ANTIBIOTICS

Antibiotics are secondary metabolites produced by microorganisms that inhibit or kill a wide spectrum of other microorganisms.

Most of the useful ones are produced by molds and bacteria of the genus Streptomyces. Some antibiotic like substances are produced by Bacillus spp., and at least one, nisin, is produced by some strains of Lactococcus lactis. Three antibiotics have been investigated extensively as heat adjuncts for canned foods: Subtilin, Tylosin, and Nisin. 60 to 80 percent of all livestock and poultry will receive antibiotics in feed or water or by systemic injection at some time during their production lifespan.

Preservatives Ethyl formate Caprylic acid Sodium nitrite Dehydroacetic acid Nisin Sodium diacetate Ethylene/ propylene oxides SO2/ sulfites

Maximum Tolerance 15-120 ppm ² 120 ppm 65 ppm 1% 0.32% 700 ppm 200-300 ppm

Organisms Affected Yeasts and molds Molds Clostridia Insects Lactics, clostridia Molds Yeasts, molds,

Foods Dried fruits, nuts Cheese wraps Meat-curing preparations Pesticide on strawberries, Squash Certain pasteurized cheese spreads Bread vermin Fumigant for spices, nuts

Insects, microorganisms Molasses, dried fruits, wine making, lemon juice (not to be used in meats or other foods recognized as sources of thiamine) Yeasts and molds Yeasts and molds Bakery products, soft drinks, pickles, salad dressings Margarine, pickle relishes, apple cider, soft drinks, tomato ketchup, salad dressings Hard cheeses, figs, syrups, salad dressings, jellies, cakes Bread, cakes, some cheeses, rope inhibitor in bread dough

Parabens Benzoic acid/ benzoates

0.1 %f 0.1 %

Sorbic acid/ sorbates Propionic acid/ propionates

0.2% 0.32%

Molds Molds

THE ANTIBIOTIC ISSUE ‡ Antibiotics are used in food animals for
± treatment or prevention of disease ± for increased production performance or increased efficiency of use of feed consumed by the animal for growth, product output, or ± modifying the nutrient composition of an animal product ± Enhance their growth and production performance first demonstrated for poultry. ± fighting subclinical diseases and bolstering health defense processes

‡ Various nutritional studies in chicks showed that antibiotic-fermentation products influenced the growth of chicks. ‡ By 1951, the addition of growth-promoting antibiotics to feed had become standard practice.

Some 15 considerations on the use of antibiotics as food preservatives were noted by Ingram et al., and several of the key ones are summarized as follows: The antibiotic agent should kill, not inhibit, the flora and should ideally be destroyed on cooking for products that require cooking. The antibiotic should not be inactivated by food components or products of microbial metabolism. The antibiotic should not readily stimulate the appearance of resistant strains. The antibiotic should not be used in foods if used therapeutically or as an animal feed additive.

Nisin 
Nisin was discovered in 1928 by Rogers and his workgroup.  Nisin is a lantibiotic (contains the rare amino acids, meso-lanthionine and 3-methyl-lanthionine instead of cysteine and methionine), it is a bacteriocin.  Nisin has approved bactericidal effect against most LAB; S. aureus; L. monocytogenes, Bacillus and Clostridium vegetative forms.  Nisin ihibits synthesis of peptidoglycan: Inhibition of cell wall synthesis
Nisin E234 Nisaplin (Danisco)
GRAS status 1969 WHO and FDA 1983 EU 1988 US Food and Drug Agency 

The first food use of nisin was by Hurst to prevent the spoilage of Swiss cheese by Clostridium butyricum.  Most widely used compound for food preservation, with around 50 countries.  Approved in 1988 for food use in the United States.  The compound is effective against gram-positive bacteria, primarily spore formers, and is ineffective against fungi and gram-negative bacteria. Among some of its desirable properties as a food preservative are the following: It is nontoxic. It is produced naturally by Lactococcus lactis strains. It is heat stable and has excellent storage stability. It is destroyed by digestive enzymes.  Nisin as a heat adjunct in canned foods or as an inhibitor of heat-shocked spores of Bacillus and Clostridium strains. Nisin, however, is used most widely in cheeses.  Usable levels are in the range of about 2.5-100 ppm. Nisin is most often employed in dairy products processed cheeses, condensed milk, pasteurized milk, and so on. Some countries permit its use in processed tomato products and canned fruits and vegetables. It is most stable in acidic foods.

Monensin
‡ Monensin, isolated from Streptomyces cinnamonensis. ‡ First described by Agtarap et al. in 1967. ‡ This antibiotic was approved by the FDA as a cattle feed additive in the 1970s, and it is used primarily to improve feed efficiency in ruminants. ‡ It inhibits gram-positive bacteria. ‡ Destroys selective permeability of cell membranes. ‡ Monensin is used extensively in the beef and dairy industries

Natamycin
‡ It is a polyene antibiotic that is quite effective against yeasts and molds but not bacteria, i.e, employed as a food fungistat. ‡ Natamycin, the name, as it was isolated from Streptomyces natalensis. ‡ Binds to ergosterol, a major component of the fungal cell membrane. When present in sufficient concentrations, it forms pores in the membrane that lead to K+ leakage and death of the fungus. Ergosterol is fairly unique to fungi, so the drug does not have such catastrophic effects on animals. ‡ Its use is limited as a clinical agent, and it is not used as a feed additive. ‡ To control fungi on strawberries and raspberries, natamycin was compared with rimocidin and nystatin, and it, along with rimocidin, was effective at levels of 10-20 ppm, whereas 50 ppm of nystatin were required for effectiveness.

Tetracyclines
‡ Tetracyclines are both heat sensitive and storage labile in foods. ‡ Chlortetracycline (CTC) and oxytetracycline (OTC) were approved by the FDA in 1955 and 1956, respectively, at a level of 7 ppm to control bacterial spoilage in uncooked refrigerated poultry. ‡ The efficacy of this group of antibiotics in extending the shelf life of refrigerated foods was first established by Tarr and associates working with fish in Canada. ‡ Delay bacterial spoilage of fish and seafoods, poultry, red meats, vegetables, raw milk, and other foods. ‡ CTC is generally generally more effective than OTC. ‡ When CTC is combined with sorbate, a natural organic compound to delay the spoilage of fish, the combination has been shown to be effective for up to 14 days. ‡ Used to treat diseases in animals and are also used in feed supplements.

Effects of Antibiotics on the Shelflife of Prepeeled Potatoes at Room Temperature E. A. M. ASSELBERGS, W. E. FERGUSON, AND W. P. MOHR, Plant Research Institute, Research Branch, Canada Department of Agriculture, Ottawa,Canada AND K. F. MACQUEEN Commercial Products Division, Atomic Energy of Canada, Ltd., Ottawa, Canada

‡ The quality of commercially prepeeled potatoes deteriorates rapidly during nonrefrigerated transportation and storage. ‡ A major factor in this deterioration is the development of microorganisms. ‡ Ceponis and Friedman have shown that the shelflife of prepeeled potatoes at room temperature is less than 24 hr. ‡ Similar results were reported by Feustel and Harrington. ‡ The application of antibiotics in prepacked vegetables has been reported to delay bacterial spoilage by Carroll and co-workers. ‡ Recently, Francis et al. reported that the addition of oxytetracycline and chlortetracycline to a sodium bisulphite dipping solution increased the shelflife of prepeeled packaged potatoes.

Subtilin 
Discovered and developed by scientists at the Western Regional Laboratory of the USDA  Its properties were described by Dimick et al.  Produced by some strains of Bacillus subtilis.  Effective against gram-positive bacteria.  Subtilin is effective in canned foods at levels of 5-20 ppm in preventing the outgrowth of germinating endospores.  Used as a food preservative as it reduces the thermal resistance of bacterial spores and so permits a reduction in the processing time.

Tylosin
‡ This antibiotic is a nonpolyene macrolide, as are the clinically useful antibiotics erythromycin, oleandomycin, and others. ‡ It is more inhibitory than nisin or subtilin. ‡ Denny et al. were apparently the first to study its possible use in canned foods. ‡ Used in animal feeds and also to treat some diseases of poultry. ‡ As a growth promotant in food producing animals. ‡ Used as a treatment of colitis, an inflammation of colon, in small animals. ‡ Most effective against gram-positive bacteria. ‡ It inhibits protein synthesis by associating with the 50S ribosomal subunit.

Fluoroquinolones in poultry luoroquinolones
± Enrofloxacin ± Sarafloxacin
Quinolones inhibit the bacterial DNA gyrase enzyme, thereby inhibiting DNA replication and transcription. Campylobacter Resistance to Fluoroquinolones Increasing ‡ Most common food-borne infection in US ‡ 2.5 million case of diarrhea and 100 deaths per year ‡ 13% in 1998, 18% in 1999 ‡ Fluoroquinolone use up 40% over same period  Oct. 2000: FDA proposed to ban fluoroquinolones for therapeutic use in poultry 
Major poultry producers -- Perdue, Tyson Foods, Foster Farms, ConAgra, Gold Kist, Claxton, and Wayne Farms say they have reduced or eliminated routine uses of medically important antibiotics or the use of fluoroquinolones in sick birds

Nitrofurans, particularly furazolidone (FZD), furaltadone (FTD), nitrofurantoin (NFT) and nitrofurazone (NFZ), belong to a class of synthetic broad spectrum antibiotics . Nitrofurans were commonly employed as feed additives for growth promotion, and mainly used for livestock (i.e. poultry, swine and cattle), aquaculture (i.e. fish and shrimp) and bee colonies in the prophylactic and therapeutic treatment of bacterial and protozoan infections such as gastrointestinal enteritis caused by Escherichia coli and Salmonella spp., fowl cholera and coccidiosis black heads. Furazolidone was broadly used in European countries as an effective veterinary antibiotic, especially in pig husbandry. In 1995, the use of nitrofurans for livestock production was completely prohibited in the EU due to concerns about the carcinogenicity of the drug residues and their potential harmful effects on human health. The use of nitrofurans for livestock has also been prohibited in countries such as Australia, USA, Philippines, Thailand and Brazil.

Medically important antibiotics used as non-therapeutic feed additives
Macrolides Penicillins Tetracyclines Streptogramins Aminoglycosides Lincomycin Sulfonamides Bacitracin

Rx

Where is the danger?
40% of the antibiotics used each year in the United States are for animals

Heavy use of antibiotics in factory farming of animals has caused bacteria to develop antibiotic resistant strains. Campylobacter bacteria ± the most common cause of food-borne illness in the US ± increased its drug resistance from 0% in 1991 to 20% in 1999. The rate at which microorganisms in food-animal populations become resistant to antibiotics is slow because of the short lifespans and high turnover of these animal populations (Walton 1986).

Consequences of Agricultural Antibiotic Use
‡ Campylobacter fluoroquinolone resistance ‡ VREF (due to avoparcin use in chickens) ‡ MRSA in pork, chickens ‡ Gentamycin- and Cipro-resistant E. coli in chickens

RESISTANCE TO ANTIBIOTIC DRUGS
‡ Use of drugs in food animals - Microorganisms can mutate to develop or acquire resistance to antibiotic drugs. ‡ Is the microorganism zoonotic; that is, can it cause a human disease by moving from the animal to a human, more virulent, treatable with other antibiotics? ‡ Passage of antibiotic-resistant bacteria from animals to humans occurs from
± direct contact with animals or their manure, ± through indirect exposure to food contaminated with animal-derived bacteria, ± from person-to-person contact after a primary exposure of nonfarm persons.

‡ The resistance of microorganisms arising from subtherapeutic use of penicillin, tetracyclines, and sulfa drugs in agriculture is suggested by WHO (WHO 1997) to be a high- priority issue. ‡ World Health Organization in 1997- The use of any antimicrobial agent for growth promotion in animals should be terminated if it is used in human therapeutics or known to select for cross-resistance to antimicrobials used in human medicine. ‡ zoonotic pathogens:
± Salmonella DT-104 and ± Campylobacter jejuni

‡ Vancomycin and Ciprofloxacin resistance is linked to antibiotic use in animals. ‡ In Europe there is strong evidence that one type of the vancomycin-resistant enterococci (VRE - vanA) developed in animals fed an antibiotic called avoparcin (a glycopeptide or vancomycin-like antibiotic). ‡ Vancomycin-resistance genes can spread from VRE to bacteria that are much more common and aggressive such as the multi-resistant strains of Staphylococcus aureus (MRSA). ‡ Use of enrofloxacin has resulted in the development of ciprofloxacin-resistant strains of Salmonella spp and Campylobacter spp. These resistant bacteria have subsequently caused human infections. ‡ When the glycopeptide, avoparcin, was used as a growth promoter in food animals in Europe this resulted in the development and amplification of vancomycin resistant enterococcus (VRE) and subsequent colonisation by a significant percentage of the human population via the food chain (between 2 and 17%).

Antibiotic-Resistant Bacteria Have Been Transferred from Animals to Humans (statistical data)
‡ Epidemiologic studies suggest transference of antibiotic-resistant E. coli and campylobacter bacteria ‡ Examples: ± Chickens on farm fed tetracycline-supplemented feed and within 2 weeks 90% were excreting all tetracycline-resistant E. coli. Within 6 months, 7 of 11 people living on or near farm were excreting high numbers of resistant E coli. ± Percentage of Campylobacter jejuni resistant to quinolone in human isolates increased from 0.8% in 1996 to ~3% in 1998. FDA approved the use of fluoroquinolones in 1995. ‡ Epidemiologic studies with molecular subtyping show transference of antibioticresistant campylobacter and salmonella bacteria ± Fluoroquinolone-resistant Campylobacter jejuni was isolated from 14% of 91 chicken products obtained from retail markets in 1997. ± In 1998, salmonella bacteria resistant to ceftriaxone were isolated from a 12year-old boy who lived on a cattle farm.

‡ Enrofloxacin is administered to flocks of poultry through their water to control mortality from E. coli and Pasteurella multocida organisms ‡ New evidence that enrofloxacin use in poultry has not been shown safe for humans ± FDA determined that the use of enrofloxacin in poultry causes the development of a fluoroquinolone-resistant strain of campylobacter in poultry, which, when transferred to humans, is a significant cause of fluoroquinolone-resistant campylobacter infections in humans
The use of antibiotics in livestock production for increased feed efficiency is widespread. Such use may indirectly access human food in the form of residuals in products such as meat and milk. For years, scientists and health officials have warned of the risk of developing resistant pathogens from feeding antibiotics to livestock. Recently, this concern was fueled by a fatal case of salmonellosis caused by an antibiotic resistant strain linked to meat.

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Use of antibiotics in the treatment of mastitis has created problems for the milk processor and consumer. Following treatment of mastitis with antibiotics, they may be found in the milk in sufficient concentrations to inhibit dairy starter microorganisms and cause economic losses to the cheese and fermented milk industries. Penicillin in very small concentrations found in milk may cause reactions in highly sensitive individuals. When antibiotics are used to treat mastitis, dairymen should follow the prescribed recommendations for withholding milk for human use following treatment. When adulterated milk leaves the farm, it is subjected to various processes in the milk plant. Antibiotics in milk are relatively stable to pasteurization temperatures and above, as well as to low temperatures (0 10° F.). Under refrigeration temperatures up to seven days of storage, in raw and pasteurized milk there tends to be a loss in antibiotic activity. The presence of antibiotics in milk constitutes an adulteration under the Federal Food, Drug and Cosmetic Act.

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Triphenyltetrazolium Chloride as a Test for Antibiotic Substances in Milk
Summary
‡ A simple test for detection of inhibitory substances in milk is outlined.
± The test is based on the conversion of 2,3,5-triphenyltetrazolium chloride (TTC) to 1,3,5-triphenylformazan. ± The reaction is characterized by a color change from the white to red in the presence of growing bacterial cells. ± This conversion is inhibited by antibiotics at low levels of concentration. ± The total time for the test is 2½ hours.

Findings on antibiotic resistance for food animals are as follows: ‡ Increases the risk of emergence of microorganisms that are resistant to specific, and perhaps other, antibiotics. ‡ Resistance emergence should be classified with regard to each antibiotic used, the concentration and dosage administered, the blood and tissue concentrations attained, the bacterial species or strain affected, and the animal species in which the drug is used. ‡ Zoonotic spread of pathogens to humans is very low. Postfarm food processing, storage, and improper handling and cooking are major contributors to the chain of events that allows the pathogen to contaminate the product, proliferate on or in the food, and attain the large numbers that cause disease.

‡ Three basic principles of antibiotic use were adopted in the agriculture sector to reduce or eliminate antibiotic resistance:
± Antibiotics that are critical or last-line for serious human infections should not be used in food production animals or agriculture. ± The use of antibiotics for prophylactic purposes in animals should be kept to a minimum. The use of methods (other than antibiotics) to prevent infections should be expanded and developed. ± Antibiotics should not be used as growth promoters.

REFERENCES:‡ James M. Jay, Modern Food Microbiology, Sixth Edition ‡ Foodborne Threats to Health: Policies, Practices, and Global Coordination ‡ Microorganisms in food, 2nd edition, Kluwer Academic / Plenum Publishers, New York, Boston, Dordrecht, London, Moscow ‡ The Use of Drugs in Food Animals: Benefits and Risks (1999), Board on Agriculture (BOA) ‡ Khem M. Shahani and , Paul J. Whalen, Department of Food Science and Technology, University of Nebraska, Lincoln, Agricultural Uses of Antibiotics ‡ Journal of dairy sciences ‡ International journal of food microbiology ‡ Journal of food microbiology ‡ Agricultural uses of antibiotics, ACS publications

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