Introduction to Enterobacteriaceae

Taxonomy

The members of the Enterobacteriaceae family are Gram-negative bacteria in the shape of straight rods, non-spore forming, facultative anaerobic and oxidase-negative (except for the  Plesiomonas genus, which was recently transferred to this family).  Enterobacteriaceae are chemoorganotrophs with both a respiratory and fermentative metabolism. The majority ferments glucose and other carbohydrates, producing acids and gases. They are non-halophilic, produce catalase (except for Xenorhabdus and some strains of  Shigella dysenteriae) and reduce nitrate to nitrite (except for Saccharobacter fermentatus and some strains of  Erwinia and  Yersinia) (Brenner and Farmer III, 2005).

 Escherichia is the type genus of the family, which includes several other genera of importance in foods, such as  Citrobacter,  Edwardsiella,  Enterobacter,  Erwinia, Hafnia,  Klebsiella,  Morganella,  Pantoea,  Pectobacterium, Proteus,  Salmonella,  Serratia,  Shigella and  Yersinia. This family also includes the bacteria of the  total coliform and  thermotolerant coliform ( fecal coliforms) groups,. The number of genera and species of the family has continually increased, from 12 genera and 36 species in 1974, to 20 genera and 76 species in 1984, 30 genera and 107 species in 1994 and 44 genera and 176 species in 2005, according to the 2nd Edition of Bergey’s Manual of Systematic Bacteriology (Brenner and Farmer III, 2005).

 Enterobacteriaceae are widely distributed throughout nature and are found in the soil, water, plants, fruits, vegetables, meats, eggs, grains, animals, insects and in man. Several species are pathogenic to plants and animals, causing significant economic loss in agriculture and the food industry. Erwinia and  Pectobacterium, for example, seriously affect corn plants, potatoes, apples, sugarcane, pineapple and other vegetable products. Yersinia ruckeri and several species of  Edwardsiella cause disease in tropical fish, thereby directly affecting the fishery sector.  Klebsiella e  Citrobacter freundii are causing agents of mastitis in cattle (Brenner and Farmer III, 2005).

Several  Enterobacteriaceae are also pathogenic for man, posing a serious hazard to public health.  Salmonella is the most important, with poultry, eggs, sheep and swine being the major vehicles for the transmission of salmonellosis to man. Other pathogenic genera and species transmitted by foods are  Shigella,  Yersinia enterocolitica,  Yersinia pseudotuberculosis,  Cronobacter (formerly  Enterobacter sakazakii) and enteropathogenic strains of E. coli, including  enterohaemorrhagic E. coli strains (EHEC) such as E. coli O157:H7 (Brenner and Farmer III, 2005).

 Enterobacteriaceae are used as indicators of the sanitary conditions of manufacturing processes since they are easily inactivated by sanitizing agents and capable of colonizing niches of processing plants where cleaning and sanitation procedures were inappropriately per-formed (Kornachi and Johnson, 2001).

Although  Enterobacteriaceae are mostly mesophilic in nature,  psychrotrophic strains are not uncommon, particularly within the genera  Yersinia,  Citrobacter, Enterobacter, Escherichia, Klebsiella, Serratia and Hafnia (Kornachi and Johnson, 2001).

Methods of  analysis

The quantification of  Enterobacteriaceae can be achieved by the standard plate count method, using  Violet Red Bile Glucose (VRBG) Agar as culture medium. VRBG is a differential selective medium containing crystal violet and bile salts, which inhibit Gram-positive bacteria. Fermentation of glucose results in acids, detected by the red color of the pH indicator neutral red, and by the formation of a zone of bile salt precipitation surrounding the colonies.

For products with low counts, the Most Probable Number (MPN) technique is recommended, which is performed in two stages: The first consisting of selective enrichment in  Enterobacteriaceae Enrichment Broth (EEB), and the second of isolation of typical colonies on VRBG Agar. The presence of bile salts and brilliant green inhibits most of the accompanying microbiota.

Furthermore, the high buffering capacity of the medium prevents the deleterious effect of pH reduction on the Enterobacteriaceae. This procedure can also be used as a simple presence/absence test, if quantification is not necessary or required. Another method recommended in the Compendium (Kornacki and Johnson, 2001) and in the Standard Methods for the Examination of Dairy Products (Davidson et al., 2004) is the  Petrifilm Enterobacteriaceae method of the 3M Company ( AOAC Official Method 2003.1), which follows the same principles as the VRBG plate count.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .

Brenner, D.J. & Farmer III, J.J. (2005) Family I. Enterobacteriaceae. In: Brenner, D.J., Krieg, N.R. & Staley, J.T. (eds). Bergey’s Man-ual of Systematic Bacteriology. Volume 2. 2nd edition. New York, Springer Science+Business Media Inc. pp. 587–607.

Kornacki, J.L. & Johnson, J.L. (2001) Enterobacteriaceae, coliforms, and Escherichia coli as quality and safety indicators. In: Downes, F.P. & Ito, K. (eds).  Compendium of Methods for the Microbio-logical Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 8, pp. 69–82.