Background/aims Achromobacter xylosoxidans and Stenotrophomonas maltophilia are emerging corneal pathogens, which are closely related to Pseudomonas aeruginosa, and have intrinsic resistance to many commonly available antimicrobials. The purpose of this study is to compare the in vitro efficacy of 12 antimicrobial agents against A. xylosoxidans, S. maltophilia and P. aeruginosa isolates recovered from clinical cases of keratitis.
Methods Recovered corneal isolates (n=58) were identified and extracted from the Microbiology Data Bank of the Bascom Palmer Eye Institute. Comparative in vitro minimum inhibitory concentration (MIC) susceptibility profiles for fluoroquinolones, aminoglycosides, beta-lactams and miscellaneous antibiotics were recorded using the E-test methodology. Pharmacodynamic indices (Cmax/MIC) were calculated.
Results A. xylosoxidans and S. maltophilia isolates were resistant to fluoroquinolones, aminoglycosides and ceftazidime (susceptibility rate ranging from 0% to 30%) while P. aeruginosa isolates showed a susceptibility rate of 95%–100% to these antimicrobials (P<0.00001 for the various antimicrobials). Exception was moxifloxacin with 80% of susceptibility rate to S. maltophilia isolates and Cmax/MIC=10.19. Ninety to 100% susceptibility rates were found for minocycline and trimethoprim/sulfamethoxazole for both A. xylosoxidans and S. maltophilia. One hundred per cent of the A. xylosoxidans isolates were susceptible to piperacillin/tazobactam and ticarcillin/clavulanic acid.
Conclusions There is a significant difference in susceptibility patterns between A. xylosoxidans, S. maltophilia and P. aeruginosa. Fluoroquinolones and aminoglycosides may not be effective against A. xylosoxidans and S. maltophilia. Antibiotics that are not commercially available as eye drops, such as beta-lactams for A. xylosoxidans, and trimethoprim/sulfamethoxazole and minocycline for both A. xylosoxidans and S. maltophilia should be considered.
- contact lens
- experimental laboratory
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Achromobacter xylosoxidans and Stenotrophomonas maltophilia are Gram-negative, aerobic, non-glucose-fermenting bacilli that can be confused with Pseudomonas species.1 They differ from Pseudomonas aeruginosa in being less virulent and having different antibiotic resistance profiles.1 P. aeruginosa is the most commonly isolated Gram-negative microorganism causing bacterial keratitis. In large surveys from around the world, both A. xylosoxidans and S. maltophilia are included as part of a group defined as ‘other gram negative bacteria’ which are responsible to less than 5% of the total bacterial species isolated from patients with bacterial keratitis.2 It was thought that the occurrence of these pathogens may be underestimated because of the difficulty of positively identifying the organism and differentiating them from P. aeruginosa.3 In the ophthalmic literature, there are few case reports and small case series reporting of ocular infections caused by these microorganisms and the effectiveness of topical antimicrobials. Nevertheless, reports on ocular infections are increasing, partially due to improved laboratory isolation techniques.4 In a recent report, cases and lenses of patients with keratitis were tested using culture-independent methods. A. xylosoxidans and S. maltophilia were the predominant bacteria identified, drawing attention to their emerging role in keratitis.1
A. xylosoxidans and S. maltophilia infectious keratitis are hard to manage as these pathogens are relatively resistant to common topical antibiotics.5 This can lead to extensive corneal scar, endophthalmitis and the need for therapeutic corneal transplantation, despite broad topical antibiotic treatment.6 7 Generally, topical fluoroquinolones and aminoglycosides are the mainstay for the treatment of Gram-negative corneal ulcers; however, for A. xylosoxidans and S. maltophilia keratitis, there are no solid data, in vivo or in vitro, regarding optimal topical antibiotic treatment. There are also no studies comparing between antimicrobial agents in the treatment of A. xylosoxidans, S. maltophilia and P. aeruginosa infectious keratitis. With the emergence of antibiotic-resistant organisms, it is essential for clinicians to know which antibiotics are effective against ocular pathogens and there is a continual need for the search for new ones, which are not yet available for topical treatment. The purpose of this study is to investigate the in vitro efficacy of 12 antimicrobial agents in the treatment of infectious keratitis caused by A. xylosoxidans and S. maltophilia and to compare it with P. aeruginosa.
Materials and methods
The Microbiology Laboratory Data Base was searched for clinical cases of keratitis caused by A. xylosoxidans and S. maltophilia during the years 2000–2013. Organism identification was performed by skilled technicians in the Bascom Palmer Eye Institute Clinical Microbiology Laboratory. Twenty corneal isolates of A. xylosoxidans, 15 isolates of S. maltophilia and 23 isolates of P. aeruginosa which were used as a control group were retrieved and rehydrated according to standard microbiologic protocols (n=58).
A standard inoculum was prepared by inoculating three to four colonies from each plate into liquid nutrient broth and adjusted using a nephrometer (0.5 McFarland standard). The inoculum was plated on two Mueller Hinton II agar plates. E-strips (bioMérieux, Durham, NC) of six antimicrobial agents were then applied to each agar plate (a total of 12 antimicrobial agents). The E-test strips are plastic strips containing a continuous concentration gradient of different antimicrobial agents, which when applied to inoculated agar plates create ellipses of microbial inhibition. Agar plates were then incubated at 35°C for 16–20 hours. After incubation, the minimum inhibitory concentration (MIC) (mcg/mL) was read, according to the manufacturer’s instructions for each antibiotic. The MIC value was determined at the point where the ellipse of inhibition intersects the side of the E-strip (figure 1). The MIC50 and MIC90 for each antibiotic were calculated as the minimum concentration that inhibited growth of 50% and 90% of the isolates for each bacterium.
The antimicrobial activity of 12 antibiotics was tested, including fluoroquinolones (ciprofloxacin, moxifloxacin), aminoglycosides (amikacin, tobramycin), beta-lactams (ceftazidime, imipenem, piperacillin/tazobactam, ticarcillin/clavulanic acid) and miscellaneous antibiotics (polymyxin B, trimethoprim/sulfamethoxazole, chloramphenicol, minocycline). Efficacy for each antibiotic was defined by the MIC90. Breakpoints for topical antimicrobial drops for treating bacterial keratitis are not available, thus an isolate was characterised as susceptible if its MIC was lower than the systemic breakpoint, as defined by the Clinical and Laboratory Standards Institute (Wayne, PA).
Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 (American Type Culture Collection, Manassas, VA) were used as quality control strains. Each of the laboratory procedures was done at the same time for the 58 corneal isolates, assuring identical environment for all isolates.
The pharmacodynamic indices were calculated for ciprofloxacin, moxifloxacin, tobramycin, trimethoprim/sulfamethoxazole and chloramphenicol using the published maximum attainable cornea or aqueous concentration values8–16 and the obtainable MICs of a specific antimicrobial. There are no reports of the ocular peak concentrations for the other antimicrobials in the study. Pharmacodynamics index is the ratio of the achievable concentration of an antibiotic and the concentration of the antibiotic required to inhibit 50% (Cmax/MIC50) or 90% (Cmax/MIC90) of isolates of a certain organism. The Cmax is defined as the maximum attainable cornea or aqueous humour concentration using topical therapy. Optimal performance and effectiveness of an antibiotic is best achieved when Cmax/MIC90 is 8–10.17
Table 1 summarises the MIC levels and rate of susceptibilities of A. xylosoxidans, S. maltophilia and P. aeruginosa for the 12 antimicrobial agents.
One hundred per cent of A. xylosoxidans isolates were susceptible for trimethoprim/sulfamethoxazole and for the newer beta-lactam agents piperacillin/tazobactam and ticarcillin/clavulanic acid. A. xylosoxidans also showed a high susceptible rate for minocycline and imipenem (90% and 80%, respectively, P>0.05 for both when susceptibility was compared with trimethoprim/sulfamethoxazole, piperacillin/tazobactam and ticarcillin/clavulanic acid). A. xylosoxidans was highly resistant to the fluoroquinolones and the aminoglycosides that were tested. The susceptibility rate difference of the fluoroquinolones/aminoglycosides and the antimicrobials with a susceptibility rate of 80% and higher was significant (P≤0.0002 for all drugs).
One hundred per cent of S. maltophilia strains were susceptible for minocycline and trimethoprim/sulfamethoxazole and 80% were susceptible for moxifloxacin (P=0.25). S. maltophilia strains showed very low susceptibility rate to the other antimicrobials including the aminoglycosides and the beta-lactams (0%–13.3%). This difference was significant when these antimicrobials were compared with minocycline and trimethoprim/sulfamethoxazole (P=0.0005–0.0009) or with moxifloxacin (P=0.003–0.009).
Ninety-five to 100% of P. aeruginosa isolates were susceptible to fluoroquinolones, aminoglycosides and ceftazidime. Polymyxin B showed a lower rate of susceptibility (77.3%); however, this was not significant (P=0.07–0.13). Regarding the new antimicrobials, all of the isolates were susceptible to piperacillin/tazobactam but not for imipenem and ticarcillin/clavulanic acid. When imipenem and ticarcillin/clavulanic acid susceptibility rates were compared with the antimicrobials with the 95%–100% susceptibility rates there was a significant difference (P≤0.0009). All of the P. aeruginosa strains (100%) were resistant to minocycline, trimethoprim/sulfamethoxazole and chloramphenicol (P<0.0001, compared with fluoroquinolones and aminoglycosides).
Antimicrobial susceptibility comparisons: A. xylosoxidans and S. maltophilia versus P. aeruginosa
When the susceptibility rates of A. xylosoxidans, S. maltophilia and P. aeruginosa to each antimicrobial were compared, a significant difference was found for 11 antimicrobials (P≤0.0006 for all, table 1). Chloramphenicol that has shown a very low susceptibility rate for all three bacteria was the only antimicrobial that had no significant difference in susceptibility level for the three pathogens (P=0.32) (figure 2).
Pharmacodynamic indices for MIC50 and MIC90 values
Pharmacodynamic indices are listed in table 2. As for A. xylosoxidans, the Cmax/MIC90 was less than 8–10 for all antimicrobials. For S. maltophilia, Cmax/MIC90 reached a higher value than 8–10 for moxifloxacin only. For P. aeruginosa the pharmacodynamic index showed a higher value than 8–10 only for ciprofloxacin. For moxifloxacin, the Cmax/MIC90 was 7.65, lower than the recommended minimal value of 8.
In this study, we report a significant difference in the in vitro susceptibilities between A. xylosoxidans, S. maltophilia and P. aeruginosa scraped and recovered from infectious keratitis. While the common Gram-negative antimicrobials ciprofloxacin, tobramycin, amikacin and ceftazidime were highly effective against P. aeruginosa with more than 95% susceptibility rate, they were ineffective against A. xylosoxidans and S. maltophilia. Sensitivity rates of A. xylosoxidans and S. maltophilia for these antimicrobials ranged from 0% to 30% only. Nevertheless, moxifloxacin showed moderate effectiveness against S. maltophilia with 80% of susceptible strains. The differences in antibiotic sensitivity patterns between A. xylosoxidans, S. maltophilia and P. aeruginosa found in our study make an accurate differentiation between these organisms important. To our knowledge, this is the first laboratory study executed to compare susceptibility and resistance profiles between A. xylosoxidans, S. maltophilia and P. aeruginosa keratitis for various antimicrobial agents.
A. xylosoxidans is an aerobic Gram-negative bacillus that in clinical specimens can be confused with other non-fermentative, Gram-negative bacilli, especially Pseudomonas spp.3 The sparse clinical and laboratory retrospective studies of A. xylosoxidans keratitis show that the organism may be susceptible to ceftazidime18 and polymyxin B.19 This is in contradiction with the low susceptibility rates (30%–55%) we found for these antimicrobials. The previously reported resistance to aminoglycosides and chloramphenicol1 is in agreement with our results. It is not clear whether the newer or the older fluoroquinolone generations are more effective, with contradictory results.6 In the current study, A. xylosoxidans was resistant for both the second (ciprofloxacin) and fourth (moxifloxacin) generation fluoroquinolones. A. xylosoxidans isolates were susceptible to the beta-lactams imipenem, piperacillin/tazobactam and ticarcillin/clavulanic acid. In a previous report of the clinical features, antimicrobial sensitivities and visual outcomes of infectious keratitis secondary to A. xylosoxidans during a 27-year period, we found A. xylosoxidans susceptibility rates to ciprofloxacin and ceftazidime to be 46.7% and 70%, respectively.20 That study included data also from the ‘80s and ‘90s. In the current study, the susceptibility rates decreased to 0% and 30%, respectively. Although methods in both studies were different, as the former retrospective study was based on reports of the ocular microbiology database and the current laboratory study was done on recovered corneal isolates, these differences may reflect increasing resistance of A. xylosoxidans.
The clinical and laboratory data regarding resistance profiles of S. maltophilia corneal infections are limited. S. maltophilia was reported to be resistant to aminoglycosides,4 7 piperacillin,4 imipenem4 7 and some of the cephalosporins.4 It is usually resistant to fluoroquinolones, although some cases responded to treatment with ciprofloxacin or moxifloxacin.4 7 Both trimethoprim-sulfamethoxazole and ticarcillin/clavulanic were reported as partially effective in treating S. maltophilia keratitis, with 20% resistant strains.4 7 We found that S. maltophilia is resistant to aminoglycosides, ciprofloxacin and polymyxin B. Interestingly, the fourth-generation fluoroquinolone, moxifloxacin, showed 80% of susceptibility rate and a high inhibitory quotient (Cmax/MIC90). According to our results, only trimethoprim-sulfamethoxazole and minocycline have full activity against S. maltophilia (all isolates were susceptible). Yet, the actual clinical efficacy of trimethoprim-sulfamethoxazole against S. maltophilia as well as against A. xylosoxidans may be limited as its inhibitory quotient was low (0.70 and 0.92, respectively, table 2). In a retrospective review of the medical records of patients treated for S. maltophilia keratitis in our institute during a 30-year period, we found that this pathogen showed as high as 92% susceptibility rate to fluoroquinolones, represented by ciprofloxacin.21 However, in the current report susceptibility rate to ciprofloxacin was as low as 13.3%. Although methodology in both studies was different (chart review vs in vitro comparative study in the current report), this difference probably reflects increasing resistance of S. maltophilia to the first generations of fluoroquinolones. Our results are very different from those reported in Australia by Watanabe et al 22 who tested S. maltophilia isolates from contact lenses and contact lens cases. Most of their cases were not keratitis associated. Using a disc diffusion assay, they found that S. maltophilia strains were susceptible to fluoroquinolones, aminoglycosides, ceftazidime and polymyxin B. Geographical variance and methodology (disc diffusion vs the E-test we used) may partially explain these differences. In addition, the isolates we tested were retrieved from patients with an active infection in the cornea so the S. maltophilia strains are truly virulent. These isolates may have a different susceptibility pattern (higher resistance) than isolates from non-keratitis-associated contact lenses and contact lens cases, who may not be true virulent and thus be more susceptible to more antimicrobials. If this is true, it suggests that our results are more clinically relevant.
In light of increasing resistance to Gram-negative bacterial pathogens,2 there is a need to look for antimicrobials that have the potential to treat bacterial keratitis but are not yet available topically for corneal infections. This was the rationale for investigating the beta-lactams: imipenem, piperacillin/tazobactam and ticarcillin/clavulanic acid. Current reports show increasing resistance to P. aeruginosa keratitis.23 24 The Steroids for Corneal Ulcers Trial reported a sharp increase in the proportion of P. aeruginosa corneal isolates that were resistant to the fourth-generation fluoroquinolone, moxifloxacin. The resistance rate increased from 19% to 52% in 3 years.23 Regarding aminoglycosides, they were found to be less effective than ciprofloxacin in eradicating invasive P. aeruginosa strains.24 In ciprofloxacin-resistant P. aeruginosa, susceptibility rate for tobramycin was only 67%.25 In contrast, a very recent report from the USA found less than 4% resistance of P. aeruginosa to both older and newer fluoroquinolones, including ciprofloxacin and moxifloxacin.26 Other reports documented 100% of susceptibility to tobramycin for various common corneal pathogens including P. aeruginosa.26 27 Although exploring antimicrobial susceptibility patterns for P. aeruginosa was not the main purpose of our study, we can state that P. aeruginosa isolated in South Florida is still sensitive to the standard Gram-negative bacilli antimicrobials, fluoroquinolones and aminoglycosides. Nevertheless, inhibitory quotient above 8–10 was achieved for ciprofloxacin but not for moxifloxacin and tobramycin (table 2), suggesting some resistance to topical treatment with these two agents. Ciprofloxacin also demonstrated the lowest MIC90 for P. aeruginosa. It was previously suggested that ciprofloxacin is more effective than other antimicrobials for the treatment of P. aeruginosa keratitis.28 Prokosch et al published that Gram-negative bacilli exhibited 100% susceptibility rate for chloramphenicol, a widespread economical antibiotic used outside the USA.27 As opposed to that, only 3 of 58 isolates (5.2% rate) in the current study were sensitive to this drug.
The limitations of our study stem from its in vitro nature, thus any conclusions about antimicrobial treatment of Gram-negative bacterial keratitis have inherent limitations. The relatively small sample size is another limitation although A. xylosoxidans and S. maltophilia are rare corneal pathogens.
The poor prognosis associated with A. xylosoxidans 6 and S. maltophilia 7 may be partially contributed to the lack of established susceptible patterns. In this study, we provide information to clinicians to guide their empirical therapy for A. xylosoxidans and S. maltophilia keratitis, which will help in preventing the devastating ocular complications of these pathogens. Fluoroquinolones and tobramycin are the recommended antimicrobials for Gram-negative bacilli keratitis or when the organism is not known (Bacterial Keratitis PPP, 2013, American Academy of Ophthalmology). Our results suggest that this regimen may not be effective against A. xylosoxidans and S. maltophilia. Antibiotics that are not commercially available as eye drops, such as the beta-lactams imipenem, piperacillin/tazobactam and ticarcillin/clavulanic acid for A. xylosoxidans, and trimethoprim/sulfamethoxazole and minocycline for both A. xylosoxidans and S. maltophilia, should be considered.
Contributors Conception or design of the work: OS, DM, TPOB. Acquisition, analysis or interpretation of data: OS, DM, TPOB. Drafting of the work: OS. Revising the work critically: DM, TPOB. Final approval of the version published: OS, DM, TPOB.
Funding DM and TPOB were supported by NIH Center Core Grant P30EY014801, Research to Prevent Blindness unrestricted grant.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Not commissioned; externally peer reviewed.