Effect of MexXY Overexpression on Ceftobiprole Susceptibility in Pseudomonas aeruginosa

Abstract

Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, <4 g/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P. aeruginosa resistance to ␤-lactams, including cefepime and ceftazidime, is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing cefepime but not ceftazidime. In ceftobiprole clinical studies, paired isolates of P. aeruginosa from four subjects demonstrated ceftobiprole MICs of 2 to 4 g/ml at baseline but 16 g/ml posttreatment, unrelated to ␤-lactamase levels. Within each pair, the level of mexXY RNA, but not mexAB, mexCD, and mexEF, increased by an average of 50-fold from baseline to posttreatment isolates. Sequencing of the negative regulatory gene mexZ indicated that each posttreatment isolate contained a mutation not present at baseline. mexXY expression as a primary ceftobiprole and cefepime resistance mechanism was further examined in isogenic pairs by using cloned mexXY and mexZ. Expression of cloned mexXY in strain PAO1 or in a baseline isolate increased the ceftobiprole MIC to that for the posttreatment isolate. In contrast, in posttreatment isolates, lowering mexXY expression via introduction of cloned mexZ decreased the ceftobiprole MIC to that for the baseline isolates. Similar changes were observed for cefepime. A spontaneous mutant selectively overexpressing mexXY displayed a fourfold elevation in its ceftobiprole MIC, while overexpression of mexAB, -CD, and -EF had a minimal effect. These data indicate that ceftobiprole, like cefepime, is an atypical ␤-lactam that is a substrate for the MexXY efflux pump in P. aeruginosa.Ceftobiprole is a broad-spectrum, anti-methicillin-resistant Staphylococcus aureus cephalosporin with in vitro microbiological activity against many strains of Pseudomonas aeruginosa (MIC for 50% of strains tested, Յ4 g/ml) (4, 22). Resistance to antipseudomonal cephalosporins can be due to overexpression of the chromosomal AmpC cephalosporinase (17); however, decreased susceptibility of P. aeruginosa to a wide variety of antibiotics can also be attributed to drug efflux (17, 21). In particular, basal expression of the Mex pumps of the resistance nodulation division family appears to be responsible for the intrinsic resistance of P. aeruginosa to multiple antibiotics and for enhanced resistance upon overexpression (21, 23). The efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM have each been shown to be clinically relevant factors for decreased antibiotic susceptibility, with expression of the operons encoding these pumps subject to regulation (21). Pump overexpression may be due to an assortment of mutations that affect regulation of transcription from the efflux operon, such as mutations in the cognate repressor or in a global regulatory gene, or upstream of the operon, including but not limited to its promoter (21).The MexAB-OprM efflux pump is constitutively expressed in P. aerugi...

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…The weak promoter identified ahead of oprM within the mexAB-oprM operon (442) could possibly account for an excess of OprM molecules relative to MexAB, thus allowing the interaction of OprM with other extrusion systems without impacting the activity of MexAB-OprM by titration. MexXY(OprA)-overproducing mutants can be easily selected in vitro and in vivo by substrate antibiotics (393,439,443,444) or protein synthesis inhibitors (445), which is consistent with the increased prevalence, sometimes Ͼ80%, of such mutants in cystic fibrosis (436,(446)(447)(448)(449)(450)(451) and non-cystic fibrosis (384, 385, 414, 416, 417, 420-423, 429, 431, 452) patients worldwide. The abundance of reactive oxygen species in the cystic fibrosis lung environment might explain the high rates of resistant mutants with this pathology (453).…”

“…However, its activity is adversely affected by increased efflux in A. baumannii and P. aeruginosa (955). Moreover, several new cephalosporin-␤-lactamase-inhibitor combinational products in clinical trials (e.g., ceftazidime-avibactam, ceftaroline-avibactam, and ceftolozane-tazobactam) (944) are still likely to be the substrates of RND pumps, as are other ␤-lactams and ␤-lactamase inhibitors (13,390,439,545), because avibactam cannot reverse efflux-mediated ceftazidime resistance (956), and both ceftaroline and ceftolozane (a new antipseudomonal cephalosporin) are still affected by efflux pump-and/or porin-related resistance mechanisms (although ceftolozane, containing multiple charged groups, appears less impacted by Mex pumps than many other ␤-lactams and did not select in vitro for pump overproducers in P. aeruginosa, unlike other agents) (417,(957)(958)(959). These observations could also illustrate their reduced or lack of synergistic activity against multidrug-resistant A. baumannii and P. aeruginosa (960)(961)(962).…”

“…In the PA7-related strains, the MexXY proteins seem to cooperate with both OprM and OprA, as each of the latter can compensate for the genetically engineered suppression of the other. While the MexXY-OprM/OprA system is able to transport aminoglycosides, fluoroquinolones, macrolides, tetracyclines, tigecycline, and zwitterionic cephalosporins (cefepime and ceftobiprole) (393,(434)(435)(436)439), its contribution to natural resistance is restricted to those agents able to induce mexXY(oprA) expression (219, 434). This occurs when bacteria are exposed to subinhibitory concentrations of ribosome-targeting antibiotics, including chloramphenicol, a poor substrate, if at all, of the pump (440,441).…”

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

“…The presence of the oprA gene in P. aeruginosa might be more advantageous for its survival in the presence of aminoglycosides, other antimicrobials such as b-lactams (e.g. cefepime, ceftobiprole; Hocquet et al, 2006;Baum et al, 2009), tigecycline (Dean et al, 2003), LBM415 (peptide deformylase inhibitor; Caughlan et al, 2009) and/or some other sources of stress (e.g. oxidative stress; Fraud & Poole, 2011).…”

Oxidative Stress Induction of the MexXY Multidrug Efflux Genes and Promotion of Aminoglycoside Resistance Development in Pseudomonas aeruginosa

“…Upregulation of mexXY by antimicrobials or fmt/ folD mutations is dependent upon a gene, PA5471, encoding a conserved hypothetical protein whose expression is also promoted by ribosome-disrupting antimicrobials (34) and by fmt/ folD mutations (6). Despite this primary link to translation disruption, the MexXY-OprM efflux system is a significant determinant of resistance to antimicrobials in clinical isolates, particularly aminoglycosides (19,40,52) but also ␤-lactams (3,19,23,37,51). Indeed, while it is uncommon as a mechanism of aminoglycoside resistance in most clinical strains of P. aeruginosa, MexXY-OprM is the predominant mechanism of resistance to these agents in cystic fibrosis (CF) isolates (19,40,52).…”