Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa. through altering the composition of multidrug efflux pumps.

AAC Accepts, published online ahead of print on 30 January 2012 Antimicrob. Agents Chemother. doi:10.1128/aac.05574-11 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps. 3 4 Bettina Schaible 1*, Cormac T. Taylor 1 & Kirsten Schaffer 2 5 6 7 1 Conway Institute, University College Dublin, Belfield & 2 Department of Clinical Microbiology, St. Vincent s University Hospital, Dublin 4, Ireland. 8 9 Running title: Hypoxia induces antibiotic resistance in P. aeruginosa 10 11 Keywords: Hypoxia, antibiotic resistance. 12 13 14 15 16 17 Corresponding author: Bettina Schaible Conway Institute University College Dublin Belfield, Dublin 4 Ireland. 18 E-mail: bettina.schaible@ucd.ie 19 Tel: +353 1-716-6732 20 Fax: +353 1-716-6701 21 1

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Abstract Antibiotic resistance is a significant and developing problem in general medical practice and a common clinical complication in cystic fibrosis patients infected with Pseudomonas aeruginosa (P. aeruginosa). Such infections occur within hypoxic mucous deposits in the cystic fibrosis lung, however, little is known about how the hypoxic microenvironment impacts upon pathogen behavior. Here, we investigated the impact of hypoxia on antibiotic resistance in P. aeruginosa. Minimal inhibitory concentrations (MIC) of a selection of antibiotics were determined for P. aeruginosa grown under either normoxic or hypoxic conditions. Messenger RNA expression for resistance-nodulation-cell division (RND) multidrug efflux pump linker proteins were determined by real-time PCR and multidrug efflux pump activity was inhibited using Phe-Arg β naphtylamid dihydrochloride. MIC values of a subset of clinically important P. aeruginosa antibiotics were increased in bacteria incubated in hypoxia when compared with normoxia. Furthermore, hypoxia altered the stoichiometry of multidrug efflux pump linker protein subtype expression and pharmacologic inhibition of these pumps reversed hypoxia-induced antibiotic resistance. We hypothesize that hypoxia increases multidrug resistance in P. aeruginosa by shifting multidrug efflux pump linker protein expression towards a dominance of MexEF-OprN. Thus, microenvironmental hypoxia may significantly contribute to the development of antibiotic resistance in P. aeruginosa infection in cystic fibrosis patients. 44 2

45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 Introduction Hypoxia and inflammation are coincidental events in a number of chronic infectious diseases (16). The metabolic demands of infiltrating immune cells and multiplying pathogens along with vascular dysfunction associated with chronic inflammation contribute to tissue hypoxia in such conditions (4). However, rather than simply being a bystander feature of the microenvironment, hypoxia has an important effector role in influencing gene expression in host cells and invading pathogens alike and can significantly impact upon the development of both infection and inflammation (17). While the impact of decreased oxygen tension on bacterial virulence has been investigated for intestinal pathogens such as Shigella flexneri (12), little is known about it s effects on antibiotic resistance in Pseudomonas aeruginosa (P. aeruginosa). Cystic fibrosis is the most common severe autosomal recessive disease in Caucasians. Chronic lung disease is the major determinant of long-term survival in cystic fibrosis patients and P. aeruginosa, an opportunistic pathogen, can cause life threatening infections. Approximately 80% of adult cystic fibrosis patients suffer from pulmonary P. aeruginosa infection and this is associated with increased morbidity and mortality (15). In cystic fibrosis, P. aeruginosa grows within thick mucous secretions which accumulate within the airway lumen and low oxygen permeability of these biofilms results in the establishment of a hypoxic microenvironment (20). Treatment of P. aeruginosa infections in cystic fibrosis is complicated by antibiotic resistance and eradication of P. aeruginosa from the cystic fibrosis lung 3

68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 is generally unachievable in persistent infection (15). As development of new antimicrobial agents has diminished over the last decades, antimicrobial resistance is a growing global problem and new strategies to combat panresistant bacteria are required. A key mechanism of antibiotic resistance is via the expulsion of antibiotics through multidrug resistance (MDR) efflux systems belonging to the resistance nodulation division (RND). These pumps play an important role in intrinsic and acquired multidrug resistance (14). Four such pumps, MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM have been well characterized in P. aeruginosa. While increased antibiotic resistance has been previously reported for P. aeruginosa grown in anaerobic conditions or in biofilms (19), in this study we investigated whether hypoxia, independent of the complex environment of the biofilm, influences antibiotic resistance of P. aeruginosa. A more detailed understanding of the mechanisms of P. aeruginosa antibiotic resistance in cystic fibrosis lung disease will identify new therapeutic targets for antimicrobial therapy. 84 4

85 Materials and Methods 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Bacterial strains and growth conditions The P. aeruginosa control strain (ATCC 27853) and clinical strains from chronically infected cystic fibrosis patients (S8263, S8269, S8276, S8279) were cultured in cation adjusted Mueller Hinton II broth (MHB II) (Becton Dickenson, Microbiology Systems, Cockeysvill, MD). The clinical strain S8263 was resistant to all routinely used anti-pseudomonal antibiotics (Ceftazidime, Piperacillin/tazobactam, Meropenem, Aztreonam, Tobramycin, Gentamicin, Amikacin, Ciprofloxacin). The clinical strains S8269 and S8276 were susceptible to all tested antibiotics whereas S8279 showed a mixed antibiotic susceptibility pattern. All clinical isolates were cultured from sputum specimen of cystic fibrosis patients for at least 1 year. Bacteria were incubated at 30ºC (for analysis of RND multidrug efflux pump expression) or 37ºC (for antibiotic resistance) in normoxia (21% oxygen) or hypoxia (1% oxygen) in a hypoxia chamber (INVIVO 2 400 Hypoxia Workstation, Ruskinn Technology Limited, Brigend, UK). 100 101 102 103 104 105 106 Antimicrobial susceptibility testing Antibiotic susceptibility testing was performed by microbroth dilution using Sensititre susceptibility plate GNX 2F (Trek diagnostic systems, East Grinstead, UK). To determine the role of efflux pumps in antimicrobial susceptibility, the pump inhibitor, Phe-Arg β naphtylamid dihydrochloride (20 µg/ml endconcentration) (Sigma-Aldrich, Dorset, UK), was used (8). To inhibit hydroxylases, Dimethyloxalylglycine (DMOG, 1mM; Caymen Chemicals, Ann 5

107 108 Arbor, MI) was used. Dimethylsulfoxide (DMSO, Sigma-Aldrich, Dorset, UK) was used as a vehicle control. 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 Analysis of RND multidrug efflux pump expression levels 6 ml of pre-conditioned MHB II media was inoculated with 3 x 10 8 bacteria and incubated for 6-24 h in either normoxia or hypoxia. Bacteria were harvested by centrifugation for 2.5 min at 15,000 rpm. Total RNA was isolated with the RiboPureTM-Bacteria kit (Ambion, Texas, USA) according to the manufacturer s instructions. Real time quantification of cdna was carried out on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Warrington, UK) using the Syber Green PCR Master Mix (2x). Primers for partial amplification of genes encoding the membrane fusion proteins MexA, MexC, MexE, MexX and rpsl were used as puplished before (7). The PCR reaction was run with 2 min at 50ºC, 10 min at 95ºC followed by 40 cycles with 15s at 95ºC and 1 min at 60ºC. Control samples without cdna template or reverse transcriptase were run in parallel. Differences in the amount of starting material were controlled by normalization to the ribosomal rpsl gene. Data were normalized for each gene and presented as ratio of mexe/mexc and mexe/mexa. 125 126 127 128 129 Statistical analysis MIC values are presented as descriptive statistics and MICs were compared with the Kruskal-Wallis test / Duncan posttest (Prism 5) (10). RND multidrug efflux pump expression levels were compared using an unpaired, 2-tailed Student s t- test. p values < 0.05 were considered statistically significant. 6

130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 Results P. aeruginosa grown in hypoxia displays increased antibiotic resistance. P. aeruginosa formed confluent monolayers on Mueller Hinton plates during overnight incubation in either normoxia or hypoxia. Under hypoxic conditions P. aeruginosa grew as colorless colonies rather than as standard green colonies, indicating impaired pyocyanin production during growth under hypoxic conditions (Figure 1A). We investigated P. aeruginosa susceptibility to a range of antibiotics in hypoxia compared to normoxia using microbroth dilution plates. MIC values for 21 antibiotics were determined following exposure to either normoxia or hypoxia. Among antibiotics tested by the microbroth dilution method, penicillin and cephalosporin antibiotics demonstrated increased MIC values in hypoxia when compared to normoxia (Figure 1B). These antibiotics included Cefotaxime (FOT), Ceftazidime (TAZ), Cefepime (FEP) Aztreonam (AZT), Ticarcillin / clavulanic acid (TIM) and Piperacillin / tazobactam (P/T) (Figure 1B). Other antibiotics tested including aminoglycosides, carbapenems, polymyxins, quinolones and tetracyclines showed similar MIC values in both normoxic and hypoxic cultures (Figure 1B). 147 148 149 150 151 152 Hypoxia differentially alters multidrug efflux pump linker protein expression in P. aeruginosa. We have previously observed that hypoxia affects MDR1 gene expression and chemotherapeutic drug resistance in mammalian cells (5). An antimicrobial resistance mechanism exploited by P. aeruginosa and affecting various antibiotic classes is achieved through the expression of bacterial multidrug efflux pumps. 7

153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 Therefore we investigated whether hypoxia-induced multidrug efflux pump expression may contribute to the changes in antibiotic susceptibility in P. aeruginosa. For the analysis of pump expression we used quantitative real-time PCR. Correlation between mrna expression and efflux protein expression has been previously demonstrated (21). Expression levels of four MDR efflux pumps were analyzed following 6-24 hours exposure to normoxia or hypoxia. Interestingly, the stoichiometry of pump linker protein expression changed to dominance of MexE expression over MexA and MexC expression following either 6 or 24 hours hypoxia (Figure 2A and 2B). This was primarily due to decreased MexA and MexC expression with MexE demonstrating sustained expression. Expression of the MexX linker protein gene could not be detected under these conditions (data not shown). To evaluate whether clinical P. aeruginosa strains isolated from cystic fibrosis patients demonstrated a similar change in pump isoform stoichiometry, we evaluated MexA, MexC and MexE pump expression in response to hypoxia in four P. aeruginosa strains isolated from cystic fibrosis patients with different antibiotic susceptibility profiles. All cystic fibrosis patients were chronically infected with P. aeruginosa for more than 1 year. Three of the four cystic fibrosis clinical isolates investigated in this study revealed similar changes in pump stoichiometry in response to hypoxia (Figure 2C). 173 174 Increased MIC values in hypoxia are normalized by the multidrug efflux pump inhibitor Phe-Arg β naphtylamid dihydrochloride. 8

175 176 177 178 179 180 181 182 To investigate the existence of a functional link between antibiotic resistance and changed efflux pump activity in hypoxia, we used Phe-Arg β naphtylamid dihydrochloride, an inhibitor of RND efflux pumps (8). Phe-Arg β naphtylamid dihydrochloride reversed hypoxia-induced antibiotic resistance in P. aeruginosa in a selection of β -lactam antibiotics (Figure 3). This led us to hypothesize, that changes in efflux pump activity contributed to increased antibiotic resistance in hypoxia. MIC values in normoxia remained unchanged in the presence of the pump inhibitor (data not shown). 183 184 185 186 187 188 189 190 191 192 193 194 195 196 Antibiotic susceptibility changes in hypoxia are not mediated by inactivation of hydroxylase activity. In mammalian cells a key signaling event in response to hypoxia is the inhibition of oxygen-dependent hydroxylases leading to activation of the hypoxia inducible factor (HIF) and downstream activation of HIF responsive genes (9). Inhibition of such hydroxylases can be achieved by exposure of cells to DMOG, a nonspecific hydroxylase inhibitor (6). While multiple hydroxylases have been described in several microbes including P. aeruginosa (18) their role in oxygen sensing in the HIF pathway is thought to be restricted to metazoans (11). We investigated if hypoxia-induced changes in MIC values can be mimicked by exposure of bacteria to DMOG. There was no difference between antibiotic susceptibilities between untreated cells and cells treated with DMOG, indicating that 2-oxoglutarate-dependent hydroxylase inhibition did not participate in hypoxia mediated changes in antibiotic susceptibilities (Figure 4). These data are 9

197 198 consistent with the report of HIF hydroxylases not being primary oxygen sensors in bacteria (11). 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 Discussion Hypoxia has been shown to be an important modulator of bacterial virulence of the intestinal pathogen Shigella Flexneri (12). In chronic infection of the CF lung, P. aeruginosa produces alginate and grows in dense bacterial populations forming mucoid biofilms (1). Complex microbial communities including anaerobic bacteria have been detected in sputa from CF patients (3). Although decreased oxygen tensions have been demonstrated in mucous layers in the CF lung, the effects of hypoxia on antibiotic resistance has not been investigated independent of biofilm formation. Our data show that exposure to hypoxia induces selective antibiotic resistance in P. aeruginosa. Antibiotic resistance of P. aeruginosa in response to hypoxia may not only be important for the treatment of infections in CF lung disease, but also for the management of infections in other chronic respiratory diseases such as bronchiectasis and COPD. In these diseases, chronic inflammation with remodeling of lung tissue and excessive mucous production generates comparable growth conditions to CF and not surprisingly P. aeruginosa is one of the major bacterial pathogens encountered. The analysis of RND efflux pump expression levels in P. aeruginosa revealed a shift in pump expression towards MexEF-OprN under hypoxic conditions in the ATCC 27853 strain as well as in 3 out of 4 clinical isolates of P. aeruginosa from CF patients. The β-lactam antibiotics (Aztreonam, Ceftazidime), β-lactam inhibitor combinations (Piperacillin/Tazobactam), tetracyclines and trimethoprim 10

220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 have all been shown to be substrates for the MDR efflux pumps MexAB-OprM, MexCD-OprJ and MexEF-OprN (14). As aminoglycosides are believed to be exclusively transported by MexXY-OprM (14), it was not surprising that no effect of hypoxia on tobramycin susceptibilities was observed as we could not detect MexXY-OprM. The failure of hypoxia to increase Ciprofloxacin MICs was surprising, as Ciprofloxacin had been described as a substrate for all three pumps MexAB-OprM, MexCD-OprJ and MexEF-OprN (14). One potential explanation for this phenomenon could be differences in substrate specificity of pumps expressed by various P. aeruginosa strains. As altered efflux pump expression is one of a number of antibiotic resistance mechanisms used by P. aeruginosa, we cannot exclude the possibility that other bacterial resistance mechanisms are also affected by hypoxia. Normalization of raised MIC values in the presence of the efflux pump inhibitor indicates that alterations in efflux pump activities are indeed associated with the observed changes in antimicrobial susceptibility. Altered expression of bacterial RND efflux pumps has been seen in response to exposure to antimicrobials in vivo and in vitro (2, 13), where antibiotic exposure selects for mutation events in genetic regulators suppressing pump expression. Clinical isolates with increased efflux pump expression without identifiable regulatory mutations had been identified before (2), and no inducers of efflux pump expression apart from substrate exposure have been described so far. Here we describe hypoxia as a novel regulator of RND efflux pumps in P. aeruginosa. 11

243 244 245 246 247 248 249 250 251 252 253 254 255 256 Hydroxylases are key regulatory elements in the cellular response of eukaryotic cells to hypoxia. In P. aeruginosa pharmacologic hydroxylase inhibition failed to increase MIC values compared to normoxia, suggesting that raised MIC levels in hypoxia are independent of hydroxylase activity. As secondary metabolites have been postulated to be the natural substrates for the pumps (14) it is possible that efflux pump genes contain hypoxia responsive elements as part of the bacterial adaptive response to hypoxia. Thus the extrusion of antibiotics via MDR efflux pumps in response to hypoxia could be a secondary effect of the bacterial metabolism adapting to decreased oxygen values. Our finding suggests that bacterial isolates could look sensitive in vitro on testing in the laboratory, but behave resistant in vivo once they encounter hypoxic environments. This could be one factor explaining clinical treatment failure with appropriate antibiotics, which is observed frequently in the management of chronic respiratory infections. 257 258 259 260 Acknowledgements This work was supported by grants from the Marie Curie Foundation, Irish Research Council for Science, Engineering & Technology and Science Foundation Ireland. 261 262 Transparency declaration None to declare. 263 12

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340 Figure legends 341 342 343 344 345 346 347 348 349 350 351 352 353 Figure 1: Altered antibiotic resistance in P. aeruginosa under hypoxic conditions. A: Mueller Hinton agar plates were inoculated with ATCC 27853 and grown for 20h in a normoxic (N) or hypoxic (H; 1% oxygen) environment. B: MIC determination of ATCC 27853 at 21% (N) and 1% oxygen (H) by microbroth dilution of 21 antibiotics (AMI: Amikacin; GEN: Gentamicin; TOB: Tobramycin; FOT: Cefotaxime; TAZ: Ceftazidime; FEP: Cefepime; AZT: Aztreonam; TIM: Ticarcillin /clavulanic acid; P/T: Piperacillin / tazobactam; MERO: Meropenem; ETP: Ertapenem; DOR: Doripenem; IMI: Imipenem; COL: Colistin; POL: Polymicin B; LEVO: Levofloxacin; CIP: Ciprofloxacin; DOX: Doxycycline; MIN: Minocycline; TGC: Tigecycline; SXT: Trimethoprim / sulfamethoxazole) (Sensititre susceptibility plate GNX2F) Data represent mean ± SEM MIC for n=3 independent experiments. 354 355 356 357 358 359 360 361 362 Figure 2: Altered stoichiometry of RND multidrug efflux pump composition under hypoxic conditions. Investigation of the gene expression of the RND multidrug efflux pumps MexAB- OprM, MexCD-OprJ and MexEF-OprN in P. aeruginosa in normoxia (N) and hypoxia (H) by Real time PCR for the respective linker molecules (mexa, mexc and mexe). A and B: ATCC 27853 were grown in N and H for 6h (A) and 24h (B). Gene expression of mexa, mexc and mexe were detected by Real time PCR. Relative 17

363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 gene expression was normalized and expressed as a ratio of mexe/mexa and mexe/mexc. Values are expressed as mean ± SEM for n=3 independent experiments (* p < 0.05). C: clinical P. aeruginosa strains were exposed to N and H for 6h, gene expression of the linker molecules were detected by Real time PCR. Relative gene expression was normalized within each gene and expressed as a ratio of mexe/mexa and mexe/mexc. Values are expressed as mean ± SEM for n=2 independent experiments (* p < 0.05).. Figure 3: Increased antibiotic resistance in hypoxia is reversed by a multidrug efflux pump inhibitor. MIC determination of ATCC 27853 by microbroth dilution (Sensititre susceptibility plate GNX2F) in normoxia (N), hypoxia (H) and hypoxia in the presence of the efflux pump inhibitor (N+EPI) Phe-Arg β naphtylamid dihydrochloride (20µg/ml). A selection of CF relevant antibiotics where EPI decreased MIC in hypoxia is shown. Values are expressed as mean ± SEM for n=3 independent experiments (* p < 0.05). 380 381 382 383 384 385 Figure 4: Hydroxylase inhibition fails to increase antibiotic resistance MIC determination of ATCC 27853 by microbroth dilution (Sensititre susceptibility plate GNX2F) in the presence of the hydroxylase inhibitor DMOG (1 mm) or the vehicle control (DMSO). Data represent mean MIC ± SEM of n=3 independent experiments. 18