Medical School, University of East Anglia, Norwich NR4 7TJ, UK

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1 J Antimicrob Chemother 2014; 69: doi: /jac/dkt455 Advance Access publication 19 November 2013 Comparative in vitro activity of sulfametrole/ and and other agents against multiresistant Gram-negative bacteria David M. Livermore 1,2 *, Shazad Mushtaq 1, Marina Warner 1 and Neil Woodford 1 1 Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London NW9 5EQ, UK; 2 Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, UK *Corresponding author. Norwich Medical School, University of East Anglia, Norwich NR3 1RS, UK. Tel: ; d.livermore@uea.ac.uk Received 4 July 2013; returned 20 August 2013; revised 4 October 2013; accepted 18 October 2013 Objectives: Sulfamethoxazole/ is standard therapy for infections caused by opportunist nonfermenters except Pseudomonas aeruginosa and Acinetobacter. Sulfametrol(e)/ is an alternative to available in some EU countries, with possible pharmacological advantages. We compared their activities against (i) non-fermenters, (ii) multiresistant Enterobacteriaceae and (iii) reference strains with sul1 and sul2. Methods: Test isolates were recent submissions to the reference laboratory, or were Escherichia coli previously shown to have sul1 or sul2. Identification was by MALDI-ToF, by 16S rrna gene sequencing or with API20NE strips. MICs were determined by CLSI agar dilution. The Stenotrophomonas maltophilia and Burkholderia series were enhanced by inclusion of 25% -resistant isolates; other series were not enhanced. Results: MICs of sulfametrole/ for non-fermenters tracked those of, being equal in 97/170 cases, 2-fold higher in 57/170 cases and 2-fold lower in 12/170 cases. Despite supplementing the Burkholderia and S. maltophilia collections with -resistant organisms, the antifolate combinations retained better activity against these and other non-fermenters than did piperacillin/ tazobactam, moxifloxacin, ticarcillin/clavulanate, tigecycline, cefotaxime or imipenem. By contrast, few (5% 20%) of the extended-spectrum b-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae were susceptible to the sulphonamides or their combinations, probably reflecting widespread co-carriage of sul1 and sul2, which both conferred resistance. Conclusions: Antifolate combinations remain the most active antimicrobials against less common non-fermenters, importantly including S. maltophilia and Burkholderia spp., but resistance is prevalent among ESBL- and carbapenemase-producing Enterobacteriaceae. Sulfametrole/ had similar activity to against non-fermenters. Keywords: Stenotrophomonas maltophilia, non-fermenters, cystic fibrosis Introduction Intravenous (also known as co-trimoxazole) is used extensively against Pneumocystis infection. It is also the treatment of choice against non-fermentative opportunists except Pseudomonas aeruginosa, which is inherently resistant, and Acinetobacter spp., where acquired resistance is widespread. Oral is used widely in community urinary tract infections, though alone is preferred in the UK following concern about sulfamethoxazole side effects in the 1980s. 1 Important non-fermenters targeted with include Stenotrophomonas maltophilia, Burkholderia cepacia group species, Chryseobacterium spp. and Elizabethkingia spp. (the last two species were previously both assigned to Flavobacterium), Achromobacter spp. and Alcaligenes spp. These organisms are inherently resistant to many other antibiotic classes, and is often the sole agent to remain active. 2 4 Several species, notably S. maltophilia and Elizabethkingia meningoseptica, have chromosomally mediated carbapenemases. Intravenous is sometimes also used against Enterobacteriaceae infections, particularly for b-lactam-allergic patients, 5 but this use is constrained by resistance, largely mediated by plasmid-mediated dihydopteroate synthase and dihydrofolate reductase enzymes, # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oup.com 1050

2 Activity of sulfametrole/ JAC Table 1. MICs of sulphonamides, sulphonamide/ combinations and comparators for non-fermenters and for carbapenemase- and ESBLproducing Enterobacteriaceae Percentage susceptible Achromobacter sulfametrole c sulfamethoxazole 6 a c d sulfametrole/ e e ticarcillin/clavulanate b 80 f piperacillin/tazobactam f cefotaxime b 0 f imipenem f moxifloxacin g tigecycline h A. baumannii sulfametrole sulfamethoxazole b 7 sulfametrole/ b b 37 ticarcillin/clavulanate 1 29 b 0 piperacillin/tazobactam b 0 cefotaxime b 0 imipenem moxifloxacin b 7 tigecycline Alcaligenes sulfametrole sulfamethoxazole 4 a b 10 sulfametrole/ a ticarcillin/clavulanate piperacillin/tazobactam cefotaxime imipenem moxifloxacin b 80 tigecycline Burkholderia sulfametrole sulfamethoxazole b 70 sulfametrole/ ticarcillin/clavulanate b 8 piperacillin/tazobactam b 68 cefotaxime b 35 Continued 1051

3 Livermore et al. Table 1. Continued Percentage susceptible imipenem b 20 moxifloxacin b 30 tigecycline b 5 Chryseobacterium sulfametrole sulfamethoxazole sulfametrole/ ticarcillin/clavulanate b 5 piperacillin/tazobactam cefotaxime b 5 imipenem moxifloxacin tigecycline Elizabethkingia sulfametrole sulfamethoxazole sulfametrole/ ticarcillin/clavulanate b 0 piperacillin/tazobactam cefotaxime imipenem moxifloxacin tigecycline S. maltophilia sulfametrole b 75 sulfamethoxazole b b 3 sulfametrole/ b b 75 ticarcillin/clavulanate b 33 piperacillin/tazobactam b 8 cefotaxime b 10 imipenem b 0 moxifloxacin tigecycline Carbapenemase-producing Enterobacteriaceae sulfametrole sulfamethoxazole b 5 sulfametrole/ b 5 Continued 1052

4 Activity of sulfametrole/ JAC Table 1. Continued Percentage susceptible b 5 ticarcillin/clavulanate 20 b 0 piperacillin/tazobactam b 0 cefotaxime b 15 imipenem moxifloxacin b 35 tigecycline ESBL-producing Enterobacteriaceae sulfametrole sulfamethoxazole b 20 sulfametrole/ b b 10 ticarcillin/clavulanate b 0 piperacillin/tazobactam cefotaxime b 0 imipenem moxifloxacin b 25 Tigecycline a MIC indicated value. b MIC indicated value. Grey shading indicates isolates counted as resistant, based on CLSI breakpoints, with the following adaptations where no CLSI values exist: c The CLSI breakpoint of 256 mg/l sulfamethoxazole for Enterobacteriaceae was applied to all species for both sulfamethoxazole and for sulfametrole. d The CLSI breakpoint of (8 mg/l) for Enterobacteriaceae was applied generally, there being no breakpoint for non-fermenters. e The CLSI breakpoint of 2 mg/l for Enterobacteriaceaewas applied to all species for both and for sulfametrole/; MICs presented are relative to the component in a 19:1 sulphonamide/ combination. f The CLSI breakpoints for Acinetobacter/ other non-enterobacteriaceae were used for all-non-fermenters for ticarcillin/clavulanate, piperacillin/tazobactam and imipenem. g There are no CLSI breakpoints for moxifloxacin and Gram-negative bacteria, so we used 2 mg/l for non-enterobacteriaceae (as for levofloxacin) and 1 mg/ L for Enterobacteriaceae. h There are no CLSI breakpoints for tigecycline, so analysis was based on the FDA value of 2 mg/l for Enterobacteriaceae, which was applied to all species. encoded by the sul and dfr genes, respectively. These enzymes compensate for inhibition of their chromosomal counterparts, which are targeted by sulphonamides and, respectively. 6,7 Sulfametrole/ is an alternative sulphonamide/ combination available in several EU countries, including Austria and the Netherlands (the spelling sulfametrol is used in both countries). It is suggested to have pharmacological advantages over. 8 Several old papers also assert sulfametrole/ to have greater antibacterialactivity, 9,10 but there is no recent confirmation. The present study therefore compared the activity of sulfametrole against sulfamethoxazole and sulfametrole/ against using current isolates. These principally comprised clinically relevant non-fermenters, but also included multiresistant Enterobacteriaceae and those with the common sulphonamide resistance determinants, sul1 and sul2. Materials and methods Test organisms The non-fermenters and multiresistant Enterobacteriaceae were sent to Public Health England s Antimicrobial Resistance and Healthcare Associated Infections Reference Unit for investigation of resistance between 2008 and The non-fermenters comprised: 40 S. maltophilia, including 10 previously found to be resistant; 40 B. cepacia group isolates, including 10 previously found to be resistant; 20 E. meningoseptica and 20 other chryseobacteria/flavobacteria; 10 Achromobacter spp.; 10 Alcaligenes spp.; and 30 Acinetobacter baumannii. Except for the A. baumannii, most were indicated to be from cystic fibrosis patients. Identification was by MALDI-ToF, by 16S rrna gene sequencing or with API20NE strips, except for A. baumannii, which was identified by the presence of a bla OXA-51-like gene. 11 The multiresistant Enterobacteriaceae comprised 20 isolates with carbapenemases (five each with KPC, NDM, VIM and OXA-48 enzymes, 1053

5 Livermore et al. based on PCR detection of the corresponding genes) and 20 with extendedspectrum b-lactamases (ESBLs), based on synergy between cefotaxime and/or ceftazidime and clavulanic acid. To assess the effects of common antifolate resistance determinants, we used a collection of 45 Escherichia coli isolates, comprising 9 fullysusceptible to antifolates, 9 with sul1 and resistant, 10 with sul1 and susceptible, 8 with sul2 and resistant and 9 with sul2 and susceptible. Those with sul1 and sul2 were from the collection of Enne et al., 1 who described their characterization; the susceptible isolates were from the collection of McNulty et al. 12 Antibiotics and susceptibility tests MICs were determined by CLSI agar dilution using Mueller Hinton agar (Oxoid, Basingstoke, UK) 13 and the following antibiotics: sulfametrole (Rokitan, Vienna, Austria), sulfamethoxazole and (both from Sigma, Poole, UK), ticarcillin (Sigma) with 2 mg/l clavulanate (Glaxo, Brentford, UK), moxifloxacin (Bayer, Newbury, UK), tigecycline (Pfizer, Sandwich, UK), piperacillin (Sigma) with 4 mg/l tazobactam (Pfizer), cefotaxime (Sigma) and imipenem (Merck, Hoddesdon, UK). Sulfametrole/ and were tested as 19:1 combinations, with MICs expressed relative to the component. A challenge was that neither the CLSI nor the EUCAST testing has breakpoints for many agents versus non-fermenters. Accordingly, and pragmatically, we adopted: (i) the CLSI breakpoint of 256 mg/l sulfamethoxazole for Enterobacteriaceae for all species for both sulfamethoxazole and for sulfametrole; (ii) the CLSI breakpoint of 8 mg/l for Enterobacteriaceae for all species; (iii) the CLSI breakpoint of 2 mg/l for Enterobacteriaceae for all species for both and sulfametrole/; (iv) the CLSI breakpoints for Acinetobacter/ other non-enterobacteriaceae for ticarcillin/ clavulanate, piperacillin/tazobactam and imipenem; (v) 2 mg/l moxifloxacin for non-enterobacteriaceae (as for levofloxacin) and 1 mg/l for Enterobacteriaceae; and (vi) the FDA breakpoint of 2 mg/l tigecycline for Enterobacteriaceae, for all species. Control strains comprised E. coli NCTC 10418, ATCC and ATCC along with P. aeruginosa ATCC Results and discussion Non-fermenters The MIC distributions for non-fermenter groups are shown in Table 1, with direct comparison of sulfametrole versus sulfamethoxazole and sulfametrole/ versus in Figure 1. Results for sulfametrole closely resembled those for sulfamethoxazole whilst results for sulfametrole/ resembled those for, with MICs mostly identical or one doubling dilution higher for sulfametrole anditscombination(figure1). Sulphonamides and their combinations were the most broadly active agents against the non-fermenter collections (Table 1), with 70% 100% susceptibility among all groups except A. baumannii, which are unusual among non-fermenters in their propensity to acquire resistance genes more usually associated with Enterobacteriaceae, carried by plasmids or on resistance islands. 14,15 It should be stressed that the Burkholderia and S. maltophilia collections with 60% 75% susceptibility to sulphonamides and their combinations were loaded with 25% -resistant isolates. This loading sought to identify whether sulfametrole/ might overcome resistance a hypothesis refuted by the results. Sulphonamide combinations would be expected to have Count Count MIC ratio sulfametrole:sulfamethoxazole S. maltophilia Elizabethkingia Chryseobacterium Burkholderia Alcaligenes Acinetobacter Achromobacter S. maltophilia 60 Elizabethkingia 50 Chryseobacterium 40 Burkholderia Alcaligenes 10 Acinetobacter 0 Achromobacter MIC ratio sulfametrole/: Figure 1. Distribution of MIC ratios for non-fermenters. A ratio,1 indicates that the advantages lies with sulfametrole or sulfametrole/ and a ratio.1 that the advantage lies with sulfamethoxazole or. A ratio of 1 indicates equal activity for both sulphonamides or combinations. more comprehensive activity against unselected isolates; current Public Health England data suggest an 5% resistance rate to in S. maltophilia ( hpr/archives/2011/hpr2811.pdf). Mechanisms of resistance were not characterized, but it is known that S. maltophilia can acquire and express sul1 and sul2 determinants, but that these do not account forall observed resistance. 16 We can find no record of antifolate resistance mechanisms having been characterized in Burkholderia spp. The only other antibiotics to retain broad overall antinon-fermenter activity were: (i) piperacillin/tazobactam, which, however, had poor activity (8% susceptible versus 70% 75% for sulphonamide combinations) against S. maltophilia, which is the most prevalent of these pathogens; and (ii) moxifloxacin, which nevertheless lacked activity against Burkholderia and Achromobacter. Ticarcillin/clavulanate and imipenem had good activity only against Achromobacter and Alcaligenes spp., and cefotaxime only against Alcaligenes spp., whilst tigecycline had scattered activity, with 30% 70% of Achromobacter, Alcaligenes, Chryseobacterium and S. maltophilia susceptible, but with generalized resistance among Burkholderia and Elizabethkingia spp. Tigecycline was the most active antimicrobial against the Acinetobacter panel, with 47% susceptibility compared with 30% 37% for the sulphonamides and their combinations, and negligible rates for other compounds. 1054

6 Activity of sulfametrole/ JAC Table 2. MICs of sulphonamides, and sulphonamide/ combinations for E. coli with combinations of sul and resistance determinants Sulfametrole E. coli antifolate susceptible E. coli sul1 resistant 9 E. coli sul1 susceptible 10 E. coli sul2 resistant 8 E. coli sul2 susceptible 9 Sulfamethoxazole E. coli antifolate susceptible 1 a E. coli sul1 resistant 9 E. coli sul1 susceptible 10 E. coli sul2 resistant 8 E. coli sul2 susceptible 9 Trimethoprim E. coli antifolate susceptible 1 a E. coli sul1 resistant 9 b E. coli sul1 susceptible 1 a E. coli sul2 resistant 8 b E. coli sul2 susceptible 3 a Sulfametrole/ E. coli antifolate susceptible E. coli sul1 resistant 9 b E. coli sul1 susceptible E. coli sul2 resistant 8 b E. coli sul2 susceptible Sulfamethoxazole/ E. coli antifolate susceptible 6 a 2 1 E. coli sul1 resistant 9 b E. coli sul1 susceptible 1 a E. coli sul2 resistant 8 b E. coli sul2 susceptible 1 a Grey shading indicates resistance using the criteria detailed in the footnotes to Table 1. a MIC indicated value. b MIC indicated value. Enterobacteriaceae Most (80% 95%) isolates of Enterobacteriaceae with ESBLs and carbapenemases were resistant to both of the sulphonamides and their combinations; nevertheless, one Enterobacter cloacae with a KPC carbapenemase and one Klebsiella pneumoniae with OXA-48 enzyme showed some susceptibility The carbapenemase producers were also widely resistant to all the other antibiotics tested except tigecycline; the few that remained susceptible to cefotaxime had OXA-48 enzyme, whereas moxifloxacin susceptibility was scattered among all groups except those with NDM enzymes Most ESBL producers were resistant to all agents except imipenem and tigecycline. Most (95%) of the ESBL producers also appeared susceptible to piperacillin/tazobactam, though this would have fallen to 65% had EUCAST s 8 mg/l susceptibility breakpoint been used rather than the CLSI s 16 mg/l value. These Enterobacteriaceae were not selected on the basis of antifolate resistance and the poor activity of sulphonamides and their combinations almost certainly reflects the fact that ESBL- and carbapenemase-encoding plasmids often also carry sul1 and sul2 along with various dfr genes, which compromise. 17,18 In particular, sul1 is embedded in type 1 integrons, which are widespread among large enterobacterial plasmids. Nevertheless, occasional multiresistant isolates, including some with carbapenemases, remain susceptible to, which has been used effectively in such cases. 5,19 21 The ability of both sul1 and sul2 to confer resistance to sulfametrole as well as sulfamethoxazole was confirmed directly by testing E. coli isolates with and without these genes (Table 2). The results showed that both genes were associated with sulfametrole and sulfamethoxazole MICs of.1024 mg/l compared with 1055

7 Livermore et al mg/l for gene-negative isolates. Organisms that had sul1 or sul2 together with resistance determinants were resistant to both sulfametrole/ and, with MICs generally 128 mg/l. Conclusions We showed that sulphonamide/ combinations remain the most active antibacterials against less common non-fermenters other than A. baumannii, and that, against these organisms, sulfametrole and sulfametrole/ were mostly as active as sulfamethoxazole and, respectively, or were one doubling dilution less active. Sulfametrole and sulfametrole/ did not overcome resistance to sulfamethoxazole and in Enterobacteriaceae, refuting old assertions of better activity. These findings do not, of course, diminish the possibility that sulfametrole/ may have other pharmacological and toxicological advantages. 8 The metabolism of sulfametrole is less complicated than that of sulfamethoxazole and does not yield the oxidative products blamed by some authors for -associated rashes; nevertheless 7/9 patients who had experienced rashes with had recurrences when subsequently administered sulfametrole. 8 It is also suggested that the excipients used with intravenous sulfametrole/ formulations are better tolerated than those used in some intravenous preparations, though direct comparative trials are needed. 8 Acknowledgements We are grateful to Dr V. I. Enne of Queen Mary University London (now at University College, London) for the gift of the sul1 and sul2 strains. Funding This work was financed by Rokitan GmbH, Vienna. Transparency declarations D. M. L. is partly self-employed and consults for numerous pharmaceutical and diagnostic companies, including Achaogen, Adenium, Allecra, Astellas, AstraZeneca, Bayer, Basilea, biomérieux, Cubist, Curetis, Discuvra, GSK, Kalidex, Merck, Meiji Seika, Pfizer, Roche, Tetraphase, VenatoRx and Wockhardt, holds grants from Basilea, Cubist, Meiji Seika, Merck and Wockhardt, has received lecture honoraria or travel reimbursement from AstraZeneca, Bruker, Curetis, GSK, J&J, Merck, Novartis, Pfizer and Tetraphase, and holds shares in Dechra, GSK, Merck and Pfizer, collectively amounting to,10% of portfolio value. All other authors: none to declare. References 1 Enne VI, Livermore DM, Stephens P et al. Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction. Lancet 2001; 357: Gales AC, Jones RN, Andrade SS et al. 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