1 Department of Microbiology, Cornell University, Wing Hall, and 2 Agricultural Research Service, USDA, Ithaca, NY 14853, USA
Received 28 April 2003, returned 9 June 2003, revised 17 June 2003; accepted 2 July 2003
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Abstract |
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Methods: Ionophore-adapted and non-adapted cultures were assayed for their susceptibility to other classes of antibiotics (penicillin G, ampicillin, cephalosporin C, vancomycin, carbenicillin, tetracycline, chloramphenicol, erythromycin, streptomycin, linocomycin, rifampicin, trimethoprim, novobiocin, polymyxin B and bacitracin) using a broth microdilution method.
Results: Adapted cultures retained their resistance phenotype for at least 28 generations even if ionophore was no longer present. Monensin-adapted cultures were as resistant to lasalocid as those adapted to lasalocid, but lasalocid-adapted cultures lagged with 1 µM monensin. Monensin- and lasalocid-resistant C. aminophilum F cultures were as susceptible to most antibiotics as non-adapted cultures. The only antibiotic that seemed to have a common mechanism of resistance was bacitracin, and the ionophore-adapted cultures had a 32-fold greater MIC.
Conclusion: The use of ionophores in cattle feed and the selection of ionophore-resistant ruminal bacteria does not necessarily lead to other types of antibiotic resistance.
Keywords: antibiotics, ionophores, resistance, monensin, lasalocid
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Introduction |
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Ionophores have a distinctly different mode of action from therapeutic antibiotics,5 and can be placed into three main classes: carboxylic polyether ionophores, neutral ionophores and pore-forming ionophores.6 The polyether ionophores, which are used as feed additives for cattle, act as metalproton antiporters.5 By binding and shielding monovalent or divalent metal ions within a hydrophobic matrix, polyether ionophores can shuttle them across cell membranes.7 Monensin translocates monovalent metal ions (e.g. sodium and potassium), but lasalocid can also bind divalent cations (e.g. magnesium and calcium).7 The resulting electroneutral dissipation of ion and proton gradients leads to greater membrane ATPase and transporter activity. As a result, energy is diverted from growth to non-growth functions, and eventually susceptible cells are de-energized.4
Bacteria and mammalian enzymes can degrade ionophores, but these pathways are oxygen-dependent and not functional in anaerobic environments, such as the rumen or lower gastrointestinal tract.8 Early work with ruminal bacteria showed that Gram-negative species were often less susceptible to ionophores than Gram-positive bacteria,4 and this observation suggested that ionophore sensitivity and resistance are determined by the composition of the bacterial cell wall.
However, the cell wall model of ionophore resistance is not always straightforward. Some Gram-negative species need a period of adaptation before they can grow in the presence of ionophore.9,10 Conversely, some Gram-positive species are as resistant to ionophores as many Gram-negative bacteria.11,12 Clostridium aminophilum F is a Gram-positive, hyperammonia producing ruminal bacterium13,14 that contributes to wasteful ruminal amino acid degradation.15 C. aminophilum F can be inhibited by the ionophore monensin in vitro,13 but work with 16S rRNA probes showed that physiological doses of monensin do not eliminate this bacterium from the rumen.16 Recent work has demonstrated that C. aminophilum F cultures can be adapted with sublethal concentrations of monensin.17
The following experiments sought to: (1) define more precisely the ionophore resistance of C. aminophilum F, (2) determine if resistance to one ionophore confers cross-resistance to other ionophores, and (3) monitor the susceptibility of ionophore-resistant strains to other classes of antibiotics.
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Materials and methods |
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C. aminophilum strain F was grown anaerobically in a basal broth containing (per L): 292 mg K2HPO4, 292 mg KH2PO4, 480 mg (NH4)2SO4, 268 mg NH4Cl2, 480 mg NaCl, 100 mg MgSO4.7H2O, 64 mg CaCl2.2H2O, 600 mg cysteine hydrochloride, 4 g Na2CO3, 15 g Casaminoacids (Difco Laboratories, Detroit, MI, USA) and 15 g yeast extract (Difco). The culture broth was prepared under O2-free CO2 and dispensed into tubes (18 x 150 mm) that were sealed with butyl rubber stoppers. Cultures were maintained in basal broth and incubated at 39°C for 1248 h in a water bath. Ionophore-adapted cultures were kept in basal broth supplemented with 1 µM monensin or lasalocid. Samples were withdrawn from the culture tubes with a sterile hypodermic syringe, and growth was assessed from the increase in optical density (1 cm cuvette, 600 nm).
Cultures were also grown anaerobically in a glove box (Coy Laboratory Products, Ann Arbor, MI, USA). Agar plates (1.5% w/v) containing basal medium were spread with 100 µL of stationary phase culture that had been diluted 100 000 fold with sterile basal broth. The plates were incubated at 39°C for 48 h. Colonies were then transferred to basal medium with or without 1 µM monensin or lasalocid.
Growth with ionophore
Non-adapted C. aminophilum F cells were inoculated (1% v/v) into basal broth supplemented with either 1 µM monensin or lasalocid and incubated at 39°C for 48 h. Adapted cultures were then re-inoculated (1% v/v) into basal broth containing 1 µM monensin or lasalocid, and growth was monitored. Growth was assessed from the increase in optical density (1 cm cuvette, 600 nm).
Adapted and non-adapted cells were also inoculated (1% v/v) into basal broth supplemented with different amounts of monensin or lasalocid (0.6, 1, 2, 5 and 10 µM) and incubated at 39°C for 72 h. Growth was assessed from the increase in optical density (1 cm cuvette, 600 nm).
Enumeration
Stationary phase ionophore-adapted and non-adapted C. aminophilum F cultures were serially diluted (10-fold increments) into basal medium supplemented with 1 µM monensin or lasalocid to determine viable cell number. The dilution tubes were incubated at 39°C for 96 h, and growth was assessed from the increase in optical density (1 cm cuvette, 600 nm).
Antibiotics
All antibiotics were prepared anaerobically under O2-free N2 and added to bacterial cultures with a sterile hypodermic syringe. Monensin, lasalocid and erythromycin were dissolved in sterile 95% ethanol. Chloramphenicol, trimethoprim, rifampicin and tetracycline were dissolved in a 50:50 mixture of water and ethanol. Vancomycin, bacitracin, penicillin G, kanamycin, polymyxin B, cephalosporin C (a parent compound used to make other semisynthetic antibiotics), carbenicillin, lincomycin, streptomycin and ampicillin were dissolved in water. The antibiotic stock solutions were then sterile filtered (0.2 µm). The final concentration of ethanol in culture tubes was never >2% (v/v), and preliminary experiments indicated that this concentration of ethanol did not affect the growth of C. aminophilum F. All antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Susceptibility testing
Antibiotic susceptibility testing was performed using a broth microdilution method, adhering as closely as possible to the recommendation of the NCCLS.18 The test medium was basal broth, and final concentrations of the antibiotics were in the range 0.0041024 mg/L. The C. aminophilum F inoculum (100 µL, 5 x 105 cfu/mL) was added to 96-well microtitre plates containing antibiotics that had been diluted in two-fold increments. The plates were incubated anaerobically (35°C, 24 h), and the optical density of each well was determined (600 nm). The MIC was defined as the lowest concentration of antibiotic under which bacterial growth was no longer detectable.
Experimental design
All experimental determinations were performed in triplicate, and the mean, S.D. and coefficient of variation were computed. If the coefficient of variation was <10% and the difference among treatment means was large, statistics were not reported. When the data did not meet these criteria, statistical significance was assessed by the use of a Students t-test.19
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Results |
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Discussion |
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Monensin is the most commonly used ionophore in the cattle industry in the USA.4 When cattle were fed a typical dose of monensin (350 mg/day), the amount of monensin needed to cause half maximal potassium efflux from mixed ruminal bacteria increased from 0.18 to 1.5 µM, and this change occurred in less than 3 days.21 If cattle were fed 350 mg lasalocid per day, the amount needed to cause half maximal potassium efflux from mixed ruminal bacteria increased from 0.14 to 0.28 µM.21 These data suggest that the bacterial population of the rumen can adapt rapidly and become resilient to these growth-promoting antibiotics.
Previous work indicated that monensin-treated C. aminophilum F cultures had a long lag time before they could adapt and grow,17 and our experiments show that cultures treated with lasalocid had an even longer lag time. Because cultures treated with either 1 µM monensin or lasalocid did not lag a second time, it appeared that ionophore resistance was being selected. However, when individual colonies were picked from an agar plate lacking ionophore and transferred into broth containing ionophore 1 µM, growth was always observed. These results suggest that virtually any C. aminophilum F cell has the capacity to become resistant.
The NCCLS indicates that broth microdilution is the appropriate method for determining MIC for anaerobes, and we used this approach to assess the susceptibility of C. aminophilum F to other classes of antibiotics. However, culture conditions and pH, along with the ability of ionophores to interact with cell membranes, have a significant effect on their activity.22 As a result, ionophore activity may not be determined accurately using MIC.
The ionophore resistance of C. aminophilum F could be assessed via lag time, or via the amount of ionophore that inhibited growth completely. Adapted cultures did not lag and tolerated much more ionophore than non-adapted cultures. In Escherichia coli, adaptive responses are sometimes mediated by stationary-phase gene amplifications,23 but the ionophore resistance of C. aminophilum was not a stable adaptation. Other workers have also noted that resistance factors are not always stable, but the nature of this instability has not been defined clearly.
Chen & Wolin10 noted that Bacteroides (now Prevotella) ruminicola GA33 could become significantly more resistant to either monensin or lasalocid, but there was no cross resistance of monensin mutants to lasalocid and vice versa. Butaye et al.24 observed that enterococcal strains isolated from pigs and poultry fed ionophores exhibited cross resistance to salinomycin and narasin, but not monensin and lasalocid. Furthermore, resistance to monensin and lasalocid was not found. Our work shows that C. aminophilum F cultures adapted with monensin had an inherent increased resistance to lasalocid. However, growth of lasalocid-adapted cells lagged when they were treated with monensin. These results suggest that cross resistance is possible in C. aminophilum F, but the mechanism of resistance is, at least in some cases, ionophore-specific. Whether other classes of microorganisms (e.g. coccidia) have a similar strategy of resistance has yet to be determined.
Therapeutic antibiotics have a variety of targets within bacterial cells (peptidoglycan synthesis, ribosome activity, DNA replication, mRNA transcription, nucleotide synthesis and membrane stability),25 but ionophores have a distinctly different mechanism of action (ion translocation across the cell membrane).5 Because only some animals can be fed ionophores safely,8 this class of antibiotics never has been, and is unlikely to be, used as an antimicrobial for humans.5 Therapeutic antibiotic resistance often involves degradative enzymes, efflux pumps, altered target sites or extracellular polysaccharide (glycocalyx) that serve as a diffusion barrier.2628
The question then arises, would an ionophore-dependent increase in polysaccharide production lead to an increased resistance to other classes of antibiotics? Ionophores are highly hydrophobic molecules,5 and previous work showed that monensin-resistant C. aminophilum F cultures had increased extracellular polysaccharide and were not agglutinized by lysozyme, a positively charged protein.17 Based on these observations, we hypothesized initially that ionophore-resistant C. aminophilum F cultures would have increased resistance to therapeutic antibiotics, but there was little support for this hypothesis. The ionophore-resistant cultures were, in virtually all cases, as susceptible as non-adapted cultures. The only antibiotic with a greater MIC value was bacitracin, and work conducted with Gram-negative bacteria indicates that extracellular polysaccharides may play a role in bacitracin resistance.29
In 1999, the Scientific Steering Committee of the EU1 stated that: regarding the use of antimicrobials as growth promoting agents, the use of agents from classes which are or may be used in human or veterinary medicine (i.e. where there is a risk of selecting for cross resistance to drugs used to treat bacterial infections) should be phased out as soon as possible and ultimately abolished. However, there has been little evidence that ionophore resistance is becoming a problem. When Aarestrup et al.30 examined indicator bacteria, zoonotic bacteria and animal pathogens from Danish food animals, monensin resistance was not encountered frequently. Butaye et al.31 did not detect monensin-resistance in 146 Enterococcus faecium and 166 Enterococcus faecalis strains isolated from farm and pet animals in Europe. However, similar work has not been carried out in North America, where animals have had a greater potential exposure to ionophores. Results with C. aminophilum F are consistent with the idea that ionophore resistance is not necessarily associated with resistance to most other classes of antibiotics, but further work will be needed to confirm this idea.
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Acknowledgement |
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Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.
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Footnotes |
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References |
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