Module 2. Classification of dairy microorganisms

Lesson 7

CHARACTERISTICS OF SPOILAGE AND PATHOGENIC MICROORGANISMS – I

In this lesson we shall be discussing the characteristics of spoilage and pathogenic microorganisms associated with dairy.

7.1 Pseudomonas

Pseudomonas is a genus belonging to the family Pseudomonadaceae containing 191 species.

The word was used early in the history of microbiology to refer to germs. The name 'fluorescens' refers to the microbe's secretion of a soluble fluorescent pigment called pyoverdin (also called fluorescein), which is a type of siderophore (iron binding protein).

Pseudomonas fluorescens has multiple flagella. It has an extremely versatile metabolism, and can be found in the soil and in water. It is an obligate aerobe, but certain strains are capable of using nitrate instead of oxygen as a final electron acceptor during cellular respiration.

P. fluorescens produces water soluble fluorescent greenish-brown pigment known as ‘Pyoverdin’. The optimum growth temperature ranges between 25-30 °C but grows at 5°C. It causes bitterness and lipolysis in refrigerated foods by elaborating proteinases and lipases and gelation in UHT products. It also produces phospholipases and glycosidase but causes no coagulation of litmus milk.

‘Pseudo’ means false; ‘monas’ means unit i.e. false species of ‘monas’. Many species of Pseudomonas produces fluorescent, diffusible pigments of greenish, yellow or yellow green colors and black or blue colors. The optimum growth temperature of it varies between 25-30°C. These are nutritionally versatile and grow well on solid media. Presence of these organisms in milk and milk products is highly undesirable as these:

· May produces heat stable proteases and lipases even at low temperature

· Are versatile spoilage causing microbial agents attacking fats and proteins

· Include pathogens that cause milk borne illness.

· Have very little fermentative activity on carbohydrates

· May produce phosphatases and if grown in pasteurized milk they can cause false positive tests.

Optimal temperatures for growth of Pseudomonas fluorescens are 25-30°C. It tests positive for the oxidase test. Pseudomonas fluorescens is also a nonsaccharolytic bacterium. Heat-stable lipases and proteases are produced by Pseudomonas fluorescens and other similar pseudomonads. These enzymes cause milk to spoil, by causing bitterness, casein breakdown, and ropiness due to production of slime and coagulation of proteins.

Pseudomonas fragi is a psychrophile, Gram-negative bacteria that is responsible for dairy spoilage. Unlike many other members of the Pseudomonas, P. fragi does not produce siderophores. Pseudomonas fragi rarely produces pigments but sometimes a diffusible brown pigment is seen. It grows at 5°C; however, the optimum growth temperature is between 20 to 25°C. These are mostly lipolytic in nature and rarely proteolytic. It also produces fruit like odor resembling to that of apple or strawberries compounds are ‘ethyl butyrate and ethyl hexanoate’. 

Pseudomonas aeruginosa is increasingly recognized as an emerging opportunistic pathogen of clinical relevance. Several different epidemiological studies indicate antibiotic resistance is increasing in clinical isolates. P. aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized natural and artificial environments. It uses a wide range of organic material for food; in animals, the versatility enables the micro-organism to infect damaged tissues or those with reduced immunity.

This organism produces a greenish blue pigment called ‘pyocyanin’ and ‘pyoverdin’ and non-caratenoid pigments. P. aeruginosa does not grow at 5°C but grows well at 41-42°C. The optimum growth temperature of it is 37°C. It is an opportunistic pathogen and causes mastitis in dairy animals and human infection. 

7.2 Alcaligenes

Alcaligenes is Gram-negative, pathogenic, opportunistic bacteria found in soil or water. Alcaligenes faecalis is significant because it converts the most toxic form of arsenic, arsenite (AsO₂^-, oxidation state +3) to its less dangerous form, arsenate (AsO₄^-, oxidation state +5). These are rods or cocci, motile, obligate aerobe, psychrotrophs and some of the species are thermoduric. It causes ropiness in milk and milk products (Alcaligenes viscosus, Alcaligenes tolerans)

7.3 Brucella

Brucella is Gram-negative bacteria. These are small (0.5 to 0.7 by 0.6 to 1.5 µm), non-motile, non-encapsulated coccobacilli, that function as facultative intracellular parasites. It causes brucellosis that is a zoonosis. It is transmitted by ingesting infected food, direct contact with an infected animal, or inhalation of aerosols. Transmission from human to human, example from mother to child, is rare, but possible. Minimum infectious exposure of it is between 10 - 100 micro-organisms. Brucellosis primarily occurs through occupational exposure (e.g. exposure to cattle, sheep, pigs), but also by consumption of unpasteurized milk products. There are a few different species of Brucella, each with slightly different host specificity. B. melitensis infects goats and sheep, B. abortus infects cattle, B. suis infects pigs and B. ovis infects sheep.

7.4 Coliforms

Coliforms are used as indicator of sanitary quality of food and water, which belong to the family of Enterobacteriaceae. These are rod-shaped, Gram-negative, non-spore former that can ferment lactose with the production of acid and gas, when incubated at 35-37°C. Coliforms are found in aquatic environment, in soil and on vegetation; these are universally present in large numbers in the faeces of warm-blooded animals. Coliforms normally are not the cause of serious illness, these are easy to culture and their presence to indicate that other pathogens of faecal origin may be present.

The typical genera of coliforms include: Citrobacter, Enterobacter, Hafnia, Klebsiella, Serratia, Faecal coliforms: Escherichia

7.4.1 Escherichia

Escherichia coli are a Gram-negative, rod-shaped bacterium commonly found in the lower intestine of warm-blooded animals. Most E. coli strains are harmless, but some serotypes can cause food poisoning in humans. The harmless strains are part of the normal flora of gut, and can benefit their hosts by producing vitamin K, and by preventing the establishment of pathogens within the intestine. It is a natural inhabitant of intestine.

E. coli and related bacteria constitute about 0.1% of gut flora, and faecal-oral transmission is the major route via which pathogens cause disease. These are able to survive outside the body for a limited period of time that makes these ideal indicator micro-organisms to test environmental samples for faecal contamination. E. coli is most widely studied prokaryotic model micro-organism and an important species that is served as host for majority of recombinant DNA work.

E. coli ferment glucose and carbohydrates and produces lactic, acetic and formic acids. Due to rapid production of acid and gas the litmus milk gets coagulated faster and easily. Citric acid and its salts are not utilized as the sole source of carbon. About 35 percent of the acid produced by E. coli is lactic acid and hence, these are also called ‘pseudolactic acid bacteria’. Type of acid produced by E. coli depends on the type of sugar fermented and the nature of N₂ source used.

7.4.2 Significance

· Presence in foods is an indicative of fecal contamination

· Potent food spoilage microorganisms

· Produce gas and unclean flavor in the dairy products

· Some strains also produce ropiness in the dairy products

· Presence in pasteurized foods indicates unhygienic practices during manufacturing 

· Some strains produce enterotoxins, while some are enteropathogenic

7.4.3 Enterobacter aerogenes

Based on the following tests Enterobacter aerogenes is differentiated with E. coli (Table 7.1).

7.4.3.1 Indole test

The test is based on the ability of a microorganism to degrade amino acid tryptophan into indole. The presence of indole is detected by Kovac’s reagent. The positive test shows development of cherry red colour using reagent.

7.4.3.2  Methyl red test

The test determines the ability of a microorganism to ferment the glucose with the production and stabilization of acid end products. Methyl red is used as pH indicator to detect the concentration of acid as end product. Indicator turns red in the pH range of 4.0 and yellow in the pH range of 6.0.

7.4.3.3 Voges-Proskaur test

The test is based on the ability of a microorganism to produce acetyl methyl carbinol from glucose on fermentation. Acetylmethylcarbinol, the neutral or non acidic end product from glucose metabolism, releases diacetyl that is detected in presence of Barritt's reagent, producing a deep rose colour. Detection of acetylmethylcarbinol requires that this end product be oxidized to a diacetyl compound which occurs in the presence of α-napthol and a guanidine group of peptone present in the medium. As a result pink colour complex is formed.

7.4.3.4  Citrate test

The test determines the ability of a microorganism to use citrate as a carbon source. Citrate inside the bacterial cell undergoes enzymatic degradation and finally produces pyruvic acid and carbon dioxide. This carbon dioxide combines with sodium to form sodium carbonate and converting medium of reaction into alkaline. Bromothymol blue indicator previously incorporated into the medium turns the medium from green into deep Prussian blue. During its conversion to pyruvic acid, the medium becomes alkaline and CO₂ generated combines with sodium and water to form Na₂CO₃ whose presence is detected by bromothymol blue indicator which changes green color into deep blue.

Table 7.1 Bio-chemical differentiation between E. coli and E. aerogenes

Characteristic(s)	E. coli	E. aerogenes
Motility	Motile	Non-motile
CO2:‑H2	1:1	2:1
Origin/ Source	Fecal	Non fecal
MacConkey’s broth	Acid and gas production	Acid and gas production
BGLB broth (2%)	Gas production	Gas production
EMB agar	Dark colonies with green metallic sheen	Pink colonies without any metallic sheen
Methyl red	Positive	Negative
Voges-proskauer	Negative	Positive
Citrate utilization	Negative	Positive