Mutant prevention concentrations of doripenem and meropenem alone and in combination with colistin ( polymyxin E ) , levofloxacin and tobramycin in Pseudomonas aeruginosa

BACKGROUND: With a limited number of available antimicrobial agents to treat Pseudomonas aeruginosa infections, the prevention of emergence of antimicrobial resistance and its subsequent spread is critical. In the present study, the mutant prevention concentration (MPC) of doripenem was examined and compared with meropenem for its ability to prevent resistant mutant selection for P aeruginosa when used alone and in combination with the other antipseudomonal agents colistin (polymyxin E), levofloxacin and tobramycin. OBJECTIVE: To determine if two antimicrobial agents that possessed different mechanisms of action and separate demonstrated activities against P aeruginosa would produce a reduced MPC in combination compared with the MPC of each agent alone. METHODS: Twelve clinical isolates of P aeruginosa were plated on Mueller-Hinton agar containing 1×, 2×, 4×, 8×, 16× and 32× the doripenem, imipenem or meropenem minimum inhibitory concentration (MIC), and also on agar containing doripenem or meropenem in combination with either tobramycin (6 μg/mL), colistin (2 μg/mL or 8 μg/mL), levofloxacin (8 μg/mL) or azithromycin (0.4 μg/mL). The MPC for each antimicrobial agent-isolate combination was defined as the lowest antibiotic concentration that prevented the visible growth of mutant colonies at 48 h of incubation. The MPC/MIC (μg/mL) ratio was defined as the ratio of the MPC obtained to the original MIC. RESULTS: The MPC/MIC ratios of doripenem alone ranged from 8 to 32 for the twelve isolates tested compared with 32 for two isolates and greater than 32 for 10 isolates with imipenem, and 32 for three isolates and greater than 32 for nine isolates with meropenem. All antimicrobials tested exhibited markedly elevated MPCs compared with their original MICs with MPC/MIC ratios of 8 to 32 for doripenem, 32 to greater than 32 for imipenem, 32 to greater than 32 for meropenem, 32 to greater than 32 for colistin (tested at 2 μg/mL), 8 to 16 for levofloxacin and 8 to 32 for tobramycin. When a second antimicrobial was used in combination with doripenem, the MPC/MIC ratio decreased up to twofold for doripenem combined with colistin (2 μg/mL), decreased fourto 16-fold for doripenem combined with colistin (8 μg/mL), decreased eightto 32-fold for doripenem combined with levofloxacin, and decreased fourto 16-fold for doripenem combined with tobramycin. Adding a second antimicrobial in combination with meropenem resulted in the following decreases in MPC/MIC: no decrease for meropenem combined with colistin (2 μg/mL), four to greater than eightfold decrease for meropenem combined with colistin (8 μg/mL), fourto 16-fold decrease for meropenem combined with levofloxacin, and twoto 16-fold decrease for meropenem combined with tobramycin. For all antimicrobial combinations tested, doripenem yielded greater decreases in MPC/MIC ratios than did meropenem. CONCLUSION: The present study found that individual antipseudomonal antimicrobial agents tested against 12 clinical isolates of P aeruginosa had eightto greater than 32-fold higher MPCs than MICs, that combining doripenem or meropenem with a second active antipseudomonal agent with a different mechanism of action was more effective at preventing resistance selection than the two agents used individually, and finally, that doripenem was less likely than both imipenem and meropenem to select for spontaneous resistance mutants of P aeruginosa.

meropenem resulted in the following decreases in MPC/MIC: no decrease for meropenem combined with colistin (2 μg/mL), four to greater than eightfold decrease for meropenem combined with colistin (8 μg/mL), four-to 16-fold decrease for meropenem combined with levofloxacin, and two-to 16-fold decrease for meropenem combined with tobramycin.For all antimicrobial combinations tested, doripenem yielded greater decreases in MPC/MIC ratios than did meropenem.CONCLUSION: The present study found that individual antipseudomonal antimicrobial agents tested against 12 clinical isolates of P aeruginosa had eight-to greater than 32-fold higher MPCs than MICs, that combining doripenem or meropenem with a second active antipseudomonal agent with a different mechanism of action was more effective at preventing resistance selection than the two agents used individually, and finally, that doripenem was less likely than both imipenem and meropenem to select for spontaneous resistance mutants of P aeruginosa.

Pseudomonas aeruginosa
HISTORIQUE : Étant donné le nombre limité d'antimicrobiens disponibles pour traiter les infections à Pseudomonas aeruginosa, il est essentiel de prévenir l'émergence d'une résistance aux antimicrobiens et sa propagation subséquente.Dans la présente étude, on a examiné la concentration de prévention de mutation (CPM) du doripénem et on l'a comparée avec le méropénem pour sa capacité de prévenir la sélection d'une mutation résistante au P aeruginosa lorsqu'il est utilisé seul et en association avec les autres antipseudomonaux, soit la colistine (polymyxine E), la lévofloxacine et la tobramycine.OBJECTIF : Déterminer si deux antimicrobiens aux mécanismes d'action différents et aux activités démontrées distinctes contre le P aeruginosa assurent une réduction de la CPM s'ils sont associés, par rapport à la CPM de chaque agent utilisé seul.MÉTHODOLOGIE : Douze isolats cliniques de P aeruginosa ont été cultivés sur une gélose Mueller-Hinton contenant une, deux, quatre, huit, 16 et 32 fois la concentration minimale inhibitrice (CMI) de doripénem, d'imipénem ou de méropénem, et également sur une gélose contenant du suite page suivante P seudomonas aeruginosa continues to be an important cause of infections in Canadian and American hospitals (1,2).With a limited number of available antimicrobial agents to treat P aeruginosa infections, the prevention of emergence of antimicrobial resistance and its subsequent spread is critical (3).Carbapenems such as meropenem and imipenem/cilastatin are used alone and in combination with other agents to treat patients with P aeruginosa infections (4).A recent in vitro study (5) reported that when antipseudomonal antimicrobials are tested alone against P aeruginosa, resistant subpopulations can be selected, leading to dramatic increases in minimum inhibitory concentrations (MICs).
It has been suggested that manipulating antimicrobial agent dosing strategies may limit the selection of resistant mutants and preserve antimicrobial activity (6)(7)(8)(9).This concept has been well studied extensively with fluoroquinolones and is known as the mutant selection window (6)(7)(8)(9).The mutant selection window is bounded by the MIC at its lower end and the organism's mutant prevention concentration (MPC) at its upper end.The MPC is the drug concentration required to prevent the emergence of all single-step mutants in a population of approximately 10 10 bacterial cells (5,6).If antimicrobial dosing strategies maintain concentrations above the MPC for a sufficient duration (ideally the entire dosing interval), a reduced risk of selecting antimicrobial-resistant mutants may result.The MPC has recently been validated in vivo (10).
The use of two antimicrobial agents in combination compared with one agent alone has been reported to lower an organism's MPC (5,11).To survive exposure to two antimicrobial agents, an organism must develop simultaneous resistance to both agents, assuming that both agents act via different modes of action and that the organism is initially susceptible to both agents (5,11).
In the present study, we examined the MPC of doripenem, and compared it with meropenem in its ability to prevent resistant mutant selection for P aeruginosa when used alone and in combination with the other antipseudomonal agents colistin (polymyxin E), levofloxacin and tobramycin.Our hypothesis was that two antimicrobial agents that possessed different mechanisms of action and separately displayed activity against P aeruginosa would demonstrate a reduced MPC in combination compared with each individual antimicrobial agent MPC.

RESULTS
The MICs of the antimicrobial agents tested against the 12 isolates of P aeruginosa and MPC/MIC ratios are displayed in Table 1.The MPC/MIC of doripenem ranged from 8 to 32.For imipenem, MPC/MIC ratios were 32 for two isolates and greater than 32 for 10 isolates.Similarly, for meropenem, the MPC/MIC were 32 for three isolates and greater than 32 for nine isolates.For each isolate tested, doripenem demonstrated a twofold to greater than fourfold lower MPC/MIC ratio than both imipenem and meropenem.For all antimicrobials tested alone, MPCs were markedly elevated compared with MICs with MPC/MIC ratios of 8 to 32 for doripenem, 32 to greater than 32 for imipenem, 32 to greater than 32 for meropenem, 32 to greater than 32 for colistin (tested at 2 μg/mL), 8 to 16 for levofloxacin, and 8 to 32 for tobramycin.
When a second antimicrobial was used in combination with doripenem, the MPC/MIC ratio did not decrease for the combination of doripenem and azithromycin (data not shown), decreased up to twofold for doripenem combined with colistin (2 μg/mL), decreased four-to 16-fold for doripenem combined with colistin (8 μg/mL), decreased eight-to 32-fold for doripenem combined with levofloxacin, and decreased four-to 16-fold for doripenem combined with tobramycin (Table 2).Adding a second antimicrobial in combination with meropenem resulted in the following decreases in MPC/MIC: no decrease for meropenem combined with azithromycin (data not shown), no decrease for meropenem combined with colistin (2 μg/mL), four-to greater than eightfold decrease for meropenem combined with colistin (8 μg/mL), four-to 16-fold decrease for meropenem combined with levofloxacin, and twoto 16-fold decrease for meropenem combined with tobramycin.For all antimicrobial combinations tested, doripenem yielded greater decreases in MPC/MIC ratios than did meropenem.

DISCUSSION
The present study generated three important observations.First, our results demonstrated that when individual antipseudomonal antimicrobial agents are tested alone against P aeruginosa, all agents have markedly increased MPCs (MPC/MIC ratios eight-to greater than 32-fold) compared with their MIC (Table 1).This suggests that using an agent alone to treat P aeruginosa infections may result in elevated MPC/MIC ratios in vivo with the potential for resistance development and bacteriological failure.Elevated MPC/MIC ratios and the potential for resistance may be less likely to occur when treating patients with urinary tract infections because high concentrations of carbapenems, levofloxacin and aminoglycosides are attained in the urinary tract, which may prevent resistance development.
The second observation was that combination treatment with either doripenem or meropenem and a second antimicrobial agent (each agent possessing independent activity against P aeruginosa and acting with a different mechanism of action) was more effective at preventing mutant selection for P aeruginosa than the two agents used individually (Table 2).This was demonstrated by large decreases in MPC/MIC ratios (four-to 32-fold) when doripenem or meropenem were used in combination with colistin (8 μg/mL), levofloxacin or tobramycin.This observation that dual-drug therapy for isolates of P aeruginosa that are susceptible to both antimicrobial agents may prevent the selection of resistant mutants has been previously reported both in vitro and in vivo (5,16).It should be mentioned that combination therapy with two antimicrobial agents with different mechanisms of action is not a new idea; it has been reported to result in reduced mortality in P aeruginosa bacteremia (16).What is novel about combination MPC is the concept of using specific combinations to not simply increase bacterial killing, but to actually maximize resistance prevention using a specific combination of antimicrobial agents.
The third observation was that doripenem was less likely than both imipenem and meropenem to select for resistance in P aeruginosa as manifested by its twofold to greater than fourfold lower MPC/MIC ratios compared with imipenem and meropenem.This observation was consistent and occurred whether doripenem and meropenem were used alone or in combination with colistin (8 μg/mL), levofloxacin or tobramycin.Why would doripenem be less likely to select for resistance in P aeruginosa than imipenem or meropenem?Microbiologically, studies have demonstrated that MIC 90 s (concentration that inhibits 90% of isolates) for doripenem and meropenem tested against P aeruginosa are similar (8 μg/mL and 16 μg/mL, respectively) and that both agents are more potent than imipenem (4).Davies et al (17) recently reported that doripenem demonstrated greater affinity than imipenem for penicillin-binding protein (PBP)2 and PBP3 in P aeruginosa.Doripenem also has slightly greater affinity than meropenem against both PBP2 and PBP3 with one of the two P aeruginosa strains (ATCC 27853) studied (17).Mushtaq et al (18) performed single-step mutational studies (analogous to our MPC studies) and reported that mutant selection with doripenem occurred with fewer strains versus other agents including meropenem and imipenem, and that the final multiple of the MIC killing all cells was lower with doripenem than meropenem and imipenem and never exceeded four times the MIC.Sakyo et al (19) assessed the potencies of doripenem, imipenem and meropenem in preventing the emergence of carbapenem-resistant mutants of P aeruginosa.In performing mutational studies, these investigators documented both lower mutational frequencies of doripenem compared with both imipenem and meropenem as well as lower MICs of doripenem versus imipenem and meropenem in the carbapenem-resistant P aeruginosa selected (19).In fact, these investigators could not select doripenem-resistant P aeruginosa on agar plates containing doripenem but could select doripenem-resistant strains on plates containing imipenem and meropenem (19).Their carbapenem-resistant P aeruginosa exhibited reductions in an outer membrane protein with a molecular weight of approximately 48,000 Da, which the authors speculated was outer membrane protein D2.

CONCLUSIONS
The present study found that individual antipseudomonal antimicrobial agents tested against 12 clinical isolates of P aeruginosa had eight-to greater than 32-fold higher MPCs than MICs, that combining doripenem or meropenem with a second active antipseudomonal agent with a different mechanism of action was more effective at preventing resistance selection than the two agents used individually, and finally, that doripenem was less likely than both imipenem and meropenem to select for spontaneous resistance mutants of P aeruginosa.