Markedly different rates and resistance profiles exhibited by seven commonly used and newer ß-lactams on the selection of resistant variants of Enterobacter cloacae

Wai C. Chana, Ronald C. Lia,*, Julia M. Lingb, Augustine F. Chengb and Jerome J. Schentagc

a Department of Pharmacy, The Chinese University of Hong Kong, Shatin, Hong Kong; b Department of Microbiology, The Chinese University of Hong Kong, Shatin, Hong Kong; c Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo, NY, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Seven ß-lactam antibiotics (cefepime, cefoperazone, ceftazidime, ceftriaxone, cefamandole, imipenem and meropenem) were tested for their potential to select resistance in standard and clinical strains of Enterobacter cloacae (n = 9). The strains were subcultured daily with the test antibiotics at doubling concentrations starting at 0.125 x MIC. Development of resistance throughout the passages was detected by a disc diffusion test. Ceftazidime, ceftriaxone and cefamandole selected resistance at a faster rate than cefoperazone, cefepime and meropenem. Imipenem did not select resistance in the nine strains tested and was the only antibiotic that eradicated all the strains during selection. The resistance patterns of strains selected by meropenem, cefepime and the other cephalosporins were markedly different, although cross-resistance to the early generation cephalosporins was common. The resistance phenotypes of most strains remained stable upon serial passages in antibiotic-free medium. The findings of this study highlight the importance of the choice of antibiotic for therapy not only on the basis of its antibacterial activity, but also on its potential to select resistance to itself and other antibiotics.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Selection pressure causing the development of antibiotic resistance in both Gram-positive and Gram-negative organisms has been increasing constantly as the amount of antibiotic use continues to rise. 1,2 Gram-positive organisms such as Staphylococcus, Enterococcus and Streptococcus spp., and Gram-negative organisms such as Escherichia coli, Pseudomonas, Enterobacter and Proteus spp. are common causes of serious infections. 3 Successful treatment of systemic infections caused by these bacteria requires the proper choice of antibiotic which depends heavily on antibiotic susceptibility data. Much evidence suggests a causal relationship between antibiotic usage and resistance development. 4,5 Interestingly, animal models have demonstrated the development of antimicrobial resistance during or following antibiotic therapy. 6,7 Similar observations have also been documented in many prospective clinical studies. 8,9 However, few studies have systematically analysed and compared the rate of resistance development upon exposure to different antibiotics and the possibility of different resistance phenotypes of the resistant variants selected. In addition, cross-resistance is a common phenomenon for antibiotics that share a similar mechanism of action, e.g. penicillins and cephalosporins; such resistance can also take place in antibiotics with no or minimal structural similarity. It is therefore important to investigate the long-term consequences associated with the use of a particular antibiotic apart from its immediate antimicrobial effect; these include the ability of an antibiotic to select resistance to itself and to other antibiotics.

More rational use of antibiotics may minimize the development of resistance: the antibiotic chosen should have adequate antimicrobial activity yet the lowest resistance selection potential. Recently, Stapleton et al. 10 showed that different ß-lactams exhibited different potential in the selection of resistant mutants of Citrobacter freundii. Lability of the antibiotic to ß-lactamase is also an important factor in determining whether the antibiotic will select resistant mutants. On the other hand, Fung-Tomc et al. 11 demonstrated that different cephalosporins exhibited varied rates of resistance selection in E. cloacae, but only four cephalosporins, i.e. ceftriaxone, ceftazidime, cefepime and cefpirome, were studied. Since more than 50% of infections locally are caused by Gram-negative bacteria, we studied E. cloacae, which is increasingly implicated in episodes of hospital-acquired infections. 12 Both antimicrobial activity and resistance selection potentials of seven ß-lactam antibiotics were analysed, along with the nature of resistance phenotypes that arose during the selection process in an attempt to generate more meaningful information on antibiotic selection and use.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics and bacterial strains

Seven commonly used and newer ß-lactam antibiotics were studied. These included cefepime (Bristol-Myers Squibb, Princeton, NJ, USA), ceftazidime (Glaxo, Research Triangle Park, NC, USA), cefamandole (Sigma, St Louis, MO, USA), cefoperazone (Sigma), ceftriaxone (Sigma), imipenem (Merck, West Point, PA, USA) and meropenem (Zeneca, Wilmington, DE, USA). The fourth-generation cephalosporin, cefpirome, was also studied by analysing the nature of the resistance phenotypes for strains it selected, but the rate at which resistance developed was not determined as antibiotic discs were not commercially available. Both clinical and standard strains of E. cloacae were employed. The clinical strains studied were isolated in the Microbiology Laboratory from blood cultures of patients at the Prince of Wales Hospital, Hong Kong and identified by the API 20E system (bioMérieux, Lyon, France). The eight clinical and one ATCC standard (23355) strains of E. cloacae included in the present study were initially susceptible to all the seven antibiotics tested.

MIC determination

The antibiotic susceptibility of the strains was assessed by determining the baseline MIC of the test antibiotics for the isolates by use of an agar dilution method in accordance with the procedures described by the National Committee for Clinical Laboratory Standards. 13 Briefly, a multiple inoculator (Dynatech, Alexandria, VA, USA) was used for the inoculation of approximately 104 bacteria per spot on to Mueller– Hinton Agar (Oxoid, Basingstoke, UK) containing doubling concentrations of the antibiotic. E. coli strains NCTC 10418 and ATCC 25922 were used as controls. The MIC was taken as the lowest concentration of the antibiotic at which there was no visible growth after an overnight incubation at 37°C. On the basis of their MICs, strains susceptible to individual test antibiotics were employed in the resistance selection experiments.

Selection of antibiotic resistance

To select the resistant bacterial sub-populations, each strain was exposed to increasing (doubling) concentrations of the antibiotic by daily passages in Mueller– Hinton Broth (MHB) (Oxoid) starting at 0.125 = MIC. At each passage, the overnight culture was diluted to a concentration of approximately 108 cfu/mL. A volume of 200 µL of this bacterial suspension was re-inoculated into 5 mL of MHB containing the antibiotic at twice its previous concentration and the culture was again incubated overnight at 37°C. Susceptibility of the daily samples to the antibiotics was assessed using a disc diffusion test in which antibiotic discs (Oxoid) were placed on a bacterial lawn swabbed with a bacterial suspension at a density of 107 cfu/mL. After incubation at 37°C, the size of the inhibition zone was measured and recorded. The MICs of the test antibiotics against the selected resistant strains were determined to assess the resistance levels and profiles of the strains. The resistance selection potential of an antibiotic was indicated by the rate at which the antibiotic selected resistance in the E. cloacae strains tested. The selection potential of each individual antibiotic is indicated by the overall rate of resistance development and was calculated as the percentage of strains acquiring resistance per passage. The stability of the resistance phenotypes of the selected strains was assessed. Briefly, the resistant strains were subcultured on antibiotic-free MHB daily for 20 days and the antibiotic susceptibility test was repeated in the same manner as before.


    Results
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Figure shows the relative potential of each of the six antibiotics (cefamandole, cefepime, cefoperazone, ceftazidime, ceftriaxone and meropenem) to select resistance to itself. The rates of resistance development for the test antibiotics, expressed as the percentage of strains that developed resistance per passage, were as follows: cefoperazone (6%), cefepime (7%), meropenem (9%), ceftazidime (13%), cefamandole (14%) and ceftriaxone (15%). Interestingly, none of the nine strains developed resistance against imipenem during selection. Table I shows the MICs of the test antibiotics to representative strains after resistance selection. Susceptibility to cefuroxime was tested but this antibiotic was omitted from the selection study as all eight clinical strains exhibited at least partial resistance to this drug. Table II summarizes the antimicrobial activity and resistance selection potentials of different antibiotics. For each test antibiotic, the concentrations at which 50% and the maximum number of strains became resistant are listed. As shown, the seven ß-lactams not only selected resistance at different rates, but the resistant strains they selected also exhibited varied resistance profiles and degrees of resistance.



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Figure. Resistance selection potentials of six antibiotics: {blacklozenge}, cefamandole; {blacksquare}, ceftazidime; {circ}, ceftriaxone; {blacktriangleup} cefoperazone; •, cefepime; {triangleup} , meropenem.

 

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Table I. MICs (mg/L) of nine ß-lactam antibiotics for the E. cloacae strains tested before selection experiments and for representative resistant strains selected by the antibiotics.
 

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Table II. Summary of the antimicrobial activity and resistance selection potentials of different classes of ß-lactam antibiotics
 
For the two carbapenems, resistance to meropenem began to emerge when the drug concentration was increased to 32 + MIC. At this concentration, two of nine strains developed resistance to meropenem. When the concentration was increased to 128 + MIC, all nine strains became resistant (Table II). These meropenem-resistant strains were cross-resistant to the cephalosporins, although to a lesser extent to cefepime and cefpirome (Table I). Meropenem also selected low-level resistance to imipenem (MIC = 8 mg/L). Significantly, two meropenem-resistant strains were susceptible to cephalosporins, including ceftazidime and ceftriaxone (Table I).

Despite its low original MICs for the clinical strains tested, the MICs of meropenem after selection were significantly increased (500-fold). Resistance to meropenem was found to be stable after 20 passages on antibiotic-free medium, with resistance profiles and MICs being unchanged or slightly lowered.

Imipenem appeared to exhibit good antibiotic activity as well as low selection potential. It was the only antibiotic tested that did not select resistance to itself in all nine strains. This drug selected resistance only to cefuroxime and cefamandole in two of nine strains and was able to eradicate all nine strains at 32 + MIC.

For the six cephalosporins (cefepime, cefpirome, cefamandole, ceftazidime, ceftriaxone and cefoperazone), the selection potential of cefamandole, ceftazidime and ceftriaxone appeared similar, but higher than that of cefoperazone. Cefamandole showed the highest potential to select its own resistance, with all nine strains becoming resistant at 8 + MIC (Table II). Resistance to cefamandole as well as to cefuroxime developed earliest even when selected by other ß-lactam antibiotics; however, cefamandole-selected resistant strains were not highly resistant to other cephalosporins (Table I).

Cross-resistance was common for strains selected by the early generation cephalosporins (Table I and II). The resistance profiles of resistant strains selected by different cephalosporins were similar. The MICs of the test antibiotics for the strains after selection were often more than 100-fold higher than those to the original susceptible strains (Table I). Almost all the selected resistant strains were highly resistant to the second-generation cephalosporins (cefuroxime and cefamandole) while variations were evident between the degree of resistance to other cephalosporins (Table I). A significant finding is that the cross-resistance observed among the second- and third-generation cephalosporins did not extend to the fourth-generation cephalosporins (cefepime and cefpirome) or to the carbapenems (imipenem and meropenem).

The fourth-generation cephalosporin, cefepime, started to select resistance at drug concentrations above 32 + MIC. At 512 + MIC, eight of nine strains became resistant to cefepime (Table II). The rate of selection was lower than that of other cephalosporins (Figure and Table II). Both cefepime and cefpirome selected resistance to all second- and third-generation cephalosporins but not imipenem and meropenem. Resistance to all cephalosporins was generally stable even after 20 passages in antibiotic-free medium.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Conceivably, the number of passages required for each antibiotic to select resistance depends on the following factors: (i) the potential of the antibiotic to induce development of resistance mechanisms in bacteria; (ii) the intrinsic ability of the bacterial species as well as individual strains to develop the resistance mechanisms; (iii) the antimicrobial activity of the test antibiotic; and (iv) the ability of the antibiotic itself to withstand the resistance mechanism. Hence, findings in this study highlight the importance of selecting an antibiotic for antimicrobial chemotherapy not only on the basis of its activity, but also on the above factors that influence the development of resistance to the antibiotic. This is especially true when the subsequent resistance profiles and mechanisms of the antibiotic-resistant strains are examined. For example, imipenem and ceftazidime, despite having similar antimicrobial activity against the susceptible strains, exhibited drastically different ability to select resistance in E. cloacae strains.

Overall, the seven antibiotics can be divided into three categories according to their resistance selection potentials and the resistance profiles of the resistant strains selected. The first group, comprising the cephalosporins, cefamandole, cefoperazone, ceftriaxone and ceftazidime, exhibited relatively fast selection of resistance when compared with the newer antibiotics, including meropenem, imipenem and cefepime. The resistant strains selected by these cephalosporins also exhibited similar resistance profiles, suggesting that they were likely to select mutants with similar mechanisms of resistance. It is likely that these resistant mutants overproduced an AmpC-like cephalosporinase as a result of mutations in its regulatory machinery, including the AmpD gene.14 Cefamandole and cefuroxime appeared to be most vulnerable to the resistance mechanisms developed during selection since resistance to these two antibiotics often emerged at an early stage during selection by all test antibiotics. However, cefamandole and cefuroxime did not have the highest selection potential when selection of resistance to other antibiotics was concerned, although vulnerability of these two drugs to bacterial resistance mechanisms might lead to a false impression that they had high selection potential. Alternatively, it can be concluded that these drugs only had high potential to select their own resistance. The notion that lability to ß-lactamase is an important factor in determining the ability of a compound to select derepressed mutants independent of its ability to induce ß-lactamase synthesis has been documented.10

In the second category, the two carbapenems showed good antimicrobial activity and both exhibited low resistance selection potentials. Imipenem was the only antibiotic that failed to select E. cloacae resistance in vitro. This property may be attributed partly to its ability to resist hydrolysis by ß-lactamases.10 The fact that imipenem did not effectively select resistance to other cephalosporins suggested that this antibiotic, unlike meropenem, did not readily induce ß-lactam resistance in E. cloacae. This has also been reported previously. 15,16 Compared with imipenem, meropenem demonstrated a higher potential to select resistance than its ability to eradicate the test organisms. In addition, meropenem selected resistance not only to itself, but also to the early generation ß-lactam antibiotics, indicating that meropenem is a good inducer of ß-lactamase. However, meropenem did not readily select resistance to cefepime and cefpirome, despite its ability to select resistance to imipenem, suggesting that different resistance mechanisms are involved in the bacterial resistance to carbapenems and the fourth-generation cephalosporins.

A significant finding was that some meropenem-resistant strains remained susceptible to the early generation cephalosporins, including ceftazidime. Examples are the resistant strains 2434MER512X and 23355MER256X (Table I). Such a resistance profile is characteristic of strains that produce the Imi-I or Nmc-A type carbapenemase previously found in E. cloacae.17 Further characterization of these strains is necessary to determine the nature of the carbapenem-resistant phenotype as resistance to carbapenems remains rare among clinical isolates.

The MIC used as an endpoint in the present study will affect interpretation of the study. For example, the highest resistance level selected by meropenem was only 16 mg/L, as compared with >128 mg/L for the cephalosporins. This indicates that even if two antibiotics select resistance at similar rates, the endpoint MIC may be different. Hence eradication of bacteria may still be achieved for the one with a lower endpoint by increasing the antibiotic concentration.

The third category included the fourth-generation cephalosporins, cefepime and cefpirome, the use of which may be more desirable than the earlier cephalosporins in that cross-resistance to these antibiotics rarely developed upon selection by other ß-lactams, including the carbapenems, although they inevitably selected their own resistance at higher drug concentrations. Overall, the potentials of the ß-lactam antibiotics to select their own resistance were found to be in the following order: cefamandole > ceftazidime {approx} ceftriaxone > cefoperazone > meropenem {approx} cefepime > imipenem. This order appears to correlate with the MICs of the test antibiotics against the selected resistant strains (Tables I and Table II). A previous study reported a similar high rate of development of resistance to ceftriaxone, followed by ceftazidime, cefpirome and cefepime. 11

In conclusion, as the level of antibiotic resistance continues to rise, antibiotics with high antimicrobial activity and low resistance selection potential, such as imipenem, should be used sparingly and their use should be closely monitored to minimize the development of resistance.


    Notes
 
* Corresponding author. Tel: +852-2609-7983; Fax: 852-2603-5295; E-mail: ronli{at}cuhk.edu.hk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 7 October 1997; accepted 28 July 1998