Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-014-2101-3

ARTICLE

Activity of colistin in combination with tigecycline or rifampicin against multidrug-resistant Stenotrophomonas maltophilia J. W. Betts & L. M. Phee & N. Woodford & D. W. Wareham

Received: 23 February 2014 / Accepted: 25 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The antimicrobial treatment of Stenotrophomonas maltophilia infections is complicated by intrinsic multidrug resistance and a lack of reliable susceptibility data. We assessed the activity of colistin (COL), rifampicin (RIF) and tigecycline (TGC) alone and in combination using a range of in vitro susceptibility testing methodologies and a simple invertebrate model of S. maltophilia infection (Galleria mellonella). Synergy [fractional inhibitory concentration indices (FICIs) ≤0.5] between COL and either RIF or TGC was observed against 92 % and 88 % of 25 S. maltophilia isolates, respectively, despite resistance to one or another of the single agents alone. In time–kill assays, COL combined with either RIF or TGC was superior to single agents, but only the COL/ RIF regimen was reliably bactericidal. The in vitro findings correlated with treatment outcomes in G. mellonella, with heightened survival observed for larvae treated with COL/ RIF or COL/TGC compared with COL, RIF or TGC alone. COL combined with RIF was the most effective combination overall in both in vitro and in vivo (p2 used as the cut-off for species identification. Routine susceptibility testing was conducted using the MicroScan WalkAway System and the Neg MIC panel type 36 (Siemens Healthcare Diagnostics Limited, Camberley, UK). G. mellonella caterpillars were obtained from Livefood UK Limited (Rooks Bridge, Somerset, UK) and stored at 15 °C in wood shavings prior to use.

Disc diffusion assays Disc diffusion assays on Iso-Sensitest agar were used in a phenotypic screen for synergy using COL (25 μg), RIF (5 μg), TGC (15 μg) discs and SXT (23.75/1.25 μg). Synergy was assessed by applying RIF/TGC/SXT discs at increasing distances (5–25 mm) from COL discs. Plates were inoculated with a 0.5 McFarland standard of an overnight culture of S. maltophilia and incubated at 35 °C±1 °C for 18–24 h. Due to a lack of validated zone diameter breakpoints for S. maltophilia disc diffusion testing (other than for SXT), synergy was recorded as the presence of a characteristic keyshaped zone of inhibition (>5 mm), between discs set up adjacently.

Broth microtitre dilution assays Methods Antibiotics, culture media, bacterial isolates and animal subjects Antibiotic powders of colistin sulfate (COL) and rifampicin (RIF) used in this study were purchased from Sigma Aldrich (Dorset, UK), tigecycline (TGC) was provided by Pfizer Inc. (USA; batch no. Pf–05208753–00). Rifampicin was made up in dimethyl sulfoxide (DMSO) and diluted in distilled water so that the final concentration of DMSO was 0.05 %. TGC and COL were both made up in distilled water. Antibiotic discs and dehydrated culture media were purchased from Oxoid (Basingstoke, UK). S. maltophilia clinical isolates (n=17) were obtained from Barts Health NHS Trust (London, UK), originally isolated from a range of sites, including bloodstream, respiratory, and wound and soft tissue infections. Seven environmental isolates (Stm 51, Stm 52, Stm 54 and Stm 57–60) isolated from lake water samples and donated by Ladoke Akintola

The MICs of COL, RIF and TGC, alone and in combination, were determined in Iso-Sensitest broth in 96-well microtitre plates (Corning, Amsterdam, Netherlands). In synergy studies, plates were set up in a checkerboard-style with decreasing concentrations of COL (16–0 μg/mL) in vertical and TGC (32–0 μg/mL) or RIF (64–0 μg/mL) in horizontal wells. Plates were incubated at 35 °C±1 °C for 20 h and the MIC was determined as the lowest concentration where turbidity could not be observed. Cell viability was confirmed by the addition of 20 μL of PrestoBlue® (Invitrogen, Paisley, UK), a resazurin dye, to each well across the turbidity/non-turbidity interface and any colour change to pink observed. Fractional inhibitory concentration indices (FICIs) were calculated [21], using the following equation: FICI =FIC of A (MIC of antibiotic A in combination with antibiotic B/MIC of antibiotic A alone) + FIC of B (MIC of antibiotic B in combination with antibiotic A/MIC of antibiotic B alone). Synergy, an indifferent effect or antagonism were defined as FICI values ≤0.5, >0.5–4.0 or >4.0, respectively.

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Time–kill assays

G. mellonella–S. maltophilia treatment assay

Time–kill assays were performed on the NCTC 10258 type strain and on isolates SmCF 01 (cystic fibrosis), Stm 59 and Stm 60 (environmental), which were selected to represent isolates with elevated MICs to TGC, RIF and COL, but exhibiting potent synergy in checkerboard assays. A 1/1,000 dilution of an overnight culture, equating to approximately 106 CFU/mL, was used as the starting inoculum. Antibiotics were added at final concentrations of 2 mg/L (COL) [22], 1 mg/L (TGC) [23] and 8 mg/L (RIF) [24] in an attempt to mimic peak serum levels obtained with optimised dosing regimens in vivo. Falcon tubes inoculated with S. maltophilia supplemented with RIF, TGC, COL, RIF+COL or TGC+COL were incubated and continuously agitated at 35 °C. At 0, 2, 4, 6 and 24 h, 100-μL aliquots were taken from each tube and serial dilutions plated onto Iso-Sensitest agar for viable counts. Colonies on all plates were counted after incubation at 35 °C for 20 h. Synergy was defined as bactericidal activity (≥2 log10 difference in CFU/ml) of the combination compared with the single agent after 24 h of incubation.

The effects of treatment with COL, TGC, RIF, COL/TGC and COL/RIF were assessed against G. mellonella caterpillars (n= 16) inoculated with 105 CFU/larva of S. maltophilia Stm 59 and Stm 60. Antibiotics were administered in 10-μL injections into a right proleg within 20 min of infection. Antibiotic doses were chosen to mimic those used to treat human infections and consisted of COL at 2.5 mg/kg, TGC at 1 mg/kg and RIF at 10 mg/kg. Combinations of 2.5 mg/kg of COL with either 1 mg/kg of TGC or 10 mg/kg of RIF were also used. Caterpillars were selected by weight, with only those within the range of 250 mg±25 mg being used. Controls, consisting of 16 caterpillars injected with 10 μL of sterile PBS and 16 caterpillars injected with 1.25 % DMSO in place of antibiotics, were also used. Trauma to the insects related to multiple injections was accounted for by inoculating uninfected caterpillars twice with 10 μL of PBS via right and left prolegs. G. mellonella deaths were scored over 96 h of incubation at 37 °C in filter paper-lined petri dishes. All experiments were performed three times on separate occasions. Statistical analysis

S. maltophilia virulence factors The presence of S. maltophilia genes previously identified as contributing to virulence in G. mellonella was investigated in all 25 isolates. DNA was extracted from overnight cultures using an Ultrapure Microbial DNA kit (Cambio, Cambridge, UK) and used in polymerase chain reaction (PCR) assays targeting the smf-1 (type 1 fimbriae), stmPr1, stmPr2 (extracellular proteases) and Smlt3773 (esterase) sequences [25]. All PCR reactions were performed using ThermoPrime Plus ReddyMix (Thermo Fisher), with amplicons visualised after electrophoresis through 1.5 % agarose gels. G. mellonella–S. maltophilia killing assay Overnight cultures of S. maltophilia grown in LuriaBertani (LB) broth were washed three times in sterile phosphate-buffered saline (PBS). To establish the inoculum required to provide staggered killing of G. mellonella over 24 to 96 h, ten caterpillars were inoculated with S. maltophilia at concentrations spanning 104, 105 and 10 6 CFU/larva. Bacterial suspensions (10 μL) were injected directly into the hemocoels of the caterpillars via a proleg using 25-μL Hamilton syringes (ColeParmer, London, UK). Larvae were incubated in filter paper-lined petri dishes at 37 °C for 96 h and scored for survival daily. Insects were considered dead if they failed to respond to touch.

Survival curves were analysed using the log rank test, with a p-value of≤0.05 indicating statistical significance. The percentage survival of all antibiotic-treated caterpillars after 96 h was calculated using data combined across the three replicated experiments and analysed using one-tailed, unpaired t-tests of means [26].

Results Disc diffusion assays Disc diffusion assays suggested that synergy occurred when COL was combined with RIF versus S. maltophilia, as characteristic key-shaped zones of inhibition were observed when discs were placed within 12.5 mm of each other on IsoSensitest agar (Fig. 1a). Synergy between COL and TGC could not be determined in disc diffusion assays, as the zones of inhibition merged and suggested possible antagonism between the compounds at the margins of the zone diameters (Fig. 1b). In disc diffusion assays using COL and SXT, only a weak enhancement (1–2 mm) of the zone of inhibition was observed. Broth microdilution studies The MIC of COL was >2 mg/L for 88 % of the isolates, whereas 92 % of isolates had TGC MICs >1 mg/L and RIF MICs >2 mg/L (Table 1). These values are above the target

Eur J Clin Microbiol Infect Dis Fig. 1 Disc diffusion assay showing synergy between colistin (COL) and rifampicin (RIF) (a) and slight antagonism between COL and tigecycline (TGC) (b) against Stenotrophomonas maltophilia Stm 60

serum levels and equate to clinical resistance to all three agents according to non-species-specific European Committee on Antibiotic Susceptibility Testing (EUCAST) breakpoints and those set for Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. Synergy between COL and RIF was observed in checkerboard studies for 23 isolates (92 %), with no interaction seen against the remaining two (Table 1). Synergy also occurred between COL and TGC for 22 isolates (88 %), with no interaction against four. No antagonistic effects were seen even at concentrations 2 mg/L) was seen in 92 % of our isolates, similar to rates reported by others [14]. We also found resistance to TGC to be comparable with previous reports [11]. Significant variation has been reported in the susceptibility of S. maltophilia to COL. In a study of cystic fibrosis isolates, only 7 % were found to have MICs >2 mg/L using an Etest method [28], which may, again, reflect differences in the methods used for determining MICs. Microtitre-based assays may result in higher COL MICs due to the ability of polymyxins to bind to many laboratory plastics, even with the addition of dispersants such as Tween 80 to the culture media [29]. The COL/RIF combination was superior to monotherapy in both checkerboard and time–kill assays, with synergy noted against 23/25 isolates. This differs from the results found in a study using Etests, where antagonism (FICIs of 4 or 5) was frequently seen [14]. Time–kill studies suggested that the

Fig. 4 Survival curves for G. mellonella infected with S. maltophilia (a, b) Stm 59 and (c, d) Stm 60 treated with COL, TGC, RIF, TGC/COL and RIF/ COL combinations

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combination was bactericidal against S. maltophilia, with a sustained reduction of CFU/mL over the 24-h period. This confirms data from Giamarellos-Bourboulis et al., who also identified synergy between COL combined with RIF or SXT against S. maltophilia [18]. Synergy between COL and TGC occurred against 22 of the 25 isolates. The potential for COL/TGC combinations against S. maltophilia was highlighted in a recent study [17], but only three strains were studied and these were not evaluated by time–kill assays. Data from the killing assays performed here confirmed that, although synergy may exist, the combination of COL/TGC remains bacteriostatic. This may be an important parameter to consider when choosing a combination regimen for use in the treatment of S. maltophilia infections in the immunocompromised or critically ill patient. The mechanism behind the synergistic effect when COL is combined with RIF or TGC against S. maltophilia is likely to be similar to that described for other organisms [30], whereby COL disrupts outer membrane permeability, allowing the entry of hydrophobic drugs that are otherwise excluded or actively effluxed. Studies in vivo revealed that S. maltophilia strains Stm 60 and Stm 59 were both able to kill G. mellonella, regardless of their complement of known fimbrial, protease and esterase genes. This would indicate that other virulence factors are likely to be involved in G. mellonella, but also that the model can be used to investigate the pathogenesis and therapeutics of S. maltophilia infections. The COL/RIF combination was the most successful in treatment assays, mirroring the synergy predicted in vitro. The COL/TGC combination was also effective against both strains in vivo, but only superior to monotherapy versus strain Stm 59, which exhibited slightly slower killing kinetics than Stm 60 in Galleria. This may reflect the fact that COL/TGC was only bacteriostatic in vitro and highlights the need for future work to focus on the optimisation of combination dosing regimens using pharmacokinetic studies. In summary, marked synergy was demonstrated when COL was combined with either RIF or TGC against S. maltophilia both in vitro and using a simple animal model. Both combinations were superior to each compound used alone, with COL/RIF demonstrating the most potent killing and a bactericidal effect. The use of COL + TGC/RIF regimens could be considered as a treatment for use in difficult-to-treat S. maltophilia infections. Acknowledgements We would like to gratefully acknowledge Pfizer for the supply of tigecycline. Funding No specific funding was available for this study. Competing interests All authors declare no conflict of interest. Ethical approval Not applicable.

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