History of Microorganisms in Food

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Preface

The 7^thedition of Modern Food Microbiology, like previous editions, focuses on the general biology of the microorganisms that are found in foods. All but one of the 31 chapters have been extensively revised and updated. The new material in this edition includes over 80 new bacterial and 10 new genera of fungi. This title is suitable for use in a second or subsequent course in a microbiology curriculum, or as a primary food microbiology course in a food science or food technology curriculum. Although organic chemistry is a desirable prerequisite, it is not necessary for one to get a good grasp of most of the topics covered.

When used as a microbiology text, the following sequence may be used. A synopsis of the information in Chapter 1 will provide students with a sense of the historical developments that have shaped this discipline and how it continues to evolve. Memorization of the many dates and events is not recommended since much of this information is presented again in the respective chapters.

The material in Chapter 2 includes a synopsis of modern methods currently used to classify bacteria, taxonomic schemes for yeasts and molds, and brief information on the genera of bacteria and fungi encountered in foods. This material may be combined with the intrinsic and extrinsic parameters of growth in Chapter 3 as they exist in food products and as they affect the common foodborne organisms.

Chapters 4 to 9 deal with speciﬁc food products, and they may be covered to the extent desired with appropriate reviews of the relevant topics in Chapter 3. Chapters 10 to 12 cover methods for culturing and identifying foodborne organisms and/or their products, and these topics may be dealt with in this sequence or just before foodborne pathogens. The food protection methods in Chapters 13 to 19 include some information that goes beyond the usual scope of a second course, but the principles that underlie each of these methods should be covered.

Chapters 20 and 21 deal with food sanitation, indicator organisms, HACCP, and FSO systems; and coverage of these topics is suggested before dealing with the pathogens. Chapters 22 to 31 deal with the known (and suspected) foodborne pathogens including their biology and methods of control. Chapter 22 is intended to provide an overview of the chapters that follow. Some of it includes ways in which foodborne pathogens differ from nonpathogens, their behavior in biofilms, and some information on the known roles of sigma factors and quorum sensing among foodborne organisms. The other material in this chapter that deals with the mechanisms of pathogenesis is probably best dealt with when the specific pathogens are covered in their respective chapters. The new Appendix section presents a simplified scheme for grouping foodborne and some general environmental bacterial genera by use of Gram, oxidase, and calalase reactions along with colony pigmentation.

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vi Modern Food Microbiology

For most semester courses with a 3-credit lecture and accompanying 2 or 3 credit laboratory, only about 65-70% of the material in this text is likely to be covered. The remainder is meant for reference puiposes.

The following individuals assisted us by critiquing various parts or sections of this edition, and we extend special thanks to each: B. P. Hedlund, K. E. Kesterson, J. Q. Shen, and H. H. Wang. Those who assisted with the previous six editions are acknowledged in the respective editions.

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Chapter 1 History of Microorganisms in Food

Although it is extremely difﬁcult to pinpoint the precise beginning of human awareness of the presence and role of microorganisms in foods, the available evidence indicates that this knowledge preceded the establishment of bacteriology or microbiology as a science. The era prior to the establishment of bacteriology as a science may be designated the prescientiﬁc era. This era may be further divided into what has been called the food-gathering period and the food-producing period.

The former covers the time from human origin over 1 million years ago up to 8,000 years ago. During this period, humans were presumably carnivorous, with plant foods coming into their diet later in this period. It is also during this period that foods were ﬁrst cooked.

The food-producing period dates from about 8,000 to 10,000 years ago and, of course, includes the present time. It is presumed that the problems of spoilage and food poisoning were encountered early in this period. With the advent of prepared foods, the problems of disease transmission by foods and of faster spoilage caused by improper storage made their appearance. Spoilage of prepared foods apparently dates from around 6000 bc. The practice of making pottery was brought to Western Europe about 5000 bc from the Near East. The first boiler pots are thought to have originated in the Near East about 8,000 years ago.¹¹ The arts of cereal cookery, brewing, and food storage, were either started at about this time or stimulated by this new development.¹⁰ The first evidence of beer manufacture has been traced to ancient Babylonia as far back as 7000 bc.⁸ The Sumerians of about 3000 bc are believed to have been the first great livestock breeders and dairymen and were among the first to make butter. Salted meats, fish, fat, dried skins, wheat, and barley are also known to have been associated with this culture. Milk, butter, and cheese were used by the Egyptians as early as 3000 bc.

Between 3000 bc and 1200 bc, the Jews used salt from the Dead Sea in the preservation of various foods.²The Chinese and Greeks used salted fish in their diet, and the Greeks are credited with passing this practice on to the Romans, whose diet included pickled meats. Mummification and preservation of foods were related technologies that seem to have influenced each other’s development. Wines are known to have been prepared by the Assyrians by 3500 bc. Fermented sausages were prepared and consumed by the ancient Babylonians and the people of ancient China as far back as 1500 bc.⁸

Another method of food preservation that apparently arose during this time was the use of oils such as olive and sesame. Jensen⁶ has pointed out that the use of oils leads to high incidences of staphylococcal food poisoning. The Romans excelled in the preservation of meats other than beef by around 1000 bc and are known to have used snow to pack prawns and other perishables, according to Seneca. The practice of smoking meats as a form of preservation is presumed to have emerged sometime during this period, as did the making of cheese and wines. It is doubtful whether people

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4 Modern Food Microbiology

at this time understood the nature of these newly found preservation techniques. It is also doubtful whether the role of foods in the transmission of disease or the danger of eating meat from infected animals was recognized.

Few advances were apparently made toward understanding the nature of food poisoning and food spoilage between the time of the birth of Christ and ad 1100. Ergot poisoning (caused by Claviceps purpurea, a fungus that grows on rye and other grains) caused many deaths during the Middle Ages.

Over 40,000 deaths due to ergot poisoning were recorded in France alone in ad 943, but it was not known that the toxin of this disease was produced by a fungus.¹²Meat butchers are mentioned for the ﬁrst time in 1156, and by 1248 the Swiss were concerned with marketable and nonmarketable meats.

In 1276, a compulsory slaughter and inspection order was issued for public abattoirs in Augsburg.

Although people were aware of quality attributes in meats by the thirteenth century, it is doubtful whether there was any knowledge of the causal relationship between meat quality and microorganisms.

Perhaps the ﬁrst person to suggest the role of microorganisms in spoiling foods was A. Kircher, a monk, who as early as 1658 examined decaying bodies, meat, milk, and other substances and saw what he referred to as “worms” invisible to the naked eye. Kircher’s descriptions lacked precision, however, and his observations did not receive wide acceptance. In 1765, L. Spallanzani showed that beef broth that had been boiled for an hour and sealed remained sterile and did not spoil. Spallanzani performed this experiment to disprove the doctrine of the spontaneous generation of life. However, he did not convince the proponents of the theory because they believed that his treatment excluded oxygen, which they felt was vital to spontaneous generation. In 1837, Schwann showed that heated infusions remained sterile in the presence of air, which he supplied by passing it through heated coils into the infusion.⁹ Although both of these men demonstrated the idea of the heat preservation of foods, neither took advantage of his ﬁndings with respect to application. The same may be said of D. Papin and G. Leibniz, who hinted at the heat preservation of foods at the turn of the eighteenth century.

The history of thermal canning necessitates a brief biography of Nicolas Appert (1749–1841).

This Frenchman worked in his father’s wine cellar early on, and he and two brothers established a brewery in 1778. In 1784, he opened a confectioner’s store in Paris that was later transformed into a wholesale business. His discovery of a food preservation process occurred between 1789 and 1793.

He established a cannery in 1802 and exported his products to other countries. The French navy began testing his preservation method in 1802, and in 1809 a French ministry ofﬁcial encouraged him to promote his invention. In 1810, he published his method and was awarded the sum of 12,000 francs.⁷ This, of course, was the beginning of canning as it is known and practiced today.⁵ This event occurred some 50 years before L. Pasteur demonstrated the role of microorganisms in the spoilage of French wines, a development that gave rise to the rediscovery of bacteria. A. Leeuwenhoek in the Netherlands had examined bacteria through a microscope and described them in 1683, but it is unlikely that Appert was aware of this development and Leeuwenhoek’s report was not available in French.

The ﬁrst person to appreciate and understand the presence and role of microorganisms in food was Pasteur. In 1837, he showed that the souring of milk was caused by microorganisms, and in about 1860 he used heat for the ﬁrst time to destroy undesirable organisms in wine and beer. This process is now known as pasteurization.

HISTORICAL DEVELOPMENTS

Some of the more signiﬁcant dates and events in the history of food preservation, food spoilage, food poisoning, and food legislation are listed below. The latter pertains primarily to the United States.

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History of Microorganisms in Food 5

Food Preservation

1782—Canning of vinegar was introduced by a Swedish chemist.

1810—Preservation of food by canning was patented by Appert in France.

—Peter Durand was issued a British patent to preserve food in “glass, pottery, tin, or other metals, or ﬁt materials.” The patent was later acquired by Hall, Gamble, and Donkin, possibly from Appert.^1,4

1813—Donkin, Hall, and Gamble introduced the practice of postprocessing incubation of canned foods.

—Use of SO2as a meat preservative is thought to have originated around this time.

1825—T. Kensett and E. Daggett were granted a U.S. patent for preserving food in tin cans.

1835—A patent was granted to Newton in England for making condensed milk.

1837—Winslow was the ﬁrst to can corn from the cob.

1839—Tin cans came into wide use in the United States.³

—L.A. Fastier was given a French patent for the use of brine bath to raise the boiling temperature of water.

1840—Fish and fruit were ﬁrst canned.

1841—S. Goldner and J. Wertheimer were issued British patents for brine baths based on Fastier’s method.

1842—A patent was issued to H. Benjamin in England for freezing foods by immersion in an ice and salt brine.

1843—Sterilization by steam was ﬁrst attempted by I. Winslow in Maine.

1845—S. Elliott introduced canning to Australia.

1853—R. Chevallier-Appert obtained a patent for sterilization of food by autoclaving.

1854—Pasteur began wine investigations. Heating to remove undesirable organisms was introduced commercially in 1867–1868.

1855—Grimwade in England was the ﬁrst to produce powdered milk.

1856—A patent for the manufacture of unsweetened condensed milk was granted to Gail Borden in the United States.

1861—I. Solomon introduced the use of brine baths to the United States.

1865—The artiﬁcial freezing of ﬁsh on a commercial scale was begun in the United States. Eggs followed in 1889.

1874—The ﬁrst extensive use of ice in transporting meat at sea was begun.

—Steam pressure cookers or retorts were introduced.

1878—The ﬁrst successful cargo of frozen meat went from Australia to England. The ﬁrst from New Zealand to England was sent in 1882.

1880—The pasteurization of milk was begun in Germany.

1882—Krukowitsch was the ﬁrst to note the destructive effects of ozone on spoilage bacteria.

1886—A mechanical process of drying fruits and vegetables was carried out by an American, A.F.

Spawn.

1890—The commercial pasteurization of milk was begun in the United States.

—Mechanical refrigeration for fruit storage was begun in Chicago.

1893—The Certiﬁed Milk movement was begun by H.L. Coit in New Jersey.

1895—The ﬁrst bacteriological study of canning was made by Russell.

1907—E. Metchnikoff and co-workers isolated and named one of the yogurt bacteria, Lactobacillus delbrueckii subsp. bulgaricus.

—The role of acetic acid bacteria in cider production was noted by B.T.P. Barker.

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1908—Sodium benzoate was given ofﬁcial sanction by the United States as a preservative in certain foods.

1916—The quick freezing of foods was achieved in Germany by R. Plank, E. Ehrenbaum, and K.

Reuter.

1917—Clarence Birdseye in the United States began work on the freezing of foods for the retail trade.

—Franks was issued a patent for preserving fruits and vegetables under CO₂.

1920—Bigelow and Esty published the ﬁrst systematic study of spore heat resistance above 212^◦F.

The “general method” for calculating thermal processes was published by Bigelow, Bohart, Richardson, and Ball; the method was simpliﬁed by C.O. Ball in 1923.

1922—Esty and Meyer established z= 18^◦F for Clostridium botulinum spores in phosphate buffer.

1928—The ﬁrst commercial use of controlled-atmosphere storage of apples was made in Europe (ﬁrst used in New York in 1940).

1929—A patent issued in France proposed the use of high-energy radiation for the processing of foods.

—Birdseye frozen foods were placed in retail markets.

1943—B.E. Proctor in the United States was the ﬁrst to employ the use of ionizing radiation to preserve hamburger meat.

1950—The D value concept came into general use.

1954—The antibiotic nisin was patented in England for use in certain processed cheeses to control clostridial defects,

1955—Sorbic acid was approved for use as a food preservative.

—The antibiotic chlortetracycline was approved for use in fresh poultry (oxytetracycline followed a year later). Approval was rescinded in 1966.

1967—The ﬁrst commercial facility designed to irradiate foods was planned and designed in the United States. The second became operational in 1992 in Florida.

1988—Nisin was accorded GRAS (generally regarded as safe) status in the United States.

1990—Irradiation of poultry was approved in the United States.

1997—The irradiation of fresh beef up to a maximum level of 4.5 kGy and frozen beef up to 7.0 kGy was approved in the United States.

1997—Ozone was declared GRAS by the U.S. Food and Drug Administration for food use.

Food Spoilage

1659—Kircher demonstrated the occurrence of bacteria in milk; Bondeau did the same in 1847.

1680—Leeuwenhoek was the ﬁrst to observe yeast cells.

1780—Scheele identiﬁed lactic acid as the principal acid in sour milk.

1836—Latour discovered the existence of yeasts.

1839—Kircher examined slimy beet juice and found organisms that formed slime when grown in sucrose solutions.

1857—Pasteur showed that the souring of milk was caused by the growth of organisms in it.

1866—L. Pasteur’s ´Etude sur le Vin was published.

1867—Martin advanced the theory that cheese ripening was similar to alcoholic, lactic, and butyric, fermentations.

1873—The ﬁrst reported study on the microbial deterioration of eggs was carried out by Gayon.

—Lister was ﬁrst to isolate Lactococcus lactis in pure culture.

1876—Tyndall observed that bacteria in decomposing substances were always traceable to air, substances, or containers.

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1878—Cienkowski reported the ﬁrst microbiological study of sugar slimes and isolated Leuconostoc mesenteroides from them.

1887—Forster was the ﬁrst to demonstrate the ability of pure cultures of bacteria to grow at 0^◦C.

1888—Miquel was the ﬁrst to study thermophilic bacteria.

1895—The ﬁrst records on the determination of numbers of bacteria in milk were those of Von Geuns in Amsterdam.

—S.C. Prescott and W. Underwood traced the spoilage of canned corn to improper heat processing for the ﬁrst time.

1902—The term psychrophile was ﬁrst used by Schmidt-Nielsen for microorganisms that grow at 0^◦C.

1912—The term osmophile was coined by Richter to describe yeasts that grow well in an environment of high osmotic pressure.

1915—Bacillus coagulans was ﬁrst isolated from coagulated milk by B.W. Hammer.

1917—Geobacillus stearothermophilus was ﬁrst isolated from cream-style corn by P.J. Donk.

1933—Oliver and Smith in England observed spoilage by Byssochlamys fulva; ﬁrst described in the United States in 1964 by D. Maunder.

Food Poisoning

1820—The German poet Justinus Kerner described “sausage poisoning” (which in all probability was botulism) and its high fatality rate.

1857—Milk was incriminated as a transmitter of typhoid fever by W. Taylor of Penrith, England.

1870—Francesco Selmi advanced his theory of ptomaine poisoning to explain illness contracted by eating certain foods.

1888—Gaertner ﬁrst isolated Salmonella enteritidis from meat that had caused 57 cases of food poisoning.

1894—T. Denys was the ﬁrst to associate staphylococci with food poisoning.

1896—Van Ermengem ﬁrst discovered Clostridium botulinum.

1904—Type A strain of C. botulinum was identiﬁed by G. Landman.

1906—Bacillus cereus food poisoning was recognized. The ﬁrst case of diphyllobothriasis was recognized.

1926—The ﬁrst report of food poisoning by streptococci was made by Linden, Turner, and Thom.

1937—Type E strain of C. botulinum was identiﬁed by L. Bier and E. Hazen.

1937—Paralytic shellﬁsh poisoning was recognized.

1938—Outbreaks of Campylobacter enteritis were traced to milk in Illinois.

1939—Gastroenteritis caused by Yersinia enterocolitica was ﬁrst recognized by Schleifstein and Coleman.

1945—McClung was the ﬁrst to prove the etiologic status of Clostridium perfringens (welchii) in food poisoning.

1951—Vibrio parahaemolyticus was shown to be an agent of food poisoning by T. Fujino of Japan.

1955—Similarities between cholera and Escherichia coli gastroenteritis in infants were noted by S.

Thompson.

—Scombroid (histamine-associated) poisoning was recognized.

—The ﬁrst documented case of anisakiasis occurred in the United States.

1960—Type F strain of C. botulinum identiﬁed by Moller and Scheibel.

—The production of aflatoxins by Aspergillus flavus was first reported.

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1965—Foodborne giardiasis was recognized.

1969—C. perfringens enterotoxin was demonstrated by C.L. Duncan and D.H. Strong.

—C. botulinum type G was ﬁrst isolated in Argentina by Gimenez and Ciccarelli.

1971—First U.S. foodborne outbreak of Vibrio parahaemolyticus gastroenteritis occurred in Maryland.

—First documented outbreak of E. coli foodborne gastroenteritis occurred in the United States.

1975—Salmonella enterotoxin was demonstrated by L.R. Koupal and R.H. Deibel.

1976—First U.S. foodborne outbreak of Yersinia enterocolitica gastroenteritis occurred in New York.

—Infant botulism was ﬁrst recognized in California.

1977—The ﬁrst documented outbreak of cyclosporiasis occurred in Papua, New Guinea; ﬁrst in United States in 1990.

1978—Documented foodborne outbreak of gastroenteritis caused by the Norwalk virus occurred in Australia.

1979—Foodborne gastroenteritis caused by non-01 Vibrio cholerae occurred in Florida. Earlier out- breaks occurred in Czechoslovakia (1965) and Australia (1973).

1981—Foodborne listeriosis outbreak was recognized in the United States.

1982—The ﬁrst outbreaks of foodborne hemorrhagic colitis occurred in the United States.

1983—Campylobacter jejuni enterotoxin was described by Ruiz-Palacios et al.

1985—The irradiation of pork to 0.3 to 1.0 kGy to control Trichinella spiralis was approved in the United States.

1986—Bovine spongiform encephalopathy (BSE) was ﬁrst diagnosed in cattle in the United Kingdom.

Food Legislation

1890—The ﬁrst national meat inspection law was enacted. It required the inspection of meats for export only.

1895—The previous meat inspection act was amended to strengthen its provisions.

1906—The U.S. Federal Food and Drug Act was passed by Congress.

1910—The New York City Board of Health issued an order requiring the pasteurization of milk.

1939—The new Food, Drug, and Cosmetic Act became law.

1954—The Miller Pesticide Chemicals Amendment to the Food, Drug, and Cosmetic Act was passed by Congress.

1957—The U.S. Compulsory Poultry and Poultry Products law was enacted.

1958—The Food Additives Amendment to the Food Drug, and Cosmetics Act was passed.

1962—The Talmadge-Aiken Act (allowing for federal meat inspection by states) was enacted into law.

1963—The U.S. Food and Drug Administration approved the use of irradiation for the preservation of bacon.

1967—The U.S. Wholesome Meat Act was passed by Congress and enacted into law on December 15.

1968—The Food and Drug Administration withdrew its 1963 approval of irradiated bacon.

—The Poultry Inspection Bill was signed into law.

1969—The U.S. Food and Drug Administration established an allowable level of 20 ppb of aﬂatoxin for edible grains and nuts.

1973—The state of Oregon adopted microbial standards for fresh and processed retail meat. They were repealed in 1977.

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REFERENCES

1. Bishop, P.W. 1978. Who introduced the tin can? Nicolas Appert? Peter Durand? Bryan Donkin? Food Technol. 32:60–67.

2. Brandly, P.J., G. Migaki, and K.E. Taylor. 1966. Meat Hygiene, 3rd ed., chap. 1. Philadelphia: Lea & Febiger.

3. Cowell, N.D. 1995. Who introduced the tin can?—A new candidate. Food Technol. 49:61–64.

4. Farrer, K.T.H. 1979. Who invented the brine bath?—The Isaac Solomon myth. Food Technol. 33:75–77.

5. Goldblith, S.A. 1971. A condensed history of the science and technology of thermal processing. Food Technol. 25:44–50.

6. Jensen, L.B. 1953. Man’s Foods, chaps. 1, 4, 12. Champaign, IL: Garrard Press.

7. Livingston, G.E., and J.P. Barbier. 1999. The life and work of Nicolas Appert, 1749–1841. Abstract # 7-1, p. 10, Institute of Food Technol. Proceedings.

8. Pederson, C.S. 1971. Microbiology of Food Fermentations. Westport, CT: AVI.

9. Schorm¨uller, J. 1966. Die Erhaltung der Lebensmittel. Stuttgart: Ferdinand Enke Verlag.

10. Stewart, G.F., and M.A. Amerine. 1973. Introduction to Food Science and Technology, chap. 1. New York: Academic Press.

11. Tanner, F.W. 1944. The Microbiology of Foods, 2nd ed. Champaign, IL: Garrard Press.

12. Tanner, F.W., and L.P. Tanner. 1953. Food-Borne Infections and Intoxications, 2nd ed. Champaign, IL: Garrard Press.

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Chapter 2 Taxonomy, Role, and Signiﬁcance of Microorganisms in Foods

Because human food sources are of plant and animal origin, it is important to understand the biological principles of the microbial biota associated with plants and animals in their natural habitats and respective roles. Although it sometimes appears that microorganisms are trying to ruin our food sources by infecting and destroying plants and animals, including humans, this is by no means their primary role in nature. In our present view of life on this planet, the primary function of microorganisms in nature is self-perpetuation. During this process, the heterotrophs and autotrophs carry out the following general reaction:

All organic matter

(carbohydrates, proteins, lipids, etc.)

↓

Energy+ Inorganic compounds (nitrates, sulfates, etc.)

This, of course, is essentially nothing more than the operation of the nitrogen cycle and the cycle of other elements. The microbial spoilage of foods may be viewed simply as an attempt by the food biota to carry out what appears to be their primary role in nature. This should not be taken in the teleological sense. In spite of their simplicity when compared to higher forms, microorganisms are capable of carrying out many complex chemical reactions essential to their perpetuation. To do this, they must obtain nutrients from organic matter, some of which constitutes our food supply.

If one considers the types of microorganisms associated with plant and animal foods in their natural states, one can then predict the general types of microorganisms to be expected on this particular food product at some later stage in its history. Results from many laboratories show that untreated foods may be expected to contain varying numbers of bacteria, molds, or yeasts, and the question often arises as to the safety of a given food product based on total microbial numbers. The question should be twofold: What is the total number of microorganisms present per gram or milliliter and what types of organisms are represented in this number? It is necessary to know which organisms are associated with a particular food in its natural state and which of the organisms present are not normal for that particular food. It is, therefore, of value to know the general distribution of bacteria in nature and the general types of organisms normally present under given conditions where foods are grown and handled.

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BACTERIAL TAXONOMY

Many changes have taken place in the classiﬁcation or taxonomy of bacteria in the past two decades.

Many of the new taxa have been created as a result of the employment of molecular genetic methods, alone or in combination with some of the more traditional methods:

1. DNA homology and mol% G+ C content of DNA 2. 23S, 16S, and 5S rRNA sequence similarities 3. Oligonucleotide cataloging

4. Numerical taxonomic analysis of total soluble proteins or of a battery of morphological and biochemical characteristics

5. Cell wall analysis 6. Serological proﬁles 7. Cellular fatty acid proﬁles

Although some of these have been employed for many years (e.g., cell wall analysis and serological proﬁles) others (e.g., ribosomal RNA [rRNA] sequence similarity) came into wide use only during the 1980s. The methods that are the most powerful as bacterial taxonomic tools are outlined and brieﬂy discussed below.

rRNA Analyses

Taxonomic information can be obtained from RNA in the production of nucleotide catalogs and the determination of RNA sequence similarities. First, the prokaryotic ribosome is a 70S (Svedberg) unit, which is composed of two separate functional subunits: 50S and 30S. The 50S subunit is composed of 23S and 5S RNA in addition to about 34 proteins, whereas the 30S subunit is composed of 16S RNA plus about 21 proteins.

The 16S subunit is highly conserved and is considered to be an excellent chronometer of bacteria over time.⁵³Using reverse transcriptase, 16S rRNA can be sequenced to produce long stretches (about 95% of the total sequence) to allow for the determination of precise phylogenetic relationships.³¹ Alternatively, the 16S rDNA may be sequenced after ampliﬁcation of speciﬁc regions by polymerase chain reaction (PCR)-based methods.

To sequence 16S rRNA, a single-stranded DNA copy is made by use of reverse transcriptase with the RNA as template. When the single-stranded DNA is made in the presence of dideoxynucleotides,

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Taxonomy, Role, and Signiﬁcance of Microorganisms in Foods 15

DNA fragments of various sizes result that can be sequenced by the Sanger method. From the DNA sequences, the template 16S rRNA sequence can be deduced. It was through studies of 16S rRNA sequences that led Woese and his associates to propose the establishment of three kingdoms of life forms: Eukaryotes, Archaebacteria, and Prokaryotes. The last include the cyanobacteria and the eubacteria, with the bacteria of importance in foods being eubacteria. Sequence similarities of 16S rRNA are widely employed, and some of the new foodborne taxa were created primarily by its use along with other information. It appears that the sequencing of 23S rDNA will become more widely used in bacterial taxonomy.

Nucleotide catalogs of 16S rRNA have been prepared for a number of organisms, and extensive libraries exist. By this method, 16S rRNA is subjected to digestion by RNase T1, which cleaves the molecule at G(uanine) residues. Sequences (-mers) of 6–20 bases are produced and separated, and similarities SAB(Dice-type coefﬁcient) between organisms can be compared. Although the relationship between SABand percentage similarity is not good below SABvalue of 0.40, the information derived is useful at the phylum level. The sequencing of 16S rRNA by reverse transcriptase is preferred to oligonucleotide cataloging, as longer stretches of rRNA can be sequenced.

Analysis of DNA

The mol% G+ C of bacterial DNA has been employed in bacterial taxonomy for several decades, and its use in combination with 16S and 5S rRNA sequence data makes it even more meaningful. By 16S rRNA analysis, the Gram-positive eubacteria fall into two groups at the phylum level: one group with mol% G+ C >55, and the other <50.⁵³The former includes the genera Streptomyces, Propioni- bacterium, Micrococcus, Biﬁdobacterium, Corynebacterium, Brevibacterium, and others. The group with the lower G+ C values include the genera Clostridium, Bacillus, Staphylococcus, Lactobacillus, Pediococcus, Leuconostoc, Listeria, Erysipelothrix, and others. The latter group is referred to as the Clostridium branch of the eubacterial tree. When two organisms differ in G+ C content by more than 10%, they have few base sequences in common.

DNA–DNA or DNA–RNA hybridization has been employed for some time, and this technique continues to be of great value in bacterial systematics. It has been noted that the ideal reference system for bacterial taxonomy would be the complete DNA sequence of an organism.⁴⁹It is generally accepted that bacterial species can be defined in phylogenetic terms by use of DNA–DNA hybridization results, where 70% or greater relatedness and 5^◦C or less T_m(melting point) defines a species.⁵⁰When DNA–DNA hybridization is employed, phenotypic characteristics are not allowed to override except in exceptional cases.⁵⁰Although a genus is more difficult to define phylogenetically, 20% sequence similarity is considered to be the minimum level of DNA–DNA homology.⁵⁰

Even if there is not yet a satisfactory phylogenetic deﬁnition of a bacterial genus, the continued application of nucleic acid techniques, along with some of the other methods listed above, should lead ultimately to a phylogenetically based system of bacterial systematics. In the meantime, changes in the extant taxa may be expected to continue to occur.

The Proteobacteria

The Gram-negative bacteria of known importance in foods belong to the class Proteobacteria, which was established following extensive studies on the rRNA sequences of numerous genera of

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Table 2–1 Subclasses of the Proteobacteria to Which Many Foodborne Genera Belong. Campylobacter and Helicobacter Belong to the

δ-Subclass

Alpha Beta Gamma

Acetobacter Acidovorax Acinetobacter

Asaia Alcaligenes Aeromonas

Brevundimonas Burkholderia Alteromonas

Devosia Chromobacterium Azomonas

Gluconobacter Comamonas Bacteriodes

Paracoccus Delftia Carnimonas

Pseudoaminobacter Hydrogenophaga Enterobacteriaceae^a Sphingomonas Janthinobacterium Flavobacterium

Xanthobacter Pandoraea Halomonas

Zymomonas Pseudomonas (plant pathogens) Moraxella

Ralstonia Plesiomonas

Telluria Pseudoalteromonas

Variovorax Pseudomonas

Vogesella Psychrobacter

Wautersia Photobacterium

Xylophilus Shewanella

Stenotrophomonas Vibrio

Xanthomonas Xylella

aInclude Escherichia, Citrobacter, Salmonella, Shigella, Proteus, Raoultella, Proteus, Klebsiella, Edwardsiella, etc.

Gram-negative bacteria.⁴³ The class is divided into five subclasses designatedα, β, γ , etc. The subclasses are defined on the basis of their 16S rRNA sequences.^54–56 By extensive use of signature sequences (conserved inserts and deletions) of different proteins, an evolutionary relationship of the Proteobacteria has been proposed.²⁰It has been suggested that the first eubacteria were low G+ C Gram positives (e.g., Clostridium, Bacillus, Lactobacillus), followed by high G+ C Gram positives (e.g., Micrococcus, Propionibacterium, Rubrobacter), and then by Deinococcus-Thermus. Next arose three groups that are not foodborne (not listed here), and then the Proteobacteria with and σ followed byα, β, and γ .²⁰It has been stressed that these groups are related to each other in a linear rather than a tree-like manner.²⁰It can be seen from Table 2–1 that most foodborne bacteria (especially foodborne pathogens) belong to theγ -subclass. The earliest prokaryotes are estimated to have arisen 3.5–3.8 billion years ago.²⁰

Some of the important genera known to occur in foods are listed below in alphabetical order. Some are desirable in certain foods; others bring about spoilage or cause gastroenteritis. It should be noted that the bacterial genera in this list along with those in Table 2–2 are now somewhat problematic since most were deﬁned largely on phenotypic data. They are placed here mainly on historical reports but the list may be expected to change as more phylogenetic data are employed.

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Taxonomy, Role, and Signiﬁcance of Microorganisms in Foods 17

Bacteria

Acinetobacter Erwinia Proteus

Aeromonas Escherichia Pseudomonas

Alcaligenes Flavobacterium Psychrobacter

Arcobacter Hafnia Salmonella

Bacillus Kocuria Serratia

Brevibacillus Lactococcus Shewanella

Brochothrix Lactobacillus Shigella

Burkholderia Leuconostoc Sphingomonas

Campylobacter Listeria Stenotrophomonas

Carnobacterium Micrococcus Staphylococcus

Citrobacter Moraxella Vagococcus

Clostridium Paenibacillus Vibrio

Corynebacterium Pandoraea Weissella

Enterobacter Pantoea Yersinia

Enterococcus Pediococcus

Molds

Alternaria Colletotrichum Penicillium

Aspergillus Fusarium Rhizopus

Aureobasidium Geotrichum Trichothecium

Botrytis Monilia Wallemia

Byssochlamys Mucor Xeromyces

Cladosporium

Yeasts

Brettanomyces/Dekkera Issatchenkia Schizosaccharomyces

Candida Kluyveromyces Torulaspora

Cryptococcus Pichia Trichosporon

Debaryomyces Rhodotorula Yarrowia

Hanseniaspora Saccharomyces Zygosaccharomyces Protozoa

Cryptosporidium parvum Entamoeba histolytica Toxoplasma gondii Cyclospora cayetanensis Giardia lamblia

PRIMARY SOURCES OF MICROORGANISMS FOUND IN FOODS

The genera and species previously listed are among the most important normally found in food products. Each genus has its own particular nutritional requirements, and each is affected in predictable ways by the parameters of its environment. Eight environmental sources of organisms to foods are listed below, and these, along with the genera of bacteria and protozoa noted, are presented in Table 2–2 to reﬂect their primary food-source environments.

Soil and Water. These two environments are placed together because many of the bacteria and fungi that inhabit both have a lot in common. Soil organisms may enter the atmosphere by the action of wind and later enter water bodies when it rains. They also enter water when rainwater ﬂows over soils into bodies of water. Aquatic organisms can be deposited onto soils through the actions of cloud formation and subsequent rainfall. This common cycling results in soil and aquatic organisms being one and the same to a large degree. Some aquatic organisms, however, are unable to persist in soils, especially those that are indigenous to marine waters. Alteromonas spp. are aquatic forms that require seawater salinity

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Table2–2RelativeImportanceofEightSourcesofBacteriaandProtozoatoFoods SoilandFoodGastrointestinalFoodAnimalAnimalAirand OrganismsWaterPlants/ProductsUtensilsTractHandlersFeedsHidesDust Bacteria AcinetobacterXXXXXX AeromonasXXaX AlcaligenesXXXXX AlteromonasXXa ArcobacterX BacillusXXbXXXXXXX BrochothrixXXX BrevibacillusXXX BurkholderiaXX CampylobacterXXX CarnobacteriumXXX CitrobacterXXXXXX ClostridiumXXbXXXXXXXX CorynebacteriumXXbXXXXX EnterobacterXXXXXX EnterococcusXXXXXXXXX ErwiniaXXXX EscherichiaXXXXX FlavobacteriumXXXX HafniaXXXX KocuriaXXXXXX LactococcusXXXXX LactobacillusXXXXX LeuconostocXXXXX ListeriaXXXXXX MicrococcusXXXXXXXX MycobacteriumcX MoraxellaXXX Mycobacterium

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PaenibacillusXXXXXX PandoraeaX PectobacteriumXXX PantoeaXXX PediococcusXXXXX ProteusXXXXXX PseudomonasXXXXXX PsychrobacterXXXXX SalmonellaXXXX SerratiaXXXXXX ShewanellaXX SphingomonasXX ShigellaXX StenotrophomonasXXX StaphylococcusXXXX VagococcusXXXX VibrioXXX WeissellaXXX YersiniaXXX Protozoa C.cayetanensisXXX C.parvumXXXX E.histolyticaXXXX G.lambliaXXXX T.gondiiXXX Note:XXindicatesaveryimportantsource. aPrimarilywater bPrimarilysoil. cNontuberculous.

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for growth and would not be expected to persist in soils. The bacterial biota of seawater is essentially Gram-negative, and Gram-positive bacteria exist there essentially only as transients. Contaminated water has been implicated in Cyclospora contamination of fresh raspberries.

Plants and Plant Products. It may be assumed that many or most soil and water organisms con- taminate plants. However, only a relatively small number ﬁnd the plant environment suitable to their overall well-being. Those that persist on plant products do so by virtue of a capacity to adhere to plant surfaces so that they are not easily washed away and because they are able to obtain their nutritional requirements. Notable among these are the lactic acid bacteria and some yeasts. Among others that are commonly associated with plants are bacterial plant pathogens in the genera Corynebacterium, Cur- tobacterium, Pectobacterium, Pseudomonas, and Xanthomonas; and fungal pathogens among several genera of molds.

Food Utensils. When vegetables are harvested in containers and utensils, one would expect to ﬁnd some or all of the surface organisms on the products to contaminate contact surfaces. As more and more vegetables are placed in the same containers, a normalization of the microbiota would be expected to occur. In a similar way, the cutting block in a meat market along with cutting knives and grinders are contaminated from initial samples, and this process leads to a buildup of organisms, thus ensuring a fairly constant level of contamination of meat-borne organisms.

Gastrointestinal Tract. This biota becomes a water source when polluted water is used to wash raw food products. The intestinal biota consists of many organisms that do not persist as long in waters as do others, and notable among these are pathogens such as salmonellae. Any or all of the Enterobacteriaceae may be expected in fecal wastes, along with intestinal pathogens, including the ﬁve protozoal species already listed.

Food Handlers. The microbiota on the hands and outer garments of handlers generally reﬂect the environment and habits of individuals, and the organisms in question may be those from soil, water, dust, and other environmental sources. Additional important sources are those that are common in nasal cavities, the mouth, and on the skin, and those from the gastrointestinal tract that may enter foods through poor personal hygiene practices.

Animal Feeds. This is a source of salmonellae to poultry and other farm animals. In the case of some silage, it is a known source of Listeria monocytogenes to dairy and meat animals. The organisms in dry animal feed are spread throughout the animal environment and may be expected to occur on animal hides.

Animal Hides. In the case of milk cows, the types of organisms found in raw milk can be a reﬂection of the biota of the udder when proper procedures are not followed in milking and of the general environment of such animals. From both the udder and the hide, organisms can contaminate the general environment, milk containers, and the hands of handlers.

Air and Dust. Although most of the organisms listed in Table 2–2 may at times be found in air and dust in a food-processing operation, the ones that can persist include most of the Gram-positive organisms listed. Among fungi, a number of molds may be expected to occur in air and dust, along with some yeasts. In general, the types of organisms in air and dust would be those that are constantly reseeded to the environment. Air ducts are not unimportant sources.

SYNOPSIS OF COMMON FOODBORNE BACTERIA

These synopses are provided to give the reader glimpses of bacterial groups that are discussed throughout the textbook. They are not meant to be used for culture identiﬁcations. For the latter, one or

History of Microorganisms in Food

Preface

Contents

Chapter 1

History of Microorganisms in Food

Chapter 2

Taxonomy, Role, and Signiﬁcance of Microorganisms in Foods