Module 2. Classification of dairy microorganisms

Lesson 8

CHARACTERISTICS OF SPOILAGE AND PATHOGENIC MICROORGANISMS – II

8.1 Introduction

Different microorganisms possess different characteristics such as pigments, uptake and utilization of different sugars, selective medium for their growth and different colony pattern on media. All microorganisms have different biochemical and molecular characteristics which distinguish them from each other. In this lesson a number of characteristics of spoilage and pathogenic microorganisms are discussed.

8.2 Salmonella

Salmonella is of rod-shaped, Gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella (i.e. peritrichous). These are chemoorganotrophs, obtain their energy from oxidation and reduction reactions using organic sources, and are facultative anaerobes. Most species produce hydrogen sulfide that can readily be detected by growing these on media containing ferrous sulphate, such as triple sugar iron. Most isolates exist in a motile and nonmotile phases. Strains that are nonmotile upon primary culturing may be switched to the motile phase.

Salmonella is closely related to Escherichia and are found in cold- and warm-blooded animals and environment. These cause illnesses like typhoid fever, paratyphoid fever, and food-borne illness.

· Sources of salmonella include direct or indirect fecal contamination and the animals suffering from salmonellosis

· These are designated into serotypes such as ‘O’(somatic), ‘H’ (flagellar) and ‘Vi’ (virulence antigens)

· Important group of microorganisms, because of its ability to produce a variety of foodborne infections and diseases

· Produces ‘endotoxins’ after ingestion of food contaminated with these microorganisms, however 12 to 30 h may elapse before the onset of symptoms.

The following illnesses are caused by the different species of salmonella:

· Typhoid: Salmonella typhi

· Paratyphoid:  Salmonella paratyphi A, Salmonella paratyphi B, Salmonella paratyphi C

8.3 Yersinia

Yersinia is a genus of family Enterobacteriaceae. These are Gram-negative, rod shaped bacteria, a few micrometers long and fractions of a micrometer in diameter, and are facultative anaerobes. Some members of Yersinia are pathogenic to humans; for example, Y. pestis is the causative agent of plague. Rodents are the natural reservoirs of Yersinia; less frequently other mammals serve as the host. Infection may occur either through blood as of Y. pestis) or via consumption of food (especially, vegetables, milk-derived products and meat) contaminated with infected urine or feces of mouse.

The significant microbes of this genus are Y. enterocolitica, Y. pestis, and Y. psuedotuberculosis.  The Y. enterocolitica enters milk via contamination through feces, urine, and insects. These grow between 2 to 45°C with an optimum growth temperature of 30°C. Microorganisms of this genus are pathogenic in nature to humans or animals both.

8.4 Serratia

Serratia is Gram-negative, facultative anaerobic, rod-shaped bacteria of family Enterobacteriaceae. The most common species, S. marcescens, is normally the only pathogen then causes nosocomial infections. However, rare strains of S. plymuthica, S. liquefaciens, S. rubidaea, and S. odoriferae have caused diseases through infection. Members of this genus produce characteristic red pigment, prodigiosin, and can be distinguished from other members of Enterobacteriaceae by their unique production of: DNase, lipase, and gelatinase. Serratia marcescens produces a red pigment that is known as ‘prodigiosin’

8.5 Vibrio

Vibrio is a genus of Gram-negative bacteria possessing a shape of curved rod, several species of which can cause foodborne infection, usually associated with eating undercooked sea foods. These are typically found in saltwater, are facultative anaerobes that test positive for oxidase and do not form spores. All are motile and have polar flagella with sheaths. The name Vibrio derives from ‘Filippo Pacini’, who isolated these and called ‘vibrions’ from cholera patients in 1854, because of their motility. Vibrio cholerae causes ‘cholera’ in humans.

8.6 Coxiella

These are Gram-negative, short rods, ‘pleomorphic’ occurring as diplococci. For example, C. burnetii causes ‘Q (query) fever’. Milk is contaminated directly from infected animals and humans gets infection mostly via aerosol infection and less commonly by consuming contaminated milk. It shows a high resistance to chemical and physical agents, and to desiccation. It is also resistant to 0.5% formalin, 1.0% phenol and can withstand the heat treatment of 60°C for 1 hr or 61.7°C for 30 min.

8.7  Micrococcus (Micro = small; and Kokkos = means seed or grain)

Micrococcus belongs to family Micrococcaceae. These occur in a wide range of environments, including water, dust, and soil. Micrococci are Gram-positive, spherical cells ranging from about 0.5 to 3 µm in diameter and typically appear in tetrads. Micrococcus has a substantial cell wall that may comprise of nearly 50% of the cell mass. The genome of Micrococcus is rich in guanine and cytosine content (65 to 75%; GC-content). Micrococci often carry plasmids (ranging from 1 to 100 MDa) that provide the micro-organism the useful traits.

· These are aerobic, coagulase negative, mesophilic, forms tetrads

· Some of produce yellow, orange, red pigment

· Do not ferment lactose

· Optimum growth temperature is 25°C, but many species are heat resistant and can survive at 63°C/ 30 min but the true micrococci are not resistant to pasteurization. These are found in lactiferous ducts of mammary glands and in milk obtained from the udder under sterile conditions and hence, are considered as the normal microflora of milk. The contaminated equipments are the main source of micrococci in milk. These are responsible for thickening of sweetened condensed milk and causes thermoduric outbreaks in pasteurizing plants

· The example includes M. varians and M. luteus.

8.8  Staphylococcus

Staphylococcus is Gram-positive bacteria that appear round (cocci), and forms grape-like clusters.

Staphylococcus includes nearly 40 species, of these, nine have two subspecies and one has three subspecies. Most are harmless and reside normally on the skin and mucous membranes of humans and are a small component of soil microbial flora.

These are cocci and smaller than micrococci. These are non motile and may produce orange or yellow pigment. Mesophilic in nature and the optimum growth temperature is 37°C. These can ferment a variety of carbohydrates and resist up to 10% of salt in medium. These can also produce toxins (i.e. haemolysin, fibrinolysin, leucocidin, enterotoxins, thermostable nuclease etc). Haemolysins can that liberate haemoglobin from red blood cell. The β-haemolysis produces clear, colorless zone around staphylococcus colonies. The haemoglobin destroyed to produce colorless compound. The α-haemolysis converts haemoglobin to methamoglobin that produces a greenish zone.

Staphylococcus aureus, named from Greek, meaning ‘golden grape-cluster berry’, is also known as ‘golden staph’ and ‘Oro staphira’. It is facultative anaerobic Gram-positive cocci. It is part of the normal skin flora and nasal passages. As per estimates nearly 20% of the human population is long-term carriers of S. aureus. S. aureus is the most common species of staphylococcus that causes infections. This pigment produced by S. aureus called staphyloxanthin acts as a virulence factor, primarily by being a bacterial antioxidant which helps the microbe evade the reactive oxygen species, which the host immune system uses to kill pathogens.

S. aureus can cause a range of illnesses, from minor skin infections (i.e. pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses) to life-threatening diseases (i.e. pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis). Its incidence ranges from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is one of the five most common causes of nosocomial infections and often causes postsurgical infections.

Staphylococcus aureus is a facultative anaerobe, grow at 15 and 45°C and is mesophilic in nature. The colonies have yellowish tint (lemon) or orange-yellow color. It also produces α, β, γ and heamolysins. It is sensitive to antibiotic novobiocin. Most of the S. aureus strains produce enterotoxins (especially exotoxins) that are responsible for food intoxications in consumers. Five different enterotoxins (i.e. A, B, C, D, and E) are reported but ‘enterotoxins A’ are more commonly produced. These are heat stable and produce symptoms within 2 to 6 hours after ingestion of contaminated milk or its product. On Baird parker’s medium these produce black, shiny, convex colonies surrounded by a clear zone. In this clear zone a fine black precipitate (i.e. zone of opalescence) may appear. The pathogenic nature of S. aureus is confirmed with a positive coagulase and thermonuclease test.  It also produces bacteriocins viz., staphylococcin/ micrococcin that are bacteriostatic/ cidal to many bacteria and to other staphylococci. The other species of staphylococcus found in milk are S. epidermis, S. caprae and S. hyicus.

8.9 Listeria

Listeria named after the English pioneer of sterile surgery Joseph Lister in 1940, is a bacterial genus that contains seven species. Listeria species are Gram-positive bacilli. The major human pathogen is L. monocytogenes, the causative agent of the relatively rare bacterial disease, listeriosis, a serious infection caused by eating contaminated food.

Listeriosis is a serious disease for humans that have a mortality rate of about 20%. The two main clinical manifestations are sepsis and meningitis. Meningitis is often complicated by encephalitis, a pathology that is unusual for bacterial infections. Listeria ivanovii is a pathogen of mammals, specifically ruminants, and rarely causes listeriosis in humans

· Gram-positive, short rods with rounded ends; may be curved; single, short chains or V forms. Catalase positive, aerobic/ facultative anaerobic

· Colonies are bluish grey, when seen under normal illumination and bluish green sheen under obligatory transmitted light.

· The different sources of L. monocytogenes include water, mud, sewage, feces of animals and human beings.

Listeria monocytogenes, a facultative anaerobe, is the causative agent of listeriosis. It is one of the most virulent food-borne pathogens, with 20 to 30 percent of clinical infections resulting in death. Listeriosis is the leading cause of death among foodborne bacterial pathogens, with fatality rates exceeding even Salmonella and Clostridium botulinum.

L. monocytogenes is a Gram-positive bacterium, in the division Firmicutes, motile at 30°C and below, but usually not at 37°C.

L. monocytogenes also causes meningitis in newborns.

8.10  Mycobacterium

Mycobacterium is a genus of family Mycobacteriaceae that includes pathogens known to cause serious diseases in mammals for example tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The Greek prefix ‘myco—’ means fungus, alluding to the way mycobacteria have been observed to grow like mould on the surface of liquids, when cultured. It is Gram-positive, but difficult to stain due to high wax content, acid-fast, non- motile, non-spore forming and non-branching rods.

Mycobacterium tuberculosis is a pathogenic species of the genus Mycobacterium and causative agent of most cases of tuberculosis. It was discovered in 1882 by Robert Koch. M. tuberculosis has an unusual, waxy coating on its cell surface (i.e. mycolic acid), that makes the cells impervious to Gram stain, however acid-fast technique is used. The physiology of M. tuberculosis is highly aerobic and requires high levels of oxygen. The most frequently used diagnostic methods for Tuberculosis are the tuberculin skin test and acid-fast stain.

It grows quite slowly therefore requires two weeks or more to show visible growth on a special media, for example, Loeffler’s serum medium

8.11 Corynebacterium

Corynebacterium is a genus of family Corynebacteriaceae Gram-positive, rod-shaped and is widely distributed in nature and is mostly innocuous. Some strains are useful in industry like C. glutamicum. Others can cause diseases. C. diphtheriae, for example, is responsible for diphtheria. The cell shape are straight to slightly curved rods with tapered ends, club shaped forms may also appear. These are non motile, asporogenous, aerobic or facultative anaerobic in nature. These are recognized by their clubbed shaped appearance. Meta-chromatic granules are formed. They are chemo-organotrophs and optimum growth temperature is 37°C. Corynebacterium boris is not pathogenic and causes rancidity in cream while C. pyogenes causes supportive mastitis in dairy animals.

8.12 Brevibacterium

Brevibacterium is a genus of order Actinomycetales and family Brevibacteriaceae.

Brevibacterium linens are ubiquitously present on the human skin, where it causes foot odour. The same bacterium is also employed to ferment several cheeses like Limburger. Its smell also attracts mosquitoes.

B. linens is Gram positive, non-motile, obligate aerobic, chemo-organotroph with respiratory metabolism. Optimum growth temperature ranges between 20-30°C, non-thermoduric. B. linens produce yellow to deep orange red carotenoid pigments. Usually present on the exterior of surface ripened Limburger type chesses. Contributes to the surface color of such cheeses and aids in ripening by proteolysis while improving the flavor or aroma by the production of methanethiol.

8.13  Propionibacterium

Propionibacterium is a genus of bacteria named for their unique metabolism; as these are able to synthesize propionic acid by using unusual transcarboxylase enzymes. Its members are mostly facultative parasites and commensals of humans and other animals, living in and around the sweat glands, sebaceous glands, and other areas of the skin. They are ubiquitous and do not cause problems for most people, but propionobacteria have been implicated in acne and other skin conditions. The strain Propionibacterium freudenreichii subsp. shermanii is used in cheese making to create Carbon dioxide bubbles that become ‘eyes’, round holes in the cheese. Propionibacterium is:

· Gram positive, non-motile, asporogenous and anaerobic/ aerotolerant in nature.

· Pleomorphic rods, club shaped with one end rounded and the other tapered or pointed.

· Cells may be coccoid, bifid, or branched, occur singly in pairs, or short chains (in V or Y shape of configuration), carbon dioxide (5%) atmosphere is good for growth. Optimum growth temperature ranges in between 30-32°C.

· Lactic acid and carbohydrates are converted into propionic/ acetic acids and carbon dioxide.