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Bacitracin — Natural Peptide with Minimal Resistance Issues

by 5m Editor
13 January 2009, at 12:00am

A comprehensive overview about bacitracin and its advantages, including its efficacy against Clostridium perfringens. Dr Jerry Mathers, senior manager in the Government and Industry Affairs group at Alpharma Animal Health presented this paper at Alpharma's Swine Enteric Health Symposium 2008.

Bacitracin is produced by the bacterium Bacillus licheniformis. It was first described in 1945 when the producer Bacillus was isolated from a rapidly self-healing wound on a human patient. Following isolation and purification, it was commercialized for human and animal medicine.

Due to its early discovery, bacitracin was originally developed as an antibiotic drug, and companies therefore obtained human and animal approvals based on traditional FDA and European approval processes. With the current shift of focus to “natural and alternative” products since the 1990’s, however, many do not fully recognize that bacitracin shares most of their environmental and reduced antibiotic resistance benefits.

Bacitracin (active drug of BMD® and Albac®) is significantly different from other antibiotics in being: a) a “natural peptide” not an “organic chemical” b) made by an AAFCO-listed direct-fed microbe species often used in marketed animal probiotics; and c) manufactured using natural grains and salts as major ingredients in a controlled fermentation process, similar to probiotic- and enzyme-based products. Bacitracin products retain certain advantages over newer products in that: d) they have proven positive effects on animal growth; and e) control bacterial disease for which it has approvals, both alone and in combination.

As a result, BMD and Albac have legitimate, multi-study-based claims for controlling animal pathogens and enteric disease syndromes, in addition to their well-known growth promotion impacts. Indeed they are usually included as the gold standard “positive control” in studies aimed at showing positive effects for alternatives versus Clostridium perfringens, or else are routinely included in such studies aimed at showing growth promotion benefits.

As a peptide + salt molecule, bacitracin products degrade rapidly in composts and on soils to amino acids, thus have no important environmental concern. It is considered a narrow spectrum antimicrobial with no cross resistance or transferable resistances. Other than being a minor component of topical OTC drugs such as ‘triple antibiotic ointment’ used for skin irritations, there is minimal to no concern about acquired resistances somehow impacting human chemotherapies due to foodborne routes.

If we think of bacitracin as a “natural peptide” as well as its formal classification as an antibiotic, we can more fully appreciate the role of BMD and Albac products as sustainable, economically important feed and water additives that can actually benefit animal and human health.

Chemistry and Mechanisms of Activity

Bacitracin A is the major component of commercial bacitracin (Figure 1). It is usually complexed with zinc, as is the case for Albac (or with methylene disalicylate for BMD) to stabilize the molecule during manufacturing. Only bacitracin zinc is banned in the European Union.


Figure 1. Chemical structure of bacitracin.

Bacitracin methylene disalicylate is a spray dried, blended fermentation product produced by the selected industrial microorganism Bacillus licheniformis. The strain was originally selected as a specific maker of the peptide antibiotic bacitracin in the 1940s, and has since been maintained and propagated carefully in Oslo, Norway and in Chicago Heights, Illinois, USA. The technology and process for manufacturing bacitracin is well defined and controlled according to Good Manufacturing Practice (GMP), and regulated by the U.S. Food and Drug Administration (Figure 2).


Figure 2. Flow chart of cGMP manufacturing of BMD.

The biosynthesis of bacitracin was found to be a non-ribosomal synthesis involving a specific synthetase enzyme complex, and thio-activated amino acids (Frøyshøv, 1977). The relative safety of industrial uses of B. licheniformis was reviewed by deBoer, et al. (1994). Along with closely related B. subtilis, B. licheniformis is not classified an invasive or pathogenic microorganism. It is included in the AAFCO manual as a Generally Recognized as Safe (GRAS) microbe which may be used as a live direct-fed microbial (DFM) for animals.

Considerable experience concerning the industrial use of B. licheniformis has accumulated over several decades, and authorities in the United States, Europe, and Japan have approved production with and products from B. licheniformis producer strains. Bacillus licheniformis is thus a safe host for producing safe and useful bioproducts.

Bacitracin is in the class of compounds known as peptides, most of which are produced by the genus Bacillus. Polymyxin, colistin, tyrocidine, and gramicidin are examples of other peptides produced in a manner similar to bacitracin (Katz and Demain, 1977). Other related peptides include the food grade preservative nisin, produced by lactic acid bacteria fermentation (Streptococcus lactis) and routinely used for commercial cheese and vegetable preservation. Most of these peptide compounds are active against gram-positive microorganisms, thus BMD is an excellent growth promoting compound due to its narrow spectrum profile and retention in the gut, allowing continual feeding up to slaughter. No known cases of any raw material causing a toxin or toxic byproduct have ever been reported in over 5 decades of commercial manufacturing of bacitracin biomass-based feed products. Table 1 lists comparable ‘biomass’ fermentation products used in human and animal sectors.

Bacitracin – Active Against Target Pathogens

Bacitracin methylene disalicylate (BMD) and zinc bacitracin (Albac) are both approved for performance enhancement in swine while BMD is also approved for control of clostridial enteritis caused by Clostridium perfringens. Table 2 summarizes relevant published MIC values for bacitracin and other drugs over several decades. Data show that overall, C. perfringens remain sensitive to bacitracin (Figure 3).

Swine-derived strains uniformly have very low MIC90 values versus bacitracin. In contrast, other anaerobic species have variable responses to bacitracin, confirming the narrow spectrum profile of the drug. Table 3 summarizes MIC values of diverse human-origin anaerobes versus bacitracin. Many strains have either sensitivity or intrinsic tolerance to the drug as measured by in vitro testing. In summary, target pathogen (C. perfringens, S. pyogenes) sensitivities have remained low and have not significantly changed in over 50 years.

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Figure 3. Sub-MIC (0.5 ppm) bacitracin lesions on C. perfringens ATCC 13124.

Recent publications continue to find additional, favourable modes of activity for bacitracin. Weidou et al. (2007) recently found that C. perfringens alpha-toxin expression was suppressed at the transcription level. This is a new finding in addition to the known suppression of C. perfringens populations in the gut.

Bacitracin – Active in Combination with Other Antimicrobials

Chlortetracycline (CTC, Aureomycin®) and the combination of BMD plus Aureomycin is also approved for performance enhancement, control of ileitis caused by Lawsonia intracellularis, and treatment of bacterial enteritis and bacterial pneumonia. A synergistic effect of CTC+bacitracin was reported for C. perfringens, as summarized in Table 4 and Figure 4.

That study showed that 72 per cent of C. perfringens strains had an additive or synergistic response to BMD+CTC combinations in vitro. As Table 3 indicates, C. difficile has overall higher MIC values toward bacitracin compared to C. perfringens. The test data from an Alpharma study on swine strains showed however, that bacitracin in combination with CTC, could dramatically reduce the MIC of a majority of swine C. difficile strains toward CTC (Figure 5). Furthermore, about 20 per cent of the tested C. difficile were inhibited by 275 ppm or less bacitracin (data not shown).



Figure 4. Sub-MIC bacitracin+CTC lesions on C. perfringens ATCC 13124.

Another example of favourable activity of bacitracin in combination with other drugs was described by Sieradzki and Tomasz (1997), who showed that exposure of Staphylococcus aureus to early cell wall inhibitors (drugs which inhibit early stages of cell wall assembly in gram-positive microorganisms) caused significant reductions of the methicillin resistance present in the test strain. For bacitracin, the methicillin MIC of the test strain was reduced by 16-fold (from 800 mg/L to 25-50 mg/L), comparable to vancomycin and other drugs that block early-stage wall synthesis. This result when combined with other observations shows that bacitracin can in many cases synergize the activity of other drugs, or at least is neutral in not being antagonistic or associated with any coresistance toward other drugs if used at sub-MIC or subtherapeutic levels, both alone or in combination.

Bacitracin – No Cross Resistance to Other Drugs

There is no cross resistance with human medicinal antibiotics or any other antibiotics available in the market. Table 5 summarizes several studies that evaluated bacitracin for cross resistance.

Literature reveals no increase in bacitracin resistance during its more than 50 years of use as a feed additive, neither in Clostridium perfringens, staphylococci nor streptococci. In enterococci there is no cross resistance between bacitracin and the fluoroquinolone ciprofloxacin, two new macrolides (azithromycin and clarithromycin), and second- and third-generation cephalosporins (cefuroxime and ceftriaxone), according to Kahn and Mahnin, 1998 (Alpharma communication). A published study of triple antibiotic ointment actives further showed no important changes in the resistance of Staphylococcus aureus and coagulase-negative staphylococci towards bacitracin, neomycin, or polymyxin after decades of OTC human use of this commonly used product (Jones et al., 2006).




Figure 5. Reduced C. difficile MIC values towards CTC due to added bacitracin. Study involved 41 pig-derived strains and agar dilution testing with CTC alone and with BMD at two concentrations.

Thibodeau et al. (2008) evaluated the effect of subtherapeutically- fed bacitracin and virginiamycin in broiler chickens on the resistance levels for multiple drugs. After 34 days, Escherichia coli from bacitracin-fed populations had no increases in any resistance frequency, and significant declines in ampicillin, amoxicillin- clavulanic acid, and cefa - lothin resistance frequencies in comparison to controls. This study is consistent with earlier observations made by Walton et al. on bacitracin-fed swine and poultry E. coli. Among Enterococcus spp., the Thibodeau study showed no important differences in the resistance patterns for antibiotic-fed in comparison to antibiotic- free controls.

Other, secondary biological effects such as reduced gut inflammation levels, increasing vitamin and nutrient uptake, prevention of toxin formation or virulence factors, increasing phagocytosis, temperature and stress tolerance, maintaining respiratory cilia activity, reduced boar taint, and others have been described in the literature for bacitracin and other drugs. While such mechanisms are not directly provable in all field studies, they provide plausible hypotheses for empirical observations known to occur.

Bacitracin – Studies on Gram-Negative Effects Including Resistance Plasmid Transfer

There is no indication that plasmids play any role in the transfer of resistance to bacitracin itself. Bacitracin resistance has not been shown to be transferable on plasmids or other mobile genetic elements (Threlfall, 1985, and Sanchis-Bayarri et al., 1981). Walton et al. (1980) studied the effect of zinc bacitracin in the diet on antibiotic resistance and R factor transmissibility in enteric strains of E. coli in pigs. Over the duration of the study, zinc bacitracin reduced the prevalence of tetracycline resistance and by the end of the observation period all plasmid transfer had been interrupted. Walton and Wheeler had found that bacitracin administration was associated with reduced E. coli resistance frequency to tetracyclines in swine over an 11- week period (Table 6). Walton and Bird had earlier hypothesized that cell wall lesions observed in E. coli grown in the presence of bacitracin may be one mechanism that explains growth enhancement and other effects.

Walton (1984) obtained similar results in a study examining the effects of dietary zinc bacitracin (100 ppm) on the resistance status of intestinal E. coli and enterococci from broiler chickens. Decreased R plasmid transfer, especially that for ampicillin resistance, was observed in E. coli together with a decrease in resistance prevalence in enterococci.

Recent research has also shown that some antimicrobials may actually inhibit the transfer of resistance at higher concentrations, including bacitracin (Figure 6). Mathers et al. (2004) showed that bacitracin, chlortetracycline, laidlomycin, lasalocid, and salinomycin inhibited the transfer of multiresistance- conferring plasmid pBR325 into selected gram-negative strains in an in vitro transformation model.


Figure 6. Effect of increasing bacitracin concentrations on multiresistant plasmid DNA transfer frequencies in E. coli HB101 versus untreated controls.

According to Page et al. (2003) the benefits of bacitracin against gram negative bacteria are:

  • adversely affecting the cell wall of some gram negative bacteria making them more susceptible to the antimicrobial action of other antibiotics;
  • treatment of pigs and chickens resulting in decreased numbers of E. coli in the gut resistant to tetracyclines and ampicillin;
  • reducing transfer of resistance plasmids among E. coli;
  • reducing the prevalence of Salmonella in challenged birds.

In particular, bacitracin in pigs:

  • improved the growth rate and feed conversion efficiency of treated pigs
  • the treatment of pregnant sows decreased the incidence and severity of clostridial enteritis in their piglets
  • treated sows lost less weight and weaned greater numbers of heavier piglets
  • controlled proliferative enteropathy (PE) in growing-finishing pigs
  • reduced boar taint in entire male pigs.

Environmental Benefits of Bacitracin

An additional advantage to the use of zinc bacitracin is the reduction in the amount of feces and increased nitrogen retention in broilers (Huyghebaert and de Groote, 1997) and swine. Such reductions can significantly reduce the environmental impacts of facility effluents. McOrist (1998) noted that the use of low levels of oral antibiotics in livestock also have wider environmental benefits related to their effect on absorption and excretion of dietary components. It has been estimated that the removal of oral antibiotics from the European livestock diet would result in an extra 78,000 and 15,000 tonnes of nitrogen and phosphorus pollutants entering the environment each year (Frost, 1991; Avery, 1995).

According to Page (2003), a review of more than 2000 published studies confirmed the important role antibiotic growth promotants have in protecting the health of animals, improving the efficiency of food production and minimizing the environmental impacts of intensive and extensive livestock farming. Summarizing the environmental benefits:

  • the use of antibiotic growth promoters saves the equivalent of 30 million tonnes of feed through reduced methane emission;
  • for every one million pigs raised per year, environmental saving of around 221 tonnes of nitrate and 73 tonnes of phosphates are made;
  • conversion of feed to meat in chickens is improved by 3-5 per cent, saving up to 100,000 tonnes of feed per year;
  • decreased mortality of animals through control of disease;
  • grazing management of sheep and cattle can be dramatically improved.

Bacitracin has a half life of 2-4 days in swine effluents depending on conditions, and does not cause disruption of plants, fish, protozoans or composts. Bacitracin degrades to natural amino acids and salts, thus it poses no important impact on environments (Alpharma Environmental Submission, 1989).

Bacitracin and Human Health

Bacitracin was found to increase intestinal tensile strength, thereby reducing Salmonella contamination by 1-5 per cent in poultry (Duquette, 1999). Similar impacts are highly likely in bacitracin-fed swine. The reduction of inflammation in the gut results in stronger intestines that are less likely to rupture during processing. The overall reduction in the number of fecal-contaminated carcasses entering the food chain also contributes to a reduced risk of spreading resistant zoonotic bacteria from animals to humans via the food chain. Careful monitoring procedures have been in place by the USDA, FDA, and the industry to track the prevalence, types, and resistance patterns of food borne Salmonella spp., pathogenic E. coli and Campylobacter jejuni since 1996 (National Antimicrobial Resistance Monitoring System, or NARMS).

Although bacitracin has been extensively tested for use as a therapeutic agent in man, it has never had a major human therapeutic use. Due to its narrow gram-positive spectrum of activity, coupled with its nephrotoxicity when used systemically, bacitracin use in man has been limited to non-systemic applications, mainly topical, and usually in combination with other (anti-gram-negative) antibiotics in order to broaden the spectrum of activity of the topical ointment. Better alternatives have existed for systemic bacitracin human uses for several decades, and there is little evidence bacitracin would ever be re-introduced for such purposes in the future due to its nephrotoxicity (Phillips, 1999). The human use of bacitracin is very minor when compared to the animal use. Less than one per cent of the total bacitracin produced around the world is used in humans. This small share has shown a downward trend and is expected to reduce still further.

The FDA Guidance for Industry #152 (Oct. 2003) recently listed several “Critically Important Antimicrobials” for which human consequences are deemed Important, Highly Important, and “Critically” Important. Bacitracin is notable by its omission from this list.

A recent WHO/FAO/OIE expert meeting paper similarly lists bacitracin as a lower-priority agent, among a comprehensive list of all antimicrobial drugs and their potential impact on food- and potentially animal-origin bacteria.

Adverse Effects of the Ban on Antibiotic Growth Promoters

The potentially adverse effects of bans are often ignored, and the role of antibiotic growth promotants in the suppression of disease is underestimated (Phillips et al. 1999, 2004). Restrictions on the use of certain antibiotics can cause significant costs to animals. In Sweden, rates of post-weaning scours increased following restrictions of antimicrobial growth promotants (Wierup, 2001; Inborr, 1996). The year following the ban of oral antibiotics in 1986 in Sweden there were an estimated 50,000 pig deaths directly due to the removal of antibiotics. After more than a decade, despite alternative measures, “losses in production parameters…have not fully recovered on a national basis” (Wierup, 2001). In contrast no published studies have clearly indicated any particular benefits to the Swedish public of the antibiotic ban (McOrist, 1998). Similar problems were experienced in other parts of Europe which led to an increased use of therapeutic antibiotics.

In their review of published data, Phillips et al. (2004) noted that the one potentially highly undesirable effect of the growth promoter ban has been the concomitant increase in the use of therapeutic antibiotics. The case of bacitracin is of particular interest. It was predicted that if the agent were with drawn as a growth promoter, clostridial necrotic enteritis problems would emerge as a problem requiring therapy. This proved to be the case contributing to the general increases in the therapeutic use of antibiotics in animals.

Furthermore, the DANMAP report of 2001 (Bager et al., 2002) indicated no decrease in resistance to bacitracin among E. faecium since the ban. The effect of the EU ban on bacitracin can thus be considered entirely undesirable. A recent article, The European ban on growth-promoting antibiotics and emerging consequences for human and animal health by Casewell et al. 2003 also reveals that the European Union’s precautionary ban of growth-promoting antibiotics has had adverse health consequences to food animals with no diminution in the prevalence of resistant enterococcal infection in humans.

Conclusions

Bacitracin-based products have been in use for more than 50 years and are approved by authorities in numerous countries. No serious adverse effects from the use of zinc bacitracin or bacitracin methylene disalicylate have ever been reported. Baci - tracin is important for the prevention of some serious diseases in pigs and poultry. Necrotic enteritis is a common and serious disease in food producing animals, and among the choices of antibiotic or alternative products, bacitracin remains highly effective in addition to being safe and having the least risk of transfer of resistance to clinically useful (in humans) antibiotics.

Transfer of resistance, which is the greatest contemporary concern for the use of antibiotics, has not been shown with bacitracin use. To the contrary, research has shown that use of bacitracin can actually reduce the number of resistant bacteria, resistant bacteria may become more susceptible (less resistant) to other antibiotics, and bacitracin can interfere with some plasmid transfer mechanisms.

Bacteria from the intestinal flora of animals or humans test as sensitive today as they did several decades ago. The EU ban was imposed so that bacitracin could be reserved for potential human use against vancomycin-resistant enterococci (VRE) or enteric colitis. This does not seem likely to ever happen since the nephrotoxicity of bacitracin means it will almost certainly never be used as a systemic drug in the future, and other human drugs now target human VRE or C. difficile in human medicine.

Alpharma supports a scientific risk/benefit analysis process to determine whether an authorized antimicrobial drug product for animals presents an unacceptable risk to human health due to selection of resistant bacteria. Such a risk/benefit analysis would demonstrate that the importance of BMD and Albac in animal health far outweighs the virtually zero risk of adverse antimicrobial resistance in humans. With numerous approvals, legitimate claims, and a vast documented history of benefits to livestock and poultry, bacitracin-based products represent effectiveness and safety even in comparison to newer (and less well documented or safety-proven) feed additives marketed as “antibiotic alternatives” to growth promoters.

Summary – Bacitracin

  • Naturally fermented peptide
  • Produced by probiotic-class microbe (Bacillus licheniformis)
  • Narrow spectrum of activity (gram positive)
  • Prevents and controls C. perfringens related intestinal disease (NE)
  • Inhibits C. perfringens toxin formation.
  • Promotes growth, feed efficiency, daily gain rate (=AGP!)
  • Compatible with alternative products
  • No cross-resistance or co-resistance to other drugs
  • Biodegradable
  • Positive impact on environment
  • Many combinations approved
  • Has sub-MIC and synergistic effects on pathogens
  • Has favorable secondary effects
  • Safe for all approved animal and human uses

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January 2009