before subsequent identification using a range of biochemical tests.23 A further 15 previously confirmed isolates of Acinetobacter from human infections were obtained from a district hospital in Hong Kong and were cultured on TSA. Susceptibility to two commonly used antiseptics, CHX and BAK (Sigma-Aldrich USA) was determined by broth dilution minimum inhibitory concentration (MIC) method by microdilution in 96-well microtiter plates (BD Falcon USA) for both bathroom and clinical isolates. Each organism was suspended in sterile saline at a concentration equivalent to 0.5 McFarland standard. Twofold serial dilutions of CHX and BAK were prepared in 100-KL aliquots of Mueller-Hinton broth (Oxoid) in a microtiter plate to give a range of dilutions from 1- to 128-mg/L concentrations for each of the antiseptics. Fifty microliters of each bacterial suspension was added to a row on the microtiter plate, and the plate was incubated overnight at 37-C. The microtiter plate was examined using a plate reader (Benchmark Plus Microplate spectrophotometer; BioRad, Hercules, CA). The lowest concentration showing no visible growth of a particular Acinetobacter strain was considered as the MIC. DNA was extracted from each of the isolate using five to 10 colonies taken from a TSA plate and inoculated into 200 KL lysis buffer containing 184 KL Milli-Q water, 2 KL, 1 M Tris, 4 KL 0.5 M EDTA, and 10 KL lysozyme. The mixtures were incubated with agitation (Stuart Orbital Incubator S1500; Bibby Scientific Limited, Stone, UK) for 40 minutes at 37-C, followed by boiling in a water bath for 10 minutes and transferred to an ice bath for a further 10 minutes. After centrifugation at 18,000 rpm for 5 minutes, 100 KL of the supernatant was transferred to a clean Eppendorf tube for measurement of DNA concentration (NanoDrop Spectrometer ND-1000; Thermo Scientific, Waltham, MA). The DNA concentration was adjusted to 50 ng/KL by adding Milli-Q water, and the samples were stored at j30-C before performing polymerase chain reaction (PCR). Primers for qacE$1 (Invitrogen, Carlsbad, CA) were used to amplify the gene in the sample. Primers used were forward primer (P1), 5¶- TAG CGA GGG CTT TAC TAA GC-3¶; reverse primer (P2), 5¶-ATT CAG AAT GCC GAA CAC CG-3¶ based on gene sequence published in GenBank.15 The concentration of each primer was adjusted to 10 KM and was stored at -30-C. For PCR, a master mix consisting of 1 KL DNA sample, 0.5 KM primers (P1 and P2), 200 KM dNTPs, 1.5 mM MgCl2, 1 U Dream Taq DNA polymerase, 1 GoTaq buffer, and Milli-Q water to a total volume of 20 KL was used. Polymerase chain reaction was performed using a thermal cycler (GeneAmp PCR System 9700; Life Technologies, Carlsbad, CA) with initial denaturation at 93-C for 3 minutes, followed by 35 cycles of denaturation at 93-C for 3 seconds, annealing at 55-C for 30 seconds, and DNA extension at 72-C for 1 minute, and final extension at 72-C for 5 minutes. Samples were placed at 4-C after amplification. Water was used as a negative control. The PCR products were detected by electrophoresis on a 1.2% agarose gel at 80 V for 1.5 hours, stained using ethidium bromide and visualized using a UV transilluminator. The PCR product length was 300 bp on comparison with a 1-kb ladder. Eight isolates were selected based on the results of the PCR analyses, and inocula were prepared and used to test against two
Optometry and Vision Science, Vol. 91, No. 3, March 2014
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274 Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al. TABLE 1.
Information of multipurpose solutions used in the study Multipurpose solution ReNu Fresh
OPTI-FREE RepleniSH
Ingredients
MMRDT
Manufacturer
0.0001% polyaminopropyl biguanide, hydroxyalkylphosphonate, boric acid, edetate disodium, poloxamine, sodium borate, sodium chloride 0.001% polyquaternium-1, 0.0005% myristamidopropyl dimethylamine, sodium citrate, sodium chloride, sodium borate, propylene glycol, Tearglyde
4h
Bausch+Lomb Rochester, NY
6h
Alcon Laboratories Fort Worth, TX
MMRDT, manufacturer’s minimum recommended disinfection time.
popular MPS (ReNu Fresh and Optifree RepleniSH). The ingredients of these solutions are shown in Table 1. For each of the selected strains, 0.2 mL of an inoculum with a concentration of 106 CFU/mL was added to 1.8 mL of MPS in a sterile polypropylene test tube (BD Biosciences, San Jose, CA) to give a final concentration of 105 CFU/mL. These mixtures were incubated at room temperature for the manufacturer’s minimum recommended disinfection time as shown in Table 1. Each strain was tested in duplicate for each MPS. At the end of the disinfection time, 1 mL of each of the mixtures was transferred to 9 mL of Dey-Engley broth (BD Biosciences) at room temperature for 10 minutes to neutralize the effect of the disinfectant. After this, 0.1 mL of Dey-Engley broth was spread evenly on nutrient agar in duplicate, and the plates were incubated at 37-C for 18 hours. As a control, phosphate buffered saline was used in place of the MPS and the incubation in parallel for each strain used. Viable counts were performed on the nutrient agars using an automated colony counter (aCOLyte SuperCount; Symbiosis, Frederick, MD). The final CFU/mL was calculated from the viable counts and used to determine the log10 reduction, which is equivalent to the surviving organisms after exposure. Mann-Whitney U tests were used to examine the differences in MICs between solutions. Wilcoxon signed rank tests were used to test for differences in effects of exposure.
RESULTS A total of 11 strains of Acinetobacter were isolated from nine of the 30 household samples. Three samples yielded two distinguishable Acinetobacter strains on CHROMagar Acinetobacter. The results of MIC to CHX and BAK are shown in Table 2. The majority of isolates from the bathroom gave lower MICs than hospital isolates. qacE$1 was present in 24 strains, with 14 of 15 clinical isolates and 10 of 11 bathroom isolates being positive for the gene. For testing effectiveness of MPS, three clinical (A6, A7, A15) and three bathroom (B8b, B12b, B28b) isolates harboring qacE$1 and two strains (A12 and B25) that were negative for the gene were used. Samples A15 displayed the highest MIC to BAK. Both MPSs were effective against all Acinetobacter strains, achieving at least a 3-log reduction, as suggested by the US Food and Drug Administration guidelines (Table 3). Statistical analyses revealed that MICs of clinical strains to both CHX and BAK were significantly higher than bathroom isolates.
There were no statistically significant differences in efficacy of either MPS against Acinetobacter from the two sources nor between the MPS against all strains as log reductions were similar for all isolates.
DISCUSSION In parallel to the rise in users of contact lenses,24,25 there has been an increase in numbers of antiseptic-resistant microorganisms14,26,27
TABLE 2.
Summary of PCR results for qacE$1 gene Minimum inhibitory concentrations, mg/L Isolates* Chlorhexidine Benzalkonium chloride qacE$1 gene A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 B7 B8a B8b B12a B12b B18 B21 B25 B27 B28a B28b
16 16 8 16 16 16 16 16 16 16 8 16 16 16 16 2 4 2 8 8 8 4 16 4 4 4
16 16 16 16 16 16 16 16 16 16 16 16 16 16 32 8 8 4 8 8 8 8 16 4 8 8
* A, clinical strains; B, bathroom strains. + positive.
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+ + + + + + + + + + + j + + + + + + + + + + j + + +
Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al. TABLE 3.
Efficacy of multipurpose solutions at manufacturer’s minimum recommended disinfection time against eight representative Acinetobacter isolates Mean log10 reduction Isolates
ReNu Fresh
OPTI-FREE RepleniSH
A6 A7 A12 A15 B8b B12b B25 B28b Mean T SD
4.75 5.49 5.83 6.05 5.39 5.99 5.54 5.47 5.56 T 0.41
5.83 5.57 5.62 5.55 5.51 6.49 5.34 4.90 5.60 T 0.45
which has led to concern about efficacy of contact lens care products. The US Food and Drug Administration stand-alone test for contact lens solutions contains a limited number of organisms. Clearly, such a test cannot include all potential ocular pathogens, but a good disinfection capacity is essential to minimize the possibility of eye infection in contact lens users. Acinetobacter is ubiquitous in the environment and a colonizer of human skin. It is an unusual cause of microbial keratitis, but it seems to be becoming more common.10,28 It is a common cause of hospital-acquired infections and is difficult to treat because it is frequently multiresistant to antibiotics. In addition to antibiotic resistance, this organism is also reported to display reduced susceptibility to antiseptics. This study investigated the susceptibility of strains from households and hospitals to two popular contact lens solutions. Acinetobacter was isolated from the bathrooms of nine of 30 households of contact lens users, implying that the bathroom is a potential source of Acinetobacter and that cross-contamination between the hands of users and the contact lenses may occur if the user handles the lenses in the bathroom. Three samples yielded more than one Acinetobacter strain, suggesting that the environment favors the growth of this organism. As it was considered possible that hospital isolates would be more resistant than those from the community, 15 hospital isolates were also included in this study. Investigation of the strains for the presence of qacE$1 revealed that almost all strains from both sources harbored this gene, showing that there has been widespread dissemination of this resistance determinant in the population. Other workers have reported high levels of this gene in hospital isolates,29Y31 but this seems to be the first report of the presence of antiseptic resistance genes from community strains of Acinetobacter. Several studies have suggested that residual levels of disinfectants may select for strains harboring antiseptic resistance genes and that inappropriate use of antiseptics may increase selection of such strains. The high rate of antiseptic genes in hospital isolates is thought to be attributable to frequent use of disinfectants in these environments. In Hong Kong, use of disinfectants in the community increased dramatically after the outbreaks of SARS (severe acute respiratory syndrome) and bird flu.32,33 This may be a contributing factor
275
to the high rates of antiseptic resistance genes observed in community isolates. Determination of the MIC revealed that higher MICs to both CHX and BAK were observed in hospital isolates than those in the community. The levels observed for CHX were similar to those reported in a study of Acinetobacter isolates from Japan in 200834 that reported MIC50 of 10 mg/L to CHX and 5 mg/L to BAK but were higher than levels reported in US isolates.35 Both of these studies were conducted using broth dilutions as in the current study, but there is some evidence that higher MICs to BAK but not CHX are obtained if the estimation is performed by agar dilution.13 There is currently no standard method recommended for antiseptic MIC determination, making it difficult to compare results between studies. The MICs obtained showed that all the clinical and bathroom isolates remained susceptible to CHX and BAK at their in-use concentrations, which are 5000 and 2000 mg/L, respectively.13 However, concentrations of QACs and biguanide derivatives used in contact lens solutions are considerably lower than those recommended for general antiseptic use. It was therefore important to determine if the Acinetobacter strains could be effectively killed by routine care procedures with contact lens solutions. Testing of the two chosen MPSs revealed at least a 3-log reduction in viable cells was observed for all strains. This is reassuring, showing that if disinfection is correctly performed, Acinetobacter should be eliminated and would be unlikely to cause infection. However, as have been reported in numerous studies, many patients failed to comply with care procedures.13,19Y22 Such practices as reuse of MPS, topping up of lens cases without pouring away old solution, and use of expired solution may lead to a less than adequate concentration of disinfectant in the lens cases. Reduced concentrations may select for strains with antiseptic-resistant genes. This applies not only to Acinetobacter but also to other organisms including Staphylococcus aureus and Pseudomonas aeruginosa, which are also known to carry antiseptic-resistant genes.15,16,27 Repeated exposures of these strains to a low level of disinfectant can increase their nonsusceptibility to disinfectants. This may lead to development of strains that are resistant to the standard disinfecting concentration. In addition, as it has been shown that there is coselection of antibiotic resistance together with antiseptic resistance,14,35 poor compliance may also lead to the presence of antibiotic-resistant organisms on lenses and accessories. There have been increasing reports of ocular infections with antibiotic-resistant organisms.36,37 Thus, it is important for practitioners to educate their patients in the correct care procedures to minimize the risk of ocular infections.
CONCLUSIONS This study has shown that Acinetobacter is a likely contaminant of contact lens and storage cases. Despite carriage of antiseptic resistance gene qacE$1, it remains susceptible to two popular MPSs when used at the correct concentrations. However, because contact lens wear is associated with an increased risk for microbial keratitis, which can include pathogens with multiple drug resistance, it is important to consider compliance with care routine especially in immunocompromised patients
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276 Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al.
ACKNOWLEDGMENTS The authors thank Ms. Zhang Ming for technical assistance and Dr. Dominic Tsang (Consultant Pathologist, Queen Elizabeth Hospital) for the clinical isolates. Received August 14, 2013; accepted November 27, 2013.
REFERENCES 1. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538Y82. 2. Marcovich A, Levartovsky S. Acinetobacter exposure keratitis. Br J Ophthalmol 1994;78:489Y90. 3. Hsueh PR, Teng LJ, Chen CY, Chen WH, Yu CJ, Ho SW, Luh KT. Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerg Infect Dis 2002; 8:827Y32. 4. Choi CH, Lee JS, Lee YC, Park TI, Lee JC. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiol 2008;8:216. 5. Gaddy JA, Actis LA. Regulation of Acinetobacter baumannii biofilm formation. Future Microbiol 2009;4:273Y8. 6. Wroblewska MM, Sawicka-Grzelak A, Marchel H, Luczak M, Sivan A. Biofilm production by clinical strains of Acinetobacter baumannii isolated from patients hospitalized in two tertiary care hospitals. FEMS Immunol Med Microbiol 2008;53:140Y4. 7. Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PG, Geerards AJ, Kijlstra A. Incidence of contact lensYassociated microbial keratitis and its related morbidity. Lancet 1999;354:181Y5. 8. Goldstein MH, Kowalski RP, Gordon YJ. Emerging fluoroquinolone resistance in bacterial keratitis: a 5-year review. Ophthalmology 1999;106:1313Y8. 9. Alexandrakis G, Alfonso EC, Miller D. Shifting trends in bacterial keratitis in South Florida and emerging resistance to fluoroquinolones. Ophthalmology 2000;107:1497Y502. 10. Fong CF, Tseng CH, Hu FR, Wang IJ, Chen WL, Hou YC. Clinical characteristics of microbial keratitis in a university hospital in Taiwan. Am J Ophthalmol 2004;137:329Y36. 11. Lim L, Loughnan MS, Sullivan LJ. Microbial keratitis associated with extended wear of silicone hydrogel contact lenses. Br J Ophthalmol 2002;86:355Y7. 12. Henwood CJ, Gatward T, Warner M, James D, Stockdale MW, Spence RP, Towner KJ, Livermore DM, Woodford N. Aantibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936). J Antimicrob Chemother 2002;49:479Y87. 13. Kawamura-Sato K, Wachino J, Kondo T, Ito H, Arakawa Y. Correlation between reduced susceptibility to disinfectants and multidrug resistance among clinical isolates of Acinetobacter species. J Antimicrob Chemother 2010;65:1975Y83. 14. Smith K, Gemmell CG, Hunter IS. The association between biocide tolerance and the presence or absence of qac genes among hospitalacquired and community-acquired MRSA isolates. J Antimicrob Chemother 2008;61:78Y84. 15. Kazama H, Hamashima H, Sasatsu M, Arai T. Distribution of the antiseptic-resistance genes qacE and qacE delta 1 in gram-negative bacteria. FEMS Microbiol Lett 1998;159:173Y8. 16. Ku¨cken D, Feucht H, Kaulfers P. Association of qacE and qacEDelta1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria. FEMS Microbiol Lett 2000;183:95Y8.
17. Wang C, Zhan Q, Mi Z, Huang Z, Chen G. Distribution of the antiseptic-resistance gene qacEDelta1 in 283 clinical isolates of gramnegative bacteria in China. J Hosp Infect 2008;69:394Y6. 18. Sasatsu M, Shibata Y, Noguchi N, Kono M. Substrates and inhibitors of antiseptic resistance in Staphylococcus aureus. Biol Pharm Bull 1994;17:163Y5. 19. Willcox MD, Harmis N, Cowell GM, Williams T, Holden DN. Bacterial interactions with contact lenses: effects of lens material, lens wear and microbial physiology. Biomaterials 2001;22:3235Y47. 20. Corrigan KM, Harmis NY, Willcox MD. Association of Acinetobacter species with contact lens-induced adverse responses. Cornea 2001; 20:463Y6. 21. Boost MV, Cho P. Microbial flora of tears of orthokeratology patients, and microbial contamination of contact lenses and contact lens accessories. Optom Vis Sci 2005;82:451Y8. 22. Yung MS, Boost M, Cho P, Yap M. Microbial contamination of contact lenses and lens care accessories of soft contact lens wearers (university students) in Hong Kong. Ophthalmic Physiol Opt 2007; 27:11Y21. 23. Schreckenberger PC, Daneshvar MI, Hollis DG. Acinetobacter, Achromobacter, Chryseobacterium, Moraxella, and other nonfermentative gram-negative rods. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of Clinical Microbiology, 9th ed. Washington, DC: ASM Press; 2007:770Y802. 24. Charm J, Cheung SW, Cho P. Practitioners’ analysis of contact lens practice in Hong Kong. Cont Lens Anterior Eye 2010;33:104Y11. 25. Morgan PB, Woods CA, Knajian R, Jones D, Efron N, Tan KO, Pesinova A, Grei, HJ, Marx J, Santodomingo S, Runberg SE, Tranoudis IG, Chandrinos A, Itoi M, Bendoriene J, van der Worp E, Helland M, Phillips G, Gonza´lez-Me´ijome JM, Belousov V, Mack, CJ. International contact lens prescribing in 2007. Contact Lens Spectrum 2008;23(1):36Y41. 26. Wang JT, Sheng WH, Wang JL, Chen D, Chen ML, Chen YC, Chang SC. Longitudinal analysis of chlorhexidine susceptibilities of nosocomial methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Taiwan. J Antimicrob Chemother 2008;62:514Y7. 27. Zhang M, O’Donoghue MM, Ito T, Hiramatsu K, Boost MV. Prevalence of antiseptic-resistance genes in Staphylococcus aureus and coagulase-negative staphylococci colonising nurses and the general population in Hong Kong. J Hosp Infect 2011;78:113Y7. 28. Kaye S, Tuft S, Neal T, Tole D, Leeming J, Figueiredo F, Armstrong M, McDonnell P, Tullo A, Parry C. Bacterial susceptibility to topical antimicrobials and clinical outcome in bacterial keratitis. Invest Ophthalmol Vis Sci 2010;51:362Y8. 29. Zhang J, Zhu ZH, Su GL, Ping J. Detection of qacE$1-sul1 in Acinetobacter baumannii isolated from Xinjiang and its clinical significance. Chin J Nosocomiol 2009-11. 30. Rajamohan G, Srinivasan VB, Gebreyes WA. Novel role of Acinetobacter baumannii RND efflux transporters in mediating decreased susceptibility to biocides. J Antimicrob Chemother 2010; 65:228Y32. 31. Yavari SA, Rota S, Caglar K, Fidan I. Role of Acinetobacter baumannii AdeB, AdeJ and QacE efflux genes in mediating decreased susceptibility to biocides. Ann Biol Res 2013,4:144Y50. 32. Wong WCW, Lee A, Tsang KK, Wong SYS. How did general practitioners protect themselves, their family, and staff during the SARS epidemic in Hong Kong? J Epidemiol Commun H 2004;58:180Y5. 33. Wong SY, Kung K, Wong MC, Wong C, Tsui W, Chan K, Liang J, Lee NL, Cheung AW, Wong EL. Primary care physicians’ response to pandemic influenza in Hong Kong: a mixed quantitative and qualitative study. Int J Infect Dis 2012;16:687Y91.
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Multipurpose Solutions against Acinetobacter Carrying Antiseptic Resistance GenesVBoost et al. 34. Kawamura-Sato K, Wachino J, Kondo T, Ito H, Arakawa Y. Reduction of disinfectant bactericidal activities in clinically isolated Acinetobacter species in the presence of organic material. J Antimicrob Chemother 2008;61:568Y76. 35. Vali L, Davies SE, Lai LL, Dave J, Amyes SG. Frequency of biocide resistance genes, antibiotic resistance and the effect of chlorhexidine exposure on clinical methicillin-resistant Staphylococcus aureus isolates. J Antimicrob Chemother 2008;61:524Y32. 36. Pachigolla G, Blomquist P, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities: a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens 2007; 33:45Y9.
277
37. McDonald M, Blondeau JM. Emerging antibiotic resistance in ocular infections and the role of fluoroquinolones. J Cataract Refract Surg 2010;36:1588Y98.
Maureen Boost Department of Health Technology and Informatics The Hong Kong Polytechnic University Kowloon, Hong Kong SAR China e-mail: [email protected]
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