THEME:  I. Sanitary microbiology. Ecology of microorganisms. Microflora of a soil, water, air. Methods of their examination.

II. Human microflora and methods of its studying. Dysbacteriosis, causes of its appearance.

 

 

Microflora of the environment

Microbes are distributed everywhere in the environment surrounding us. They are found in the soil, water, air, in plants, animals, food products, various utensils, in the human body, and on the surface of the human body.

The relationship of micro-organisms with the environment has beeamed ecology (Gr.  oikos home, native land, logos idea, science). This is an-adaptive relationship. Micro-organisms have a remarkable ability to adapt themselves to certain environmental conditions. Individual ecology, population ecology, and association community ecology are distinguished. The study of micro-organism ecology forms the basis for understanding parasitism, zoonotic diseases, and particularly diseases with natural nidality, as well as for elaborating measures for the control of various infectious diseases.

 

Soil Microflora

Types of Microorganisms in Soil

Living organisms both plants and animals, constitute an important component of soil. The pioneering investigations of a number of early microbiologists showed for the first time that the soil was not an inert static material but a medium pulsating with life. The soil is now believed to be a dynamic or rather a living system, containing a dynamic population of organisms/microorganisms. Cultivated soil has relatively more population of microorganisms than the fallow land, and the soils rich in organic matter contain much more population than sandy and eroded soils. Microbes in the soil are important to us in maintaining soil fertility / productivity, cycling of nutrient elements in the biosphere and sources of industrial products such as enzymes, antibiotics, vitamins, hormones, organic acids etc. At the same time certain soil microbes are the causal agents of human and plant diseases.
The soil organisms are broadly classified in to two groups viz soil flora and soil fauna, the detailed classification of which is as follows.

Soil Organisms

A. Soil Flora

a) Microflora: 1. Bacteria 2. Fungi, Molds, Yeast, Mushroom 3. Actinomycetes, Stretomyces 4. Algae eg. BGA, Yellow Green Algae, Golden Brown Algae.

1. Bacteria is again classified in I) Heterotrophic eg. symbiotic & non – symbiotic N2 fixers, Ammonifier, Cellulose Decomposers, Denitrifiers II) Autrotrophic eg. Nitrosomonas, Nitrobacter, Sulphur oxidizers, etc.

b) Macroflora: Roots of higher plants

B. Soil Fauna

a) Microfauna: Protozoa, Nematodes

b) Macrofauna: Earthworms. moles, ants & others.

 

• Macroflora: Vascular plants, Mosses, etc.

• Microflora: Bacteria, Actinomycetes, Fungi, Algae

Influences on Microbial Activity

• Temperature (70°-100°F most active microbes)

• Moisture (Field capacity is optimal)

• Aeration (want a nice mix of pores filled with water and air)

• pH (optimal pH is 6-7)

• Soil organic matter

Soil Microorganisms: Bacteria

1.     Most numerous in soil

2.     Most diverse metabolism

3.     Can be aerobic or anaerobic

4.     Optimal growth at pH 6-8

5.     Examples: Nitrosomonas and Nitrobacter iitrification processes, N2 fixers, fire blight is caused by a bacterium

Soil Microorganisms: Actinomycetes

1.     Transitional group between bacteria and fungi

2.     Active in degrading more resistant organic compounds

3.     Optimal growth at alkaline pH

4.     2 important products:
– produce antibodies (streptomycin is produced by an actino)
– produce geosmin

5.     Negative impact – potato scab (Streptomyces scabies)

Soil Microorganisms: Fungi

1.     Dominate the soil biomass

2.     Obligate aerobes

3. Can survive desiccation
4. Dominate in acid soils
5. Negative impacts:
   – Apple replant disease (Rhizoctonia, Pythium, Fusarium, and Phytophtora)
   – Powdery mildew is caused by a fungus

6. Beneficials:– Penicillium

 

 

Nematode-trapping Fungus

 

Plant root / Soil / Microbial Interactions

Beneficial

• Symbiotic associations such as that found with Rhizobia (N2 fixing bacteria, ex. legumes)

• Fungi-mycorrhizal associations: important for water and P uptake; also improves soil structure

• Earthworm channels: improve permeability and aeration

Deleterious

• Agrobacterium (bacteria) cause gall formation in plants

• Fungi causing soil-borne plant rot diseases

• Rhizoctonia and Pythium (involved with replant disease)

Soil Nutrient Cycling

• Materials are broken down by macro and meso-fauna

• Nutrients are taken up and converted by lower life forms in the soil

• They convert these nutrients to organic forms within the cell or to inorganic forms released to soil

• These organisms die and are decomposed by other organisms

• This also releases inorganic ions for plant or other microbe uptake and…

• The cycle starts all over

Nitrogen Cycle: Nitrogen is the nutrient needed in largest amounts by plants and is the most commonly applied fertilizer. Excess N can have negative affects on plant growth and crop quality as well as harming the environment, especially water quality.

Nitrogen is present in one of five forms in soil:

1. Organic N: 90% of N is in organic form. It must be mineralized to become available.

2. Ammonium N (NH₄⁺): Inorganic, soluble form

3. Nitrate (NO₃^–): Inorganic, soluble form
4. Atmospheric N (N₂): 80% of atmosphere but unavailable to most plants except N-fixers

5. Nitrite (NO₂^–): only under anaerobic conditions. This form is toxic to plants and normally will not be present in significant amounts in soil.

 

N: Nutrient cycling and composting

 

Soil science was founded by V. Dokuchaev, P. Kostychev. S. Vinogradsky, V. Williams, and others. Soil fertility depends not only on the presence of inorganic and organic substances, but also on the presence of various species of micro-organisms which influence the qualitative composition of the soil. Due to nutrients and moisture in the soil the number of microbes in 1 g of soil reaches a colossal number — from 200 million bacteria in clayey soil to 5 thousand million in black soil. One gram of the ploughed layer of soil contains 1-10 thousand million bacteria.

 

 

Soil microflora

Soil microflora consists algae nitrifying nitrogen-fixing, denitrifying, cellulose-splitting  and sulfur bacteria, pigmented microbes fungi, protozoa, etc.

The blue-green algae play an important part in enrichment of the soil with nitrogen. The extent to which the soil is contaminated with microbes depends on its nature and chemical composition (Table 1).

The greatest amount of microbes (1 000000 per cu cm) is found in the top layer of soil at a depth of 5-15 cm. In deeper layers (1.5-5 m) individual microbes are found. However, they have been discovered at a depth of 17.5 m in coal, oil, and artesian water.

Table 1

Total Amount of Microbes in Different Soils according to the Direct Counting Method

 

Oil’ bacteria live in oil wells. Using paraffins (distillates of oil) as nutrients, they turn part of the oil into a thick asphalt-like mass with the formation of which natural oil reservoirs become clogged up. It has been calculated that in the ploughed layer of cultivated soil over an area of 1 hectare there may be from 5 to 6 tons of microbial mass.

The number of microorganisms in the soil depends on the extent of contamination with faeces and urine, and also on the nature of treating and fertilizing the soil. For example, ploughed soil contains 2.5 times more microbes than forest soil.

Saprophytic spores (B. cereus. B, meguterium, etc.) survive for long periods in the soil.

Pathogenic bacteria which do not produce spores due to lack of essential nutrients, and also as a result of the lethal activity of light, drying, antagonistic microbes, and phages do not live long in the soil (from a few days to a few months) (Table 2).

Usually the soil is an unfavourable habitat for most pathogenic species of bacteria, rickettsiae, viruses, fungi, and protozoa. The survival period of some pathogenic bacteria is shown in Table 2. However, the soil as a factor of transmitting a number of causative agents of infectious diseases is quite a complex substrate. Thus, for example, anthrax bacilli after falling on the soil produce spores which can remain viable for many years. In favourable conditions (in dark brown soil and chernozem) they pass through the whole cycle of development: during the summer months the spores germinate into the vegetative forms and then this cycle is repeated.

Table 2

Survival Period of Pathogenic Bacteria in the Soil

 

As is known, the spores of clostridia causing tetanus, anaerobic infections, and botulism, and of many soil microbes survive for long periods in the soil. The soil is the habitat for various animals (rodents) which are parasitized by the carriers of the causative agents of plague, tularaemia, the viruses of mosquito fever, haemorrhagic fever, encephalitis, agricultural leishmaniasis, etc. The cysts, of intestinal protozoa (amoeba, balantidium, etc.) spend a certain stage in the soil. The soil plays an important role in transmitting worm invasions (ascarids, hook-worms, nematode worms, etc.). Some fungi live in the soil. Entering the body they cause fusariotoxicosis, ergotism, aspergillosis, penicilliosis mucormycosis, etc.

Taking into consideration the definite epidemiological role played by the soil in spreading some infectious diseases of animals and man, sanitary-epidemiological practice involves measures directed at protecting the soil from pollution and infection with pathogenic species of microorganisms.

S. Vinogradsky, V. Omeliansky, N. Kholodny and others devised a method of  investigating soil microbes and used the results obtained in agriculture.

A valuable index of the sanitary condition of the soil is the discovery of the colibacillus and related bacteria, also enterococci, and Clostridium perfringens. The presence of the latter indicates an earlier faecal contamination.

 

Microflora of the Water

 

Water microbiology is concerned with the microorganisms that live in water, or can be transported from one habitat to another by water.

Water can support the growth of many types of microorganisms. This can be advantageous. For example, the chemical activities of certain strains of yeasts provide us with beer and bread. As well, the growth of some bacteria in contaminated water can help digest the poisons from the water.

However, the presence of other disease causing microbes in water is unhealthy and even life threatening. For example, bacteria that live in the intestinal tracts of humans and other warm blooded animals, such as Escherichia coli, Salmonella, Shigella, and Vibrio, can contaminate water if feces enters the water. Contamination of drinking water with a type of Escherichia coli known as O157:H7 can be fatal. The contamination of the municipal water supply of Walkerton, Ontario, Canada in the summer of 2000 by strain O157:H7 sickened 2,000 people and killed seven people.

The intestinal tract of warm-blooded animals also contains viruses that can contaminate water and cause disease. Examples include rotavirus, enteroviruses, and coxsackievirus.

Another group of microbes of concern in water microbiology are protozoa. The two protozoa of the most concern are Giardia and Cryptosporidium. They live normally in the intestinal tract of animals such as beaver and deer. Giardia and Cryptosporidium form dormant and hardy forms called cysts during their life cycles. The cyst forms are resistant to chlorine, which is the most popular form of drinking water disinfection, and can pass through the filters used in many water treatment plants. If ingested in drinking water they can cause debilitating and prolonged diarrhea in humans, and can be life threatening to those people with impaired immune systems. Cryptosporidium contamination of the drinking water of Milwaukee, Wisconsin with in 1993 sickened more than 400,000 people and killed 47 people.

Many microorganisms are found naturally in fresh and saltwater. These include bacteria, cyanobacteria, protozoa, algae, and tiny animals such as rotifers. These can be important in the food chain that forms the basis of life in the water. For example, the microbes called cyanobacteria can convert the energy of the sun into the energy it needs to live. The plentiful numbers of these organisms in turn are used as food for other life. The algae that thrive in water is also an important food source for other forms of life.

A variety of microorganisms live in fresh water. The region of a water body near the shoreline (the littoral zone) is well lighted, shallow, and warmer than other regions of the water. Photosynthetic algae and bacteria that use light as energy thrive in this zone. Further away from the shore is the limnitic zone. Photosynthetic microbes also live here. As the water deepens, temperatures become colder and the oxygen concentration and light in the water decrease. Now, microbes that require oxygen do not thrive. Instead, purple and green sulfur bacteria, which can grow without oxygen, dominate. Finally, at the bottom of fresh waters (the benthic zone), few microbes survive. Bacteria that can survive in the absence of oxygen and sunlight, such as methane producing bacteria, thrive.

Saltwater presents a different environment to microorganisms. The higher salt concentration, higher pH, and lower nutrients, relative to freshwater, are lethal to many microorganisms. But, salt loving (halophilic) bacteria abound near the surface, and some bacteria that also live in freshwater are plentiful (i.e., Pseudomonas and Vibrio). Also, in 2001, researchers demonstrated that the ancient form of microbial life known as archaebacteria is one of the dominant forms of life in the ocean. The role of archaebacteria in the ocean food chain is not yet known, but must be of vital importance

Another microorganism found in saltwater are a type of algae known as dinoflagellelates. The rapid growth and multiplication of dinoflagellates can turn the water red. This “red tide” depletes the water of nutrients and oxygen, which can cause many fish to die. As well, humans can become ill by eating contaminated fish.

Water can also be an ideal means of transporting microorganisms from one place to another. For example, the water that is carried in the hulls of ships to stabilize the vessels during their ocean voyages is now known to be a means of transporting microorganisms around the globe. One of these organisms, a bacterium called Vibrio cholerae, causes life threatening diarrhea in humans.

Drinking water is usually treated to minimize the risk of microbial contamination. The importance of drinking water treatment has been known for centuries. For example, in pre-Christian times the storage of drinking water in jugs made of metal was practiced. Now, the anti-bacterial effect of some metals is known. Similarly, the boiling of drinking water, as a means of protection of water has long been known.

Chemicals such as chlorine or chlorine derivatives has been a popular means of killing bacteria such as Escherichia coli in water since the early decades of the twentieth century. Other bacteria-killing treatments that are increasingly becoming popular include the use of a gas called ozone and the disabling of the microbe’s genetic material by the use of ultraviolet light. Microbes can also be physically excluded form the water by passing the water through a filter. Modern filters have holes in them that are so tiny that even particles as miniscule as viruses can be trapped.

An important aspect of water microbiology, particularly for drinking water, is the testing of the water to ensure that it is safe to drink. Water quality testing can de done in several ways. One popular test measures the turbidity of the water. Turbidity gives an indication of the amount of suspended material in the water. Typically, if material such as soil is present in the water then microorganisms will also be present. The presence of particles even as small as bacteria and viruses can decrease the clarity of the water. Turbidity is a quick way of indicating if water quality is deteriorating, and so if action should be taken to correct the water problem.

In many countries, water microbiology is also the subject of legislation. Regulations specify how often water sources are sampled, how the sampling is done, how the analysis will be performed, what microbes are detected, and the acceptable limits for the target microorganisms in the water sample. Testing for microbes that cause disease (i.e., Salmonella typhymurium and Vibrio cholerae) can be expensive and, if the bacteria are present in low numbers, they may escape detection. Instead, other more numerous bacteria provide an indication of fecal pollution of the water. Escherichia coli has been used as an indicator of fecal pollution for decades. The bacterium is present in the intestinal tract in huge numbers, and is more numerous than the disease-causing bacteria and viruses. The chances of detecting Escherichia coli is better than detecting the actual disease causing microorganisms. Escherichia coli also had the advantage of not being capable of growing and reproducing in the water (except in the warm and food-laden waters of tropical countries). Thus, the presence of the bacterium in water is indicative of recent fecal pollution. Finally, Escherichia coli can be detected easily and inexpensively.

See also Chlorination; Oil spills; Sewage treatment; Water pollution.

Read more: Water Microbiology – Bacteria, Microorganisms, Life, and Drinking – JRank Articles http://science.jrank.org/pages/7311/Water-Microbiology.html#ixzz2NVtehNPi

 

Read more: Water Microbiology – Bacteria, Microorganisms, Life, and Drinking – JRank Articles http://science.jrank.org/pages/7311/Water-Microbiology.html#ixzz2NVtT1OJa

 

Pseudomonas fluorescens, Micrococcus roseus, etc., are among the specific aquatic  aerobic microorganisms. Anaerobic bacteria are very rarely found in water.

The microflora of rivers depends on the degree of pollution and the quality of purification of sewage waters flowing into river beds. Micro-organisms are widespread in the waters of the seas and oceans. They have been found at different depths (3700-9000 m).

The degree of contamination of the water with organisms is expressed as saprobity which designates the total of all living matter in water containing accumulations of animal and plant remains. Water is subdivided into three zones. Polysaprobic zone is strongly polluted water, poor in oxygen and rich in organic compounds. The number of bacteria in 1 ml reaches 1 000000 and more. Colibacilli and anaerobic bacteria predominate which bring about the processes of  putrefaction and fermentation. In the mesosaprobic zone (zone of moderate pollution) the mineralization of organic substances with intense oxidation and marked nitrification takes place. The number of bacteria in 1 ml of water amounts to hundreds of thousands, and there is a marked decrease in the number of colibacilli. The ohgosaprobic zone is characteristic of pure water. The number of microbes is low, and in 1 ml there are a few tens or hundreds; this zone is devoid of the colibacillus.

Depending on the degree of pollution pathogenic bacteria can survive in reservoirs and for a certain time can remain viable. Thus, for example, in tap water, river, or well water  salmonellae of enteric fever can live from 2 days to 3 months, shigellae — 5-9 days, leptospirae — from 7 to 150 days. The cholera, vibrio El Tor lives in water for many months, the causative agent of tularaemia — from a few days to 3 months.

Tap water is considered clean if it contains a total amount of 100 microbes per ml, doubtful if there are 100-150 microbes, and polluted if 500 and more are present. In well water and in open reservoirs the amount of microbes in 1 ml should not exceed 1000. Besides, the quality of the water is determined by the presence of E. coli and its variants.

The degree of faecal pollution of water is estimated by the colititre or coli-index. The colititre is the smallest amount of water in millilitres in which one E. coli bacillus is found. The coli-index is the number of individuals off. coli found in 1 litre of water. Tap water is considered good if the colititre is within the limits of 300-500. Water is considered to be good quality if the coli-index is 2-3.

Due to the fact that Str. faecalis (enterococci) are constant inhabitants only of the intestine in man and warm-blooded animals, and are highly resistant to temperature variations and other environmental factors, they are taken into account with the colititre and coli-index for the determination of the degree of faecal pollution of water, sewage waters,  soil, and other objects. At present new standards of enterococcus indices are being worked out.

Water is an important factor for the transmission of a number of infectious diseases (enteric fever, paratyphoids, cholera, dysentery, leptospiroses, etc.).

Due to the enormous sanitary-epidemiological role of water in relation to the intestinal group of diseases, it became necessary to work out rapid indicator methods for revealing colibacillus and pathogenic bacteria in water.

These include the methods of luminescent microscopy for the investigation of water for the presence of pathogenic microbes and the determination of the increase of the titre of the phage. Upon the addition of specific phages to liquids containing a homologous microbe in 6-10 hours a considerable increase in the amount of phage particles can be observed.

For a more complete and profound study of the microflora of the soil and water capillary microscopy is used. The principle is that very thin capillary tubes are placed in the soil or water reservoirs after which their contents are exposed to microscopic investigations. This method reveals those species of micro-organisms which do not grow in ordinary nutrient media, and which for many years were unknown to microbiologists.

 

Microflora of the Air

Air Microbiology http://www.google.com.ua/url?sa=t&rct=j&q=microflora+of+the+air&source=web&cd=4&cad=rja&ved=0CEYQFjAD&url=http%3A%2F%2Fupendrats.blogspot.com%2F2009%2F08%2Fair-microbiology.html&ei=i7RBUfD1MuqN4ASqjYHoAw&usg=AFQjCNFWYo8bSilKy2HVsqzMPaVwN10XRQ

Of all environments, air is the simplest one and it occurs in a single phase gas. The relative quantities of various gases in air, by volume percentage are nitrogen 78%, oxygen 21 %, argon 0.9%, carbon dioxide 0.03%, hydrogen 0.01 % and other gases in trace amounts. In addition to various gases, dust and condensed vapor may also be found in air
Various layers can be recognized in the atmosphere upto a height of about 1000km. The layer nearest to the earth is called as troposphere. In temperate regions, troposphere extends upto about 11 km whereas in tropics up to about 16km. This troposphere is characterized by a heavy load of microorganisms. The temperature of the atmosphere varies near the earth’s surface. However, there is a steady decrease of about 1 DC per 150m until the top of the troposphere. Above the troposphere, the temperature starts to increase. The atmosphere as a habitat is characterized by high light intensities, extreme temperature variations, low amount of organic matter and a scarcity of available water making it a non hospitable environment for microorganisms and generally unsuitable habitat for their growth. Nevertheless, substantial numbers of microbes are found in the lower regions of the atmosphere.

Microbes Found in Air– In addition to gases, dust particles and water vapour, air also contains microorganisms. There are vegetative cells and spores of bacteria, fungi and algae, viruses and protozoan cysts. Since air is often exposed to sunlight, it has a higher temperature and less moisture. So, if not protected from desiccation, most of these microbial forms will die.Air is mainly it transport or dispersal medium for microorganisms. They occur in relatively small numbers in air when compared with soil or water. The microflora of air can be studied under two headings outdoor and indoor microflora.

Sources of Microorganisms in Air – Although a number of microorganisms are present in air, it doesn’t have an indigenous flora. Air is not a natural environment for microorganisms as it doesn’t contain enough moisture and nutrients to support their growth and reproduction.
Quite a number of sources have been studied in this connection and almost all of them have been found to be responsible for the air microflora. One of the most common sources of air microflora is the soil.
Soil microorganisms when disturbed by the wind blow, liberated into the air and remain suspended there for a long period of time. Man made actions like digging or plaguing the soil may also release soil borne microbes into the air. Similarly microorganisms found in water may also be released into the air in the form of water droplets or aerosols. Splashing of water by wind action or tidal action may also produce droplets or aerosols. Air currents may bring the microorganisms from plant or animal surfaces into air. These organisms may be either commensals or plant or animal pathogens. Studies show that plant pathogenic microorganisms are spread over very long distances through air. For example, spores of Puccinia graminis travel over a thousand kilometers. However, the transmission of animal diseases is not usually important in outside air.
The main source of airborne microorganisms is human beings. Their surface flora may be shed at times and may be disseminated into the air. Similarly, the commensal as well as pathogenic flora of the upper respiratory tract and the mouth are constantly discharged into the air by activities like coughing, sneezing, talking and laughing.
The microorganisms are discharged out in three different forms which are grouped on the basis of their relative size and moisture content. They are droplets, droplet nuclei and infectious dust. It was Wells, who described the formation of droplet nuclei. This initiated the studies on the significance of airborne transmission. A brief description of these agents is given below
Droplets

Droplets are usually formed by sneezing, coughing or talking. Each consists of saliva and mucus. Droplets may also contain hundreds of microorganisms which may be pathogenic if discharged from diseased persons. Pathogens will be mostly of respiratory tract origin. The size of the droplet determines the time period during which they can remain suspended.
Most droplets are relatively large, and they tend to settle rapidly in still air. When inhaled these droplets are trapped on the moist surfaces of the respiratory tract. Thus, the droplets containing pathogenic microorganisms may be a source of infectious disease.
Droplet Nuclei
Small droplets in a warm, dry atmosphere tend to evaporate rapidly and become droplet nuclei. Thus, the residue of solid material left after drying up of a droplet is known as droplet nuclei. These are small, 1-4µm, and light. They can remain suspended in air for hours or days, traveling long distances. They may serve as a continuing source of infection if the bacteria remain viable when dry. Viability is determined by a set of complex factors including, the atmospheric conditions like humidity, sunlight and temperature, the size of the particles bearing the organisms, and the degree of susceptibility or resistance of the particular microbial species to the new physical environment. If inhaled droplet nuclei tend to escape the mechanical traps of the upper respiratory tract and enter the lungs. Thus, droplet nuclei may act as more potential agents of infectious diseases than droplets.
Infectious Dust
Large aerosol droplets settle out rapidly from air on to various surfaces and get dried. Nasal and throat discharges from a patient can also contaminate surfaces and become dry. Disturbance of this dried material by bed making, handling a handkerchief having dried secretions or sweeping floors in the patient’s room can generate dust particles which add microorganisms to the circulating air. Microorganisms can survive for relatively longer periods in dust. This creates a significant hazard, especially in hospital areas. Infective dust can also be produced during laboratory practices like opening the containers of freeze dried cultures or withdrawal of cotton plugs that have dried after being wetted by culture fluids. These pose a threat to the people working in laboratories

Significance of Air Microflora – Although, when compared with the microorganisms of other environments, air microflora are very low in number, they playa very significant role. This is due to the fact that the air is in contact with almost all animate and inanimate objects.
The significance of air flora has been studied since 1799, in which year Lazaro Spallanzani attempted to disprove spontaneous generation. In t 837, Theodore Schwann, in his experiment to support the view of Spallanzani, introduced fresh heated air into a sterilized meat broth and demonstrated that microbial growth couldn’t occur. This formed the basis of modern day forced aeration fermentations. It was Pasteur in 1861, which first showed that microorganisms could occur as airborne contaminants. He used special cotton in his air sampler onto which the microorganisms were deposited.
He microscopically demonstrated the presence of microorganisms in the cotton. In his famous swaecked flask experiment, he showed that growth could not occur in sterile media unless airborne contamination had occurred.

Factors Affecting Air Microflora – A number of intrinsic and environmental factors influences the kinds and distribution of the microflora in air. Intrinsic factors include the nature and physiological state of microorganisms and also the state of suspension. Spores are relatively more abundant than the vegetative bacterial cells.
This is mainly due to the dormant nature of spores which enables them to tolerate unfavourable conditions like desiccation, lack of enough nutrients and ultraviolet radiation. Similarly fungal spores are abundant in the air since they are meant for the dispersal of fungi.
The size of the microorganisms is another factor that determines the period of time for which they remain suspended in air. Generally smaller microorganisms are easily liberated into the air and remain there for longer period. Fungal mycelia have a larger size and hence mainly fragments of mycelia will be present in air. The state of suspension plays an important role in the settling of microorganisms in air. Organisms in the free state are slightly heavier than air and settle out slowly in a quiet atmosphere. However, microorganisms suspended in air are only rarely found in the free state.
Usually they are attached to dust particles and saliva. Microorganisms embedded in dust particle settle out rapidly and in a quiet atmosphere they remain airborne only for a short period of time. Droplets which are discharged into the air by coughing or sneezing are also remain suspended in air for a short period of time. When their size decreases by evaporation they remain for a longer period in air.
Environmental factors that affect air microflora include atmospheric temperature, humidity, air current, the height at which the microorganisms are found etc. Temperature and relative humidity are the two important factors that determine the viability of microorganisms in aerosol. Studies with Serratia marcesens and E. coli show that the airborne survival is closely related to the temperature.
There is a progressive increase in the death rate with an increase in temperature from -18°C to 49°C. Viruses in aerosols show a similar behaviour. Particles of influenza, poliomyelitis and vaccinia viruses survive better at low temperature from 7 to 24°C.The optimum rate of relative humidity (RH) for the survival of most microorganisms is between 40 and 80 percent. Low and high relative humidity cause the death of most microorganisms. Almost all viruses survive better at a RH of 17 to 25 percent.
A notable exception is that of poliomyelitis which survives better at 80 to 81 percent. survival has been found to be a function of both RH and temperature. At all temperatures, survival is best at the extremes of RH. Irrespective of RH, an increase in temperature leads to decrease in survival time.Air current influences the time for which either the microorganisms or the particles laden with microorganisms remain suspended in air. In still air the particles tend to settle down. But a gentle air current can keep them in suspension for relatively long periods. Air current is also important in the dispersal of microorganisms as it carries them over a long distance.
Air currents also produce turbulence which causes a vertical distribution of air flora. Global weather patterns also influence the vertical distribution. High altitudes have a limiting effect on the air microflora. High altitudes are characterized by severe conditions like desiccation, ultraviolet radiation and low temperature. Only resistant forms like spores can survive these conditions. Thus high attitudes are characterized by the presence of spores and other resistant forms.

Distribution of Microbes in Air – No microbes are indigenous to the atmosphere rather they represent allochthonous populations transported from aquatic and terrestrial habitats into the atmosphere. Microbes of air within 300-1,000 or more feet of the earth’s surface are the organisms of soil that have become attached to fragments of dried leaves, straw or dust particles, being blown away by the wind. Species vary greatly in their sensitivity to a given value of relative humidity, temperature and radiation exposures.
More microbes are found in air over land masses than far at sea. Spores of fungi, especially Alternaria, Cladosporium, Penicillium and Aspergillus are more numerous than other forms over sea within about 400 miles of land in both polar and tropical air masses at all altitudes up to about 10,000 feet.
Microbes found in air over populated land areas below altitude of 500 feet in clear weather include spores of Bacillus and Clostridium, ascos­pores of yeasts, fragments of myceilium and spores of molds and strepto­mycetaceae, pollen, protozoan cysts, algae, Micrococcus, Corynebacterium etc.
In the dust and air of schools and hospital wards or the rooms of persons suffering from infectious diseases, microbes such as tubercle bacilli, streptococci, pneumococci and staphylococci have been demonstrated.
These respiratory bacteria are dispersed in air in the droplets of saliva and mucus produced by coughing, sneezing, talking and laughing. Viruses of respiratory tract and some enteric tract are also transmitted by dust and air. Pathogens in dust are primarily derived from the objects contaminated with infectious secretions that after drying become infectious dust.
Droplets are usually formed by sneezing, coughing and talking. Each droplet consists of saliva and mucus and each may contain thousands of microbes. It has been estimated that the number of bacteria in a single sneeze may be between 10,000 and 100,000. Small droplets in a warm, dry atmosphere are dry before they reach the floor and thus quickly become droplet nuclei.
Many plant pathogens are also transported from one field to another through air and the spread of many fungal diseases of plants can be predicted by measuring the concentration of airborne fungal spores. Human bacterial pathogens which cause important airborne diseases such as diphtheria, meningitis, pneumonia, tuberculosis and whooping cough are described in the chapter “Bacterial Diseases of Man”.

Air Microflora Significance in Hospitals – Although hospitals are the war fields for combating against diseases, there are certain occasions in which additional new infectious diseases can be acquired during hospitalization. Air within the hospital may act as a reservoir of pathogenic microorganisms which are transmitted by the patients.Infection acquired during the hospitalization are called nosocomial infections and the pathogens involved are called as nosocomial pathogens. Infections, manifested by the corresponding symptoms, after three days of hospitalization can be regarded as nosocomial infection (Gleckman & Hibert, 1982 and Bonten& Stobberingh, 1995). Nosocomial infection may arise in a hospital unit or may be brought in by the staff or patients admitted to the hospital.The common microorganisms associated with hospital infection are Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, members of Enterobacteriaceae and respiratory viruses. Development of high antibiotic resistance is a potential problem among nosocomial pathogens. For example, Methicillin Resistant Staphylococcus aureus (MRSA) and gentamicin resistant Gram-negative bacilli are of common occurrence. Even antiseptic liquids used would contain bacteria, for example Pseudomonas, due to their natural resistance to certain disinfectants and antiseptics and to many antibiotics.
Nosocomial pathogens may cause or spread hospital outbreaks. Nosocomial pneumonia is becoming a serious problem nowadays and a number of pathogens have been associated with it. (Bonten & Stobberingh, 1995). Frequent agents are Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Enterobacter, Klebsiella, Escherichia coli and Haemophilus influenzae. Other less frequent agents are enterococci, streptococci other than S. pneumoniae, Serratia marcescens, Citrobacter freundii, Acinetobacter sp. and Xanthomonas sp.
In addition Legionella, Chlamydia pneumoniae and Mycobacterium tuberculosis have also been reported. Nosocomial transmissions of tuberculosis from patients to patients and from patients to health care workers have also been well documented (Wenger et a/., 1995). There are two main routes of transmission for nosocomial pathogens, contact (either direct or indirect) and airborne spread. Airborne spread is less common than the spread by direct or indirect contact. It occurs by the following mechanisms. The source may be either from persons or from inanimate objects.
In case of spread from persons the droplets from mouth, skin scales from nose, skin exudates and infected lesion transmit diseases such as measles, tuberculosis, pneumonia, staphylococcal sepsis and streptococcal sepsis. Talking, coughing and sneezing produce droplets. Skin scales are shed during wound dressing or bed making.
In case of inanimate sources particles from respiratory equipment and air-conditioning plant may transmit diseases. These include Gram-negative respiratory infection, Legionnaire’s disease and fungal infections.

Air Microflora Significance in Human Health – The significance of air microflora in human health relies on the fact that air acts as a medium for the transmission of infectious agents. An adult man inhales about ‘5m3 of air per day. Although most of the microorganisms present in air are harmless saprophytes and commensals, less than I % of the airborne bacteria are pathogens.
Eventhough the contamination level is very low, the probability of a person becoming infected will be greatest if he is exposed to a high concentration of airborne pathogens. Carriers, either with the manifestation of corresponding symptoms or without any apparent symptoms, may continuously release respiratory pathogens in the exhaled air.
Staphylococcus aureus is the most commonly found pathogen in air since the carriers are commonly present. The number of S. aureus in air may vary between 0-l/m3 and 50/m3.
Practically speaking, outdoor air doesn’t contain disease causing pathogen in a significant number to cause any infection. The purity of outdoor air, however, is an essential part of man’s environment. Dispersion and dilution by large volume of air is an inherent mechanism of air sanitation in outside air.
In the case of indoor air chance for the spread of infectious disease is more, especially in areas where people gather in large numbers. For example, in theatres, schools etc.

Air-Borne Microorganisms and Human Diseases
Air-borne microorganisms cause dangerous diseases in human beings. A detailed study of these diseases falls under the preview of a text book of medical microbiology. A chart representing air-borne diseases is given below for ready reference :

 

The composition of the microbes of the air is quite variable. It depends on many factors: on the extent to which air is contaminated with mineral and organic suspensions, on the temperature, rainfall, locality, humidity, and other factors. The more dust, smoke, and soot in the air, the greater the number of microbes. Each particle of dust or smoke is able to adsorb on its surface numerous microbes. Microbes are rarely found on the surfaces of mountains, in the seas of Arctic lands covered with snow, in oceans, and in snow.

The microflora of the air consists of very different species which enter it from the soil, plants, and animal organisms. Pigmented saprophytic bacteria (micrococci, various sarcinae), cryptogams (hay bacillus, B. cereus, B. megaterium), actinomycetes, moulds, yeasts, etc., are often found in the air.

 

 

The number of microbes in the air vanes from a few specimens to many tens of thousands per 1 cu m. Thus, for example, the air of the Arctic contains 2-3 microbes per 20 cu m. In industrial cities large numbers of bacteria are found per 1 ml of air. In the forests, especially coniferous forests, there are few microbes because the volatile plant substances, phytoncides, have bactericidal properties which cause a lethal effect.

According to the investigations of E. Mishustin, 1 cu m of air in Moscow at an altitude of 500 m contains from 1100 to 2700 microbes while at an altitude of 2000 m only from 500 to 700. Some microbes (sporulating and moulds) were found at an altitude of 20 km, others at an altitude of 61 to 67 km. One gram of dust contains up to 1 million bacteria. Pathogenic species of microbes (pyogenic cocci, tubercle bacilli, anthrax bacilli, bacteria of tularaemia, rickettsiae of Q-fever, etc.) may be found in the surroundings of sick animals and humans, infected arthropods and insects, and in dust.

At present Streptococcus viridans serves as sanitary indices for the air of closed buildings, and haemolytic streptococci and pathogenic staphylococci are a direct epidemiological hazard.

Depending on the time of the year, the composition and the amount of microflora change. If the total amount of microbes in winter is accepted as 1, then in spring it will be 1.7, in summer— 2 and in autumn — 1.2.

The total amount of microbes in an operating room before operation should not exceed 500 per 1 cu m of air, and after the operatioot more than 1000. There should be no pathogenic staphylococci and streptococci in 250 litres of air. In operating rooms of maternity hospitals before work the number of saprophyte microflora colonies isolated from the air by precipitating microbes on meat-peptone agar within 30 minutes should not exceed 20. In 1 gram of dust in hospitals, there are up to 200000 pyogenic (haemolytic) streptococci.

The number of microbes in factories and homes is associated closely with the sanitary hygienic conditions of the building. In overcrowding,  poor ventilation and natural lighting and if the premises are not properly cleaned, the number of microbes increases. Dry cleaning processes, infrequent floor washing, the use of dirty rags and brushes, and drying them in the same room make the conditions favourable for the accumulation of microbes in air. The causative agents of influenza, measles, scarlet fever, diphtheria, whooping cough, meningococcal infections, tonsillitis, acute catarrhs of the respiratory tract, tuberculosis, smallpox, pneumatic plague, and other diseases can be transmitted through the air together with droplets of mucus and sputum during sneezing, coughing, and talking.

Microbes can be spread by air currents, by aerial dust and aerial droplets. During sneezing, coughing and talking, a sick person can expel pathogenic bacteria together with droplets of mucus and sputum into the surrounding environment within a radius of 1.0-1.5 m or more. Microorganisms contained in air can remain in three phases of the bacterial aerosol — droplet, droplet-nuclei, and dust. An aerosol is the physical system of solid or liquid particles suspended in a gaseous medium.

On the average a person breathes about 12000-14000 litres of air daily, while 99.8 per cent of the microbes contained in air are held back in the respiratory tract. The bacterial aerosol produced naturally in the nasopharynx, during sneezing and coughing is thrown into the air — up to 60000 droplets of different size. Among them almost 60 per cent consist of large droplets (100 mcm), 30 per cent — of average sized droplets (50 mcm), and 10 per cent — of small (5 mcm) droplets.

The greatest amount of bacteria is discharged during sneezing, less — during coughing, and still less — during talking. With each sneeze a man expels from 10000 to 1 000000 droplets. In one cough from 10 to 1000 droplets containing bacteria are discharged into the environment, and when a person utters 10-20 words — up to 80 droplets are expelled. The nature of the bacterial aerosol depends on the viscosity of the secretion excreted from the respiratory tract. A liquid secretion is dispersed into more minute droplets more easily than a viscous one. Near the person expelling the bacteria a more concentrated aerosol of bacterial droplets from 1 to 2000 mcm in size is produced. Most of the droplets are from 2 to 100 mcm in size. Large droplets from 100 to 2000 mcm in size are thrown out to a distance of 2-3 m and more and quickly settle on the ground. Small drops of the bacterial aerosol (1-10 mcm) can remain in a suspended condition for a long time (for hours or days).

The air is an unfavourable medium for microbes. The absence of nutrient substances, the presence of moisture, optimal temperature, the lethal activity of sunlight, and desiccation do not create conditions for keeping microbes viable and most of them perish. However, the relatively short period during which the microbes are in air is quite enough to bring about the transmission of pathogenic bacteria and viruses from sick to healthy persons, and to cause extensive epidemics of diseases such as influenza.

For the purpose of prophylaxis various methods are used in protecting humans from infection via air-borne dust. Thus, the sputum of tuberculosis patients is burned or  decontaminated, the room is frequently ventilated, and cleaned by mopping, the streets are sprayed, drainage and absorbers are used, and masks are used during sorting of wool and rags, etc. The air of operating rooms, isolating rooms, wards, and bacteriological laboratories is decontaminated by ultraviolet radiation (mercury-quartz, uviol lamps, etc.).

The laboratory investigation of air is carried out to determine the qualitative and quantitative composition of its microflora. This is achieved by using simple and complex methods. For a more accurate investigation of microbial contents of the air special apparatus are used (Rechmensky’s bacterial absorber, Krotov’s apparatus, Kiktenko’s apparatus (Fig. 1), and others).

 

 

Figure 1. Kiktenko’s apparatus for bacteriological testing of the atmosphere

At present rapid methods for the indication of microbes in the environment are being devised which will allow quick determinations of the presence of micro-organisms in the soil, the water and air.

 

Microflora of Food Products

Proteins, carbohydrates, vitamins and other nutrient substances contained in food products have a favourable effect not only on the preservation of different micro-organisms but also on their multiplication.

Products of sour milk and foodstuff’s produced by fermentation contain a great number of microbes which lend them flavour and consistency (specific microflora). Besides, micro-organisms or their spores may get into foodstuff’s from the environment (non-specific  microflora).

The reproduction of some micro-organisms may cause spoiling of food products which become unsuitable for eating. In some cases the foodstuffs may be seeded with Salmonella and Shigella organisms, staphylococci, Clostridium botulinun, Escherichia coli. Bacillus cereus, Clostridium perfringens, and other microbes which cause food toxicoinfections and other diseases among humans.

Milk may be contaminated with Mycobacterium bovis, Brucellae, Coxiella burnetti, pathogenic streptococci, and encephalitis viruses from sick animals. During transportation or when it is being bottled or treated milk may be infected with Salmonella and Shigella organisms, pathogenic streptococci and staphylococci, Corynebacterium diphtheriae. Vibrio cholerae, and other microbes by personnel who are sick or are microbe carriers.

Meat may have been contaminated when the animals or poultry were still alive but sick or it may be infected when they are slaughtered, cut, or when the carcasses are improperly stored and transported. Cl. perfringens, B. cereus, enteric bacteria. Streptococcus faecalis, Proteus, and other bacteria are usually found in meat. Meat and meat products, minced meat in particular, are most frequently contaminated during treatment when pathogenic microbes are found on the surface of the meat chopper, on the hands, and on the kitchen utensils (cutting board, etc.).

 

 

Bacteria from meat

 

 

The flesh of fish is infected with a wide variety of microbial species found in water, the scales and guts of fish, on the hands of persons involved in processing the fish products, and on various objects (knives, tables, boards used in preparing the fish, the deck of a fishing boat, etc.). The most dangerous micro-organisms are Cl. botulinum which produce an exotoxin in canned fish products and Vibrio parahaemolytica. When sanitary regimens are not observed, S. typhi, Sh. flexneri, and in some cases the El Tor vibrio are found in the flesh of fish and oysters.

Vegetables and fruit may be seeded with Shigella and Salmonella organisms, Vibrio cholerae, and microflora found in the soil and on the hands of persons who take part in their harvesting, packing, transportation, and those who sell them. Improperly canned vegetables (tomatoes, mushrooms, etc.) may sometimes be the cause of botulism.

Various microflora, pathogenic species among others (Salmonella organisms, fungi, actinomycetes), penetrate eggs quite often; egg powder may be contaminated with staphylococci.

Baker’s products are a relatively rare source of infection of man with pathogenic micro-organisms. Only those baked from grain left in the field the whole winter cause fusariotoxicosis due to pathogenic Fusarium genus moulds.

Among all food poisonings encountered among humans, 70 per cent are due to pathogenic bacteria. Salmonella organisms, staphylococci, and streptococci are most dangerous; they multiply and accumulate in the foodstuff’s without causing changes in the organoleptic properties.

In the different countries the quality norms of most foodstuffs are set by the All- State Standard (GOST) or Provisional Technical Specifications (VTU).

Microbiological methods for testing foodstuffs. Foodstuffs are tested in the following cases: (1) as a planned measure to control the observance of the sanitary and hygienic regimen in the preparation, storage, and realization of food, particularly those foodstuffs which are not subjected to treatment at high temperature; (2) when there is doubt concerning the quality of the food; (3) when food toxicosis or diseases due to the intake of food occur.

The main task of microbiological testing of a food product is the determination of the total content of microbes and the model sanitary microbes. The model sanitary microbe for most foodstuffs and water is E. coli.

Some foodstuffs are tested for the presence of Proteus vulgaris. Salmonella organisms, aerobic and anaerobic organisms and for the toxins of these microbes.

The technique of collecting the samples and the sanitary and bacteriologic examination are fixed strictly by instructions in the corresponding State Standard. It, for instance, specifies the methods for collecting samples and all stages in testing milk, cream, ice-cream, butter, koumiss (fermented mare’s milk), yoghurt, sour clotted milk, sour cream, acidophilin (sour fermented milk), cottage cheese and food prepared from it, dried dairy products, condensed milk, and cold beverages prepared from milk. For testing, liquid foodstuffs are diluted with sterile isotonic solution 1:10. Compact products are melted or ground in the mortar and diluted in sterile water 1:10.

Sanitary and bacteriologic tests of milk and dairy products consist in determination of the total microbial content (microbial count) and the coli titre. In sour dairy products (yoghurt, cottage cheese, cheese, etc.) the microbial count is not determined. The microbial count in milk is determined by a direct count and by inoculating nutrient media with 1.0 ml of different dilutions of the product that is tested. The dairy products should only contain microorganisms specific for the given food, e. g. lactic streptococci and lactobacilli in sour clotted milk, lactobacilli and yeasts in koumiss, etc.

The permissible microbial count in various dairy products ranges between 500 (children’s mixtures subjected to pasteurization and cooking) and 300000 (cow’s milk in cans and cisterns). The microbial count for pausterized milk kept in bottles and packets is 75 000, for ice-cream 250000, for condensed milk 50000, for dried cow’s milk 50000 per one millilitre.

The coli titre of dairy products is determined by a three-stage fermentation method and for most of them it ranges from 0.3 to 3; only for children’s milk mixtures (pausterized and cooked) it is above 11.1. Since  milk and dairy products may be vehicles of the causative agents of certain infectious diseases (typhoid fever, paratyphoid fevers, brucellosis, tuberculosis, Q fever, etc.), these agents are identified by special methods discussed in the corresponding sections of the special part of this textbook. If pathogenic micro-organisms are detected in dairy products it is unquestionable that these products are not fit to be eaten.

The sanitary and bacteriological testing of meat and meat products comprises  determination of the total amount of microbes per 1.0 g of the product and the presence of E. coli, Proteus vulgaris. Salmonella organisms, and anaerobes.

No stable norms have been fixed to date for the sanitary and bacteriological assessment of meat and meat products. According to the accepted provisional norms, the permissible microbial count for roast meat should be less than 500, for boiled sausage and meat jelly less than 1000. The coli titre for roast meat should be above 1 g, for boiled sausage and meat jelly more than 10. The presence of pathogenic and putrefactive microbes indicates that these products are not suitable for use.

Canned foodstuffs, such as canned meat, lard, beans, fish, vegetables and juices are also subjected to sanitary and bacteriological testing.  Canned food is tested microbiologically for aerobic and anaerobic micro-organisms and for the botulinum toxin. When there are epidemiological indications, canned food is tested for Salmonella organisms, pathogenic staphylococci, and Proteus vulgaris; the presence of these microbes shows that the canned food is spoiled and cannot be eaten. It is permissible for canned food to contaion-pathogenic sporulating microbes provided there is no bulging of the can and the organoleptic properties of the food are normal.

Fish, vegetables, and eggs are tested microbiologically usually in cases of food poisoning or diseases among humans. Tests are performed for detecting pathogenic and conditionally pathogenic microbes or their toxins by the commonly accepted methods.

Remnants of food and foodstuffs, vomit, lavage waters, faeces, blood, mucus, washings and scrapings, and autopsy material may be subjected to bacteriological testing according to the epidemiological indications or on instruction of a health officer.

The prevention of food poisoning and other diseases acquired through foodstuffs consists in the observance of sanitary and hygienic measures in preparation of food products, their storage, transportation, and realization. It is also necessary to observe strictly the rules for processing foodstuffs, especially for canning them. Since foodstuffs may be infected by the service staff among whom there may be sick persons or carriers of pathogenic microorganisms, all personnel of food-supplying establishments must be examined regularly. Control of the vectors of the causative agents of intestinal infections and health education among the population are extensively carried out.

 

Microflora of the body

Significance of the Normal Flora

The normal flora influences the anatomy, physiology, susceptibility to pathogens, and morbidity of the host.

Skin Flora

The varied environment of the skin results in locally dense or sparse populations, with Gram-positive organisms (e.g., staphylococci, micrococci, diphtheroids) usually predominating.

Oral and Upper Respiratory Tract Flora

A varied microbial flora is found in the oral cavity, and streptococcal anaerobes inhabit the gingival crevice. The pharynx can be a point of entry and initial colonization for Neisseria, Bordetella, Corynebacterium, and Streptococcus spp.

Gastrointestinal Tract Flora

Organisms in the stomach are usually transient, and their populations are kept low (10³ to 10⁶/g of contents) by acidity. Helicobacter pylori is a potential stomach pathogen that apparently plays a role in the formation of certain ulcer types. Iormal hosts the duodenal flora is sparse (0 to 10³/g of contents). The ileum contains a moderately mixed flora (10⁶ to 10⁸/g of contents). The flora of the large bowel is dense (10⁹ to 10¹¹/g of contents) and is composed predominantly of anaerobes. These organisms participate in bile acid conversion and in vitamin K and ammonia production in the large bowel. They can also cause intestinal abscesses and peritonitis.

Urogenital Flora

The vaginal flora changes with the age of the individual, the vaginal pH, and hormone levels. Transient organisms (e.g., Candida spp.) frequently cause vaginitis. The distal urethra contains a sparse mixed flora; these organisms are present in urine specimens (10⁴/ml) unless a clean-catch, midstream specimen is obtained.

Conjunctival Flora

The conjunctiva harbors few or no organisms. Haemophilus and Staphylococcus are among the genera most often detected.

Host Infection

Many elements of the normal flora may act as opportunistic pathogens, especially in hosts rendered susceptible by rheumatic heart disease, immunosuppression, radiation therapy, chemotherapy, perforated mucous membranes, etc. The flora of the gingival crevice causes dental caries in about 80 percent of the population.

Introduction

A diverse microbial flora is associated with the skin and mucous membranes of every human being from shortly after birth until death. The human body, which contains about 10¹³ cells, routinely harbors about 10¹⁴ bacteria (Fig. 6-1). This bacterial population constitutes the normal microbial flora . The normal microbial flora is relatively stable, with specific genera populating various body regions during particular periods in an individual’s life. Microorganisms of the normal flora may aid the host (by competing for microenvironments more effectively than such pathogens as Salmonella spp or by producing nutrients the host can use), may harm the host (by causing dental caries, abscesses, or other infectious diseases), or may exist as commensals (inhabiting the host for long periods without causing detectable harm or benefit). Even though most elements of the normal microbial flora inhabiting the human skin, nails, eyes, oropharynx, genitalia, and gastrointestinal tract are harmless in healthy individuals, these organisms frequently cause disease in compromised hosts. Viruses and parasites are not considered members of the normal microbial flora by most investigators because they are not commensals and do not aid the host.

Figure 6-1

Numbers of bacteria that colonize different parts of the body. Numbers represent the number of organisms per gram of homogenized tissue or fluid or per square centimeter of skin surface.

Significance of the Normal Flora

The fact that the normal flora substantially influences the well-being of the host was not well understood until germ-free animals became available. Germ-free animals were obtained by cesarean section and maintained in special isolators; this allowed the investigator to raise them in an environment free from detectable viruses, bacteria, and other organisms. Two interesting observations were made about animals raised under germ-free conditions. First, the germ-free animals lived almost twice as long as their conventionally maintained counterparts, and second, the major causes of death were different in the two groups. Infection often caused death in conventional animals, but intestinal atonia frequently killed germ-free animals. Other investigations showed that germ-free animals have anatomic, physiologic, and immunologic features not shared with conventional animals. For example, in germ-free animals, the alimentary lamina propria is underdeveloped, little or no immunoglobulin is present in sera or secretions, intestinal motility is reduced, and the intestinal epithelial cell renewal rate is approximately one-half that of normal animals (4 rather than 2 days).

Although the foregoing indicates that bacterial flora may be undesirable, studies with antibiotic treated animals suggest that the flora protects individuals from pathogens. Investigators have used streptomycin to reduce the normal flora and have then infected animals with streptomycin-resistant Salmonella. Normally, about 10⁶ organisms are needed to establish a gastrointestinal infection, but in streptomycin-treated animals whose flora is altered, fewer than 10 organisms were needed to cause infectious disease. Further studies suggested that fermentation products (acetic and butyric acids) produced by the normal flora inhibited Salmonella growth in the gastrointestinal tract. Figure 6-2 shows some of the factors that are important in the competition between the normal flora and bacterial pathogens.

Figure 6-2

Mechanisms by which the normal flora competes with invading pathogens. Compare this schematic with Figure 6-3.

The normal flora in humans usually develops in an orderly sequence, or succession, after birth, leading to the stable populations of bacteria that make up the normal adult flora. The main factor determining the composition of the normal flora in a body region is the nature of the local environment, which is determined by pH, temperature, redox potential, and oxygen, water, and nutrient levels. Other factors such as peristalsis, saliva, lysozyme secretion, and secretion of immunoglobulins also play roles in flora control. The local environment is like a concerto in which one principal instrument usually dominates. For example, an infant begins to contact organisms as it moves through the birth canal. A Gram-positive population (bifidobacteria arid lactobacilli) predominates in the gastrointestinal tract early in life if the infant is breast-fed. This bacterial population is reduced and displaced somewhat by a Gram-negative flora (Enterobacteriaceae) when the baby is bottle-fed. The type of liquid diet provided to the infant is the principal instrument of this flora control; immunoglobulins and, perhaps, other elements in breast milk may also be important.

What, then, is the significance of the normal flora? Animal and some human studies suggest that the flora influences human anatomy, physiology, lifespan, and, ultimately, cause of death. Although the causal relationship of flora to death and disease in humans is accepted, of her roles of the human microflora need further study.

Normal Flora of Skin

Skin provides good examples of various microenvironments. Skin regions have been compared to geographic regions of Earth: the desert of the forearm, the cool woods of the scalp, and the tropical forest of the armpit. The composition of the dermal microflora varies from site to site according to the character of the microenvironment. A different bacterial flora characterizes each of three regions of skin: (1) axilla, perineum, and toe webs; (2) hand, face and trunk; and (3) upper arms and legs. Skin sites with partial occlusion (axilla, perineum, and toe webs) harbor more microorganisms than do less occluded areas (legs, arms, and trunk). These quantitative differences may relate to increased amount of moisture, higher body temperature, and greater concentrations of skin surface lipids. The axilla, perineum, and toe webs are more frequently colonized by Gram-negative bacilli than are drier areas of the skin.

The number of bacteria on an individual’s skin remains relatively constant; bacterial survival and the extent of colonization probably depend partly on the exposure of skin to a particular environment and partly on the innate and species-specific bactericidal activity in skin. Also, a high degree of specificity is involved in the adherence of bacteria to epithelial surfaces. Not all bacteria attach to skin; staphylococci, which are the major element of the nasal flora, possess a distinct advantage over viridans streptococci in colonizing the nasal mucosa. Conversely, viridans streptococci are not seen in large numbers on the skin or in the nose but dominate the oral flora.

The microbiology literature is inconsistent about the density of bacteria on the skin; one reason for this is the variety of methods used to collect skin bacteria. The scrub method yields the highest and most accurate counts for a given skin area. Most microorganisms live in the superficial layers of the stratum corneum and in the upper parts of the hair follicles. Some bacteria, however, reside in the deeper areas of the hair follicles and are beyond the reach of ordinary disinfection procedures. These bacteria are a reservoir for recolonization after the surface bacteria are removed.

Staphylococcus epidermidis

S. epidermidis is a major inhabitant of the skin, and in some areas it makes up more than 90 percent of the resident aerobic flora.

Staphylococcus aureus

The nose and perineum are the most common sites for S. aureus colonization, which is present in 10 percent to more than 40 percent of normal adults. S. aureus is prevalent (67 percent) on vulvar skin. Its occurrence in the nasal passages varies with age, being greater in the newborn, less in adults. S. aureus is extremely common (80 to 100 percent) on the skin of patients with certain dermatologic diseases such as atopic dermatitis, but the reason for this finding is unclear.

Micrococci

Micrococci are not as common as staphylococci and diphtheroids; however, they are frequently present on normal skin. Micrococcus luteus, the predominant species, usually accounts for 20 to 80 percent of the micrococci isolated from the skin.

Diphtheroids (Coryneforms)

The term diphtheroid denotes a wide range of bacteria belonging to the genus Corynebacterium. Classification of diphtheroids remains unsatisfactory; for convenience, cutaneous diphtheroids have been categorized into the following four groups: lipophilic or nonlipophilic diphtheroids; anaerobic diphtheroids; diphtheroids producing porphyrins (coral red fluorescence when viewed under ultraviolet light); and those that possess some keratinolytic enzymes and are associated with trichomycosis axillaris (infection of axillary hair). Lipophilic diphtheroids are extremely common in the axilla, whereas nonlipophilic strains are found more commonly on glabrous skin.

Anaerobic diphtheroids are most common in areas rich in sebaceous glands. Although the name Corynebacterium acnes was originally used to describe skin anaerobic diphtheroids, these are now classified as Propionibacterium acnes and as P. granulosum. P. acnes is seen eight times more frequently than P. granulosum in acne lesions and is probably involved in acne pathogenesis. Children younger than 10 years are rarely colonized with P. acnes. The appearance of this organism on the skin is probably related to the onset of secretion of sebum (a semi-fluid substance composed of fatty acids and epithelial debris secreted from sebaceous glands) at puberty. P. avidum, the third species of cutaneous anaerobic diphtheroids, is rare in acne lesions and is more often isolated from the axilla.

Streptococci

Streptococci, especially β-hemolytic streptococci, are rarely seen oormal skin. The paucity of β-hemolytic streptococci on the skin is attributed at least in part to the presence of lipids on the skin, as these lipids are lethal to streptococci. Other groups of streptococci, such as α-hemolytic streptococci, exist primarily in the mouth, from where they may, in rare instances, spread to the skin.

Gram-Negative Bacilli

Gram-negative bacteria make up a small proportion of the skin flora. In view of their extraordinary numbers in the gut and in the natural environment, their scarcity on skin is striking. They are seen in moist intertriginous areas, such as the toe webs and axilla, and not on dry skin. Desiccation is the major factor preventing the multiplication of Gram-negative bacteria on intact skin. Enterobacter, Klebsiella, Escherichia coli, and Proteus spp. are the predominant Gram-negative organisms found on the skin. Acinetobacter spp also occurs on the skin of normal individuals and, like other Gram-negative bacteria, is more common in the moist intertriginous areas.

Nail Flora

The microbiology of a normal nail is generally similar to that of the skin. Dust particles and other extraneous materials may get trapped under the nail, depending on what the nail contacts. In addition to resident skin flora, these dust particles may carry fungi and bacilli. Aspergillus, Penicillium, Cladosporium, and Mucor are the major types of fungi found under the nails.

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Oral and Upper Respiratory Tract Flora

The oral flora is involved in dental caries and periodontal disease, which affect about 80 percent. of the population in the Western world. The oral flora, its interactions with the host, and its response to environmental factors are thoroughly discussed in another Chapter. Anaerobes in the oral flora are responsible for many of the brain, face, and lung infections that are frequently manifested by abscess formation.

The pharynx and trachea contain primarily those bacterial genera found in the normal oral cavity (for example, α-and β-hemolytic streptococci); however, anaerobes, staphylococci, neisseriae, diphtheroids, and others are also present. Potentially pathogenic organisms such as Haemophilus, mycoplasmas, and pneumococci may also be found in the pharynx. Anaerobic organisms also are reported frequently. The upper respiratory tract is so often the site of initial colonization by pathogens (Neisseria meningitides, C. diphtheriae, Bordetella pertussis, and many others) and could be considered the first region of attack for such organisms. In contrast, the lower respiratory tract (small bronchi and alveoli) is usually sterile, because particles the size of bacteria do not readily reach it. If bacteria do reach these regions, they encounter host defense mechanisms, such as alveolar macrophages, that are not present in the pharynx.

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Gastrointestinal Tract Flora

The stomach is a relatively hostile environment for bacteria. It contains bacteria swallowed with the food and those dislodged from the mouth. Acidity lowers the bacterial count, which is highest (approximately 10³ to 10⁶ organisms/g of contents) after meals and lowest (frequently undetectable) after digestion. Some Helicobacter species can colonize the stomach and are associated with type B gastritis and peptic ulcer disease. Aspirates of duodenal or jejunal fluid contain approximately 10³ organisms/ml in most individuals. Most of the bacteria cultured (streptococci, lactobacilli, Bacteroides) are thought to be transients. Levels of 10⁵ to about 10⁷ bacteria/ml in such aspirates usually indicate an abnormality in the digestive system (for example, achlorhydria or malabsorption syndrome). Rapid peristalsis and the presence of bile may explain in part the paucity of organisms in the upper gastrointestinal tract. Further along the jejunum and into the ileum, bacterial populations begin to increase, and at the ileocecal junction they reach levels of 10⁶ to 10⁸ organisms/ml, with streptococci, lactobacilli, Bacteroides, and bifidobacteria predominating.

Concentrations of 10⁹ to 10¹¹ bacteria/g of contents are frequently found in human colon and feces. This flora includes a bewildering array of bacteria (more than 400 species have been identified); nonetheless, 95 to 99 percent belong to anaerobic genera such as Bacteroides, Bifidobacterium, Eubacterium, Peptostreptococcus, and Clostridium. In this highly anaerobic region of the intestine, these genera proliferate, occupy most available niches, and produce metabolic waste products such as acetic, butyric, and lactic acids. The strict anaerobic conditions, physical exclusion (as is shown in many animal studies), and bacterial waste products are factors that inhibit the growth of other bacteria in the large bowel.

Although the normal flora can inhibit pathogens, many of its members can produce disease in humans. Anaerobes in the intestinal tract are the primary agents of intra-abdominal abscesses and peritonitis. Bowel perforations produced by appendicitis, cancer, infarction, surgery, or gunshot wounds almost always seed the peritoneal cavity and adjacent organs with the normal flora. Anaerobes can also cause problems within the gastrointestinal lumen. Treatment with antibiotics may allow certain anaerobic species to become predominant and cause disease. For example, Clostridium difficile, which can remain viable in a patient undergoing antimicrobial therapy, may produce pseudomembranous colitis. Other intestinal pathologic conditions or surgery can cause bacterial overgrowth in the upper small intestine. Anaerobic bacteria can then deconjugate bile acids in this region and bind available vitamin B₁₂ so that the vitamin and fats are malabsorbed. In these situations, the patient usually has been compromised in some way; therefore, the infection caused by the normal intestinal flora is secondary to another problem.

More information is available on the animal than the human microflora. Research on animals has revealed that unusual filamentous microorganisms attach to ileal epithelial cells and modify host membranes with few or no harmful effects. Microorganisms have been observed in thick layers on gastrointestinal surfaces (Fig. 6-3) and in the crypts of Lieberkuhn. Other studies indicate that the immune response can be modulated by the intestinal flora. Studies of the role of the intestinal flora in biosynthesis of vitamin K and other host-utilizable products, conversion of bile acids (perhaps to cocarcinogens), and ammonia production (which can play a role in hepatic coma) show the dual role of the microbial flora in influencing the health of the host. More basic studies of the human bowel flora are necessary to define their effect on humans.

Figure 6-3

(A) Scanning electron micrograph of a cross-section of rat colonic mucosa. The bar indicates the thick layer of bacteria between the mucosal surface and the lumen (L) (X 262,) (B) Higher (more…)

Urogenital Flora

The type of bacterial flora found in the vagina depends on the age, pH, and hormonal levels of the host. Lactobacillus spp. predominate in female infants (vaginal pH, approximately 5) during the first month of life. Glycogen secretion seems to cease from about I month of age to puberty. During this time, diphtheroids, S. epidermidis, streptococci, and E. coli predominate at a higher pH (approximately pH 7). At puberty, glycogen secretion resumes, the pH drops, and women acquire an adult flora in which L. acidophilus, corynebacteria, peptostreptococci, staphylococci, streptococci, and Bacteroides predominate. After menopause, pH again rises, less glycogen is secreted, and the flora returns to that found in prepubescent females. Yeasts (Torulopsis and Candida) are occasionally found in the vagina (10 to 30 percent of women); these sometimes increase and cause vaginitis.

In the anterior urethra of humans, S. epidermidis, enterococci, and diphtheroids are found frequently; E. coli, Proteus, and Neisseria (nonpathogenic species) are reported occasionally (10 to 30 percent). Because of the normal flora residing in the urethra, care must be taken in clinically interpreting urine cultures; urine samples may contain these organisms at a level of 10⁴/ml if a midstream (clean-catch) specimen is not obtained.

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Conjunctival Flora

The conjunctival flora is sparse. Approximately 17 to 49 percent of culture samples are negative. Lysozyme, secreted in tears, may play a role in controlling the bacteria by interfering with their cell wall formation. When positive samples show bacteria, corynebacteria, Neisseriae, and Moraxellae are cultured. Staphylococci and streptococci are also present, and recent reports indicate that Haemophilus parainfluenzae is present in 25 percent of conjunctival samples.

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Host Infection by Elements of the Normal Flora

This chapter has briefly described the normal human flora; however, the pathogenic mechanisms of various genera or the clinical syndromes in which they are involved was not discussed. Although such material is presented in other chapters, note that a breach in mucosal surfaces often results in infection of the host by members of the normal flora. Caries, periodontal disease, abscesses, foul-smelling discharges, and endocarditis are hallmarks of infections with members of the normal human flora (Fig. 6-4). In addition, impairment of the host (for example, those with heart failure or leukemia) or host defenses (due to immunosuppression, chemotherapy, or irradiation) may result in failure of the normal flora to suppress transient pathogens or may cause members of the normal flora to invade the host themselves. In either situation, the host may die.

Figure 6-4

Clinical conditions that may be caused by members of the normal flora

Microbiocenosis is microbial community of different bacterial populations, which colonize certain biotope. 

Biotope is an area with relatively homogenous conditions where microbial population can survive.

Ecological niche is the  place  or status of microbes in their biotic environment.  

Constant (obligate, resident, indigenous, autochthonous) microflora is native, no imported  one of different biotopes.

Transient (temporary, facultative, allochthonous) microflora is not aboriginal, it is acquired one.

Before birth, the human body has no normal flora. During the birth process, the body comes in contact with microbes in the external environment. Later with the initial feedings and exposure to an expanding environment some microorganisms find their way to a permanent residence in many parts of the body

Most organisms in the external environment apparently do not find the body to be a favourable habitat. Charactenstic features of different body areas, such as temperature, oxygen availability, nutrient availability, natural inhibitors and pH influence the population that is able to survive and establish itself. Because these conditions vary from site to site in the body different sites acquire considerably different organisms as their normal flora. Once the normal flora is established it benefits the body by preventing the overgrowth of undesirable organisms. Destruction of the normal flora frequently disrupts the status quo resulting in the growth of harmful organisms. This can be seen after the prolonged administration of broad spectrum antibiotics. For example if the normal flora of the intestinal tract and vagina  are largely destroyed, the yeast Candida albicans which is unaffected by these antibacterial antibiotics can grow unchecked to become the major organism in these areas. It then cm infect the mucous membranes and the skin, causing a severe inflammation. Another complication of antibiotic therapy is a severe gastroenteritis known as pseudomembranous colitis. This syndrome has been associated with several antimicrobial agents but the antibiotics clindamycin and lincomycin have been incriminated most often. The mechanism of this diarrhoea was elucidated when it was observed that the use of these antibiotics resulted in an overgrowth in the intestine of an organism identified is Clostridium difficile. This organism produces in enterotoxin that causes the gastroenteritis but it can do so only when antibiotic therapy destroys much of the other normal intestinal flora permitting it to grow unchecked.

Our normal flora can be categorized is helpful (mutualutic symbionts) harmless (commensals) or potentially harmful (opportunists) However these groups are not mutually exclusive. Under certain circumstances even a mutualism cause harm and, thus, become a pathogen. Therefore these categories are of value only in describing the usual role of the organism in relation to its host.

In a mutualistic relationship the microbe and the host benefit one another. This type of relationship is common in the plant kingdom and is essential in ruminants such as cattle in which microbes are necessary for digestion of the cellulose in plant material. Few such relation ships exist in humans, however. Probably the only good example of mutualism in humans is found in the normal flora of the large intestine where enteric organisms synthesize vitamin K and the vitamins of the B complex,  enabling them to be absorbed through the intestinal wall and contribute to humautrition. However considering that our normal flora provides us with protection by interfering with the growth of potentially harmful organisms much of our normal flora could be considered mutualistic symbionts.

The microbe that lives on and benefits from its host without either benefiting or harming the host is called a commensal. Most of the organisms that make up the normal flora of a healthy individual could be categorized as commensals.

Opportunists (microbes that are potential pathogens) are of greatest interest to us. These organisms seem to lack the ability to invade and cause disease in healthy individuals but may be able to colonize as pathogens in ill or injured persons. Staphylococcus aureus is a good example of an opportunist. Many people (about 25 %) carry staphylococci in their nasopharynx without suffering any illness. However if these people acquire respiratory tract infections such as measles or influenza the staphylococci can invade the lung and cause severe pneumonia. Accidental contamination of the bladder with E coli or Enterococcus faecalis during a catheterization procedure also can lead to opportunistic infection. Both these organisms ire part of the normal flora of the large intestine and usually do not produce urinary tract infections. How ever if they gain access to the urethra or are transplanted mechanically to an environment in which they can grow they can cause disease. The most startling examples of opportunistic infections are associated with the viral infection known as acquired immunodeficiency syndrome (AIDS). This syndrome is caused by a virus that destroys certain subsets of T cells inhibiting the body’s ability to mount in immune response. As a result infection by the virus causing AIDS is characterized by severe and eventually fatal infections or malignancies that do not occur in individuals with functional immune systems.

Because much of our normal flora can cause disease under the proper conditions these organisms could be considered opportunists. This is particularly true in elderly and debilitated individuals and in patients receiving immunosuppressive drug therapy to prevent rejection of organ transplants. Opportunists are especially important as causes of nosocomial infections (ic those acquired during hospitalization).  Under appropriate circumstances most of the organisms that constitute our normal flora can cause disease.

Another group of bacteria that is not really part of our normal flora consists of pathogenic organisms that can exist in a large percentage of the population without causing disease. This group includes such organisms is Neisseria meningitidis (also called the meningococcus) the causative agent of epidemic meningitis. Many individuals carry this organism in their respiratory tracts without ever having meningitis, yet they can spread the bacterium to nonimmune individuals and cause disease. Streptococcus pneumoniae (the pneumococcus) the major cause of lobar pneumonia also is carried by 10 % to 20 % of normal healthy individuals. Persons who harbor bacteria such is these without ever exhibiting overt symptoms of disease are referred to as carriers.

Thus, although our normal flora can be beneficial by preventing the growth of potential pathogens, it also can be a reservoir from which endemic and epidemic diseases are spread.

A diverse microbial flora is associated with the skin and mucous membranes of every human being from shortly after birth until death. The human body, which contains about 10¹³ cells, routinely harbors about 10¹⁴ bacteria.This bacterial population constitutes the normal microbial flora. The normal microbial flora is relatively stable, with specific genera populating various body regions during particular periods in an individual’s life. Microorganisms of the normal flora may aid the host (by competing for microenvironments more effectively than such pathogens as Salmonella spp or by producing nutrients the host can use), may harm the host (by causing dental caries, abscesses, or other infectious diseases), or may exist as commensals (inhabiting the host for long periods without causing detectable harm or benefit). Even though most elements of the normal microbial flora inhabiting the human skin, nails, eyes, oropharynx, genitalia, and gastrointestinal tract are harmless in healthy individuals, these organisms frequently cause disease in compromised hosts. Viruses and parasites are not considered members of the normal microbial flora by most investigators because they are not commensals and do not aid the host.

 

 

 

Human microflora is the result of a mutual adaptation of micro- and macro-organisms in the process of evolution. Most bacteria of the normal and constant microflora of the human body have adapted themselves to life in certain parts of the body (tabl. 1, 2, 3). Besides, there are some microbes which make up a temporary (casual) microflora.

With the development of virology and the improvement of virological technique, our concepts on the microflora of the human body were increased. It has been established that not only the open cavities, but the tissues of the human organism are inhabited by numerous persisting viruses which are excreted into the environment with milk, saliva, sputum, perspiration, urine, and faeces.

There are different methods of human microbiocenosis studying. Among them, biopsy, pad method, impression method, sticky film method, swab-washing method, scrub-washing technique etc. After receiving of tested material it should be inoculated on different nutrient media.

Etiology of different human infections

 

Microflora of the skin. Staphylococci, streptococci, moulds and yeasts, diphtheroids, and also certain pathogenic and conditionally pathogenic bacteria live on the surface of the skin. They receive their nutrition from the secretions of the sebaceous and sweat glands, dead cells, and waste products.

The total number of microbes on the skin of one person varies from 85000000 to 1212000000.

When the human body comes into contact with the soil, the clothes and skin are seeded with spores of different species of microbes (organisms responsible for tetanus, anaerobic infections, etc.).

Most frequently the exposed parts of the human body are infected, e. g. the hands, on the surface of which colibacilli, staphylococci, streptococci, enterococci, moulds, yeasts, fungi imperfecti, and spores of aerobic and anaerobic bacilli are found.

Pustular and fungal infections of the skin and gastrointestinal diseases often occur owing to violation of sanitary-hygienic conditions and normal conditions of the work and life of people.

Microbes of the mouth cavity. In the mouth cavity there are more than 100 species of microbes. There are the natural inhabitants (acidophilic bacillus, Treponema microdentium, diplococci, Streptococcus salivarius, Entamoeba gingivalis, etc.). Besides, in the mouth cavity there are foreign microbes or those which have been carried in from the environment together with food, water, and air.

Pathogenic and conditionally pathogenic microbes (staphylococci, streptococci, diphtheria bacilli, diphtheroids, Borrelia organisms, spindle-shaped bacteria), protozoa (amoebae and trichomonads) are found on the mucous membrane of the mouth.

The mouth cavity is a favourable medium for many microbes; it has an optimal temperature, a sufficient amount of food substances, and has a weakly alkaline reaction.

The greatest amount of microbes can be found at the necks of the teeth and in the spaces between teeth. Streptococci and diplococci are found on the tonsils. There are many microbes in other parts of the mouth cavity, which are inaccessible to the bathing action of saliva and the action of lysozyme (an enzyme found in the saliva, lacrimal fluid and sputum). The presence of carious teeth is a condition for increasing the microflora in the mouth cavity, for the appearance of decaying processes and unpleasant odours.

The microflora of the gastrointestinal tract (Table 3, 4). When the stomach functions normally, it is almost devoid of microflora due to the marked bactericidal properties of gastric juice.

The gastric Juice is considered to be a reliable defense barrier against the penetration of  pathogenic and conditionally pathogenic microbes into the intestine. However, the degree of acidity of the gastric juice is not always constant. It varies according to the character of the food and the amount of water consumed.

 

Table 3.

BACTERIA CONSIDERED TO BE NORMAL FLORA OF HUMANS

AND THEIR ANATOMICAL LOCATIONS

 

“++” – prominent; “+” – common; “+/-” –“irregular, occasional or transient.

 

Together with food, lactic acid bacteria, Sarcina ventriculi. hay bacillus, yeasts, etc., enter the stomach from the mouth. In some cases, dysentery, enteric fever, and paratyphoid bacilli and other pathogenic microbes are capable of penetrating into the stomach and then the intestine.

Table 3

EXAMPLES OF TISSUE SPECIFICITIES OF SOME BACTERIA ASSOCIATED WITH HUMANS

 

 

Enterococci, fungi, and various other microbes are relatively rarely found in the duodenum. There are few microbes in the small intestine. Enterococci are found more often than others – In the large intestine there are large amounts of micro-organisms. Almost one-third of the dry weight of the faeces of certain animal species is made up of microbes. Daily, an adult human excretes about 17 million billion micro-organisms with the excrements (Table 4, 5).

Table 4.

EXAMPLES OF SPECIFIC ATTACHMENTS OF BACTERIA

TO HOST CELL OR TISSUE SURFACES

Bacterium	Ligand	Receptor	Attachment site	Disease
Streptococcus pyogenes	Protein F	Amino terminus of  fibronectin	Pharyngeal epithelium	Sore throat
Streptococcus mutans	Glycosyl transferase	Salivary glycoprotein	Pellicle of tooth	Dental caries
Streptococcus salivarius	Lipoteichoic acid	Unknown	Buccal epithelium of tongue	None
Streptococcus pneumoniae	Cell-bound protein	N-acetylhexosamine-galactose disaccharide	Mucosal epithelium	pneumonia
Staphylococcus aureus	Cell-bound protein	Amino terminus of fibronectin	Mucosal epithelium	Various
Neisseria gonorrhoeae	N-methylphenyl- alanine pili	Glucosamine-galactose carbohydrate	Urethral/cervical epithelium	Gonorrhea
Enterotoxigenic E. coli	Type-1 fimbriae	Species-specific carbohydrate(s)	Intestinal epithelium	Diarrhea
Uropathogenic E. coli	Type 1 fimbriae	Complex carbohydrate	Urethral epithelium	Urethritis
Uropathogenic E. coli	P-pili (pap)	Globobiose linked to ceramide lipid	Upper urinary tract	Pyelonephritis
Bordetella pertussis	Fimbriae (“filamentous hemagglutinin”)	Galactose on sulfated glycolipids	Respiratory epithelium	Whooping cough
Vibrio cholerae	N-methylphenylalanine pili	Fucose and mannose carbohydrate	Intestinal epithelium	Cholera
Treponema pallidum	Peptide in outer membrane	Surface protein(fibronectin)	Mucosal epithelium	Syphilis
Mycoplasma	Membrane protein	Sialic acid	Respiratory epithelium	Pneumonia
Chlamydia	Unknown	Sialic acid	Conjunctival or urethral epithelium	Conjunctivitis or urethritis

 

 

Table 5.

BACTERIA FOUND IN THE LARGE INTESTINE OF HUMANS

 

The intestinal microflora undergoes essential changes with the age of man. The intestinal tract of the newly-born baby in the first hours of life is sterile. During the first days it becomes inhabited by temporary microflora from the environment, mainly from breast milk. Later on, in the intestine of the newly-born baby a specific bacterial flora is established consisting of lactic acid bacteria (biphidobacteria, acidophilic bacillus), which is retained during the year. It has antagonistic properties in relation to many microbes capable of causing intestinal disorders in breastfed children, and remains during the whole period of breast feeding. However, on the 3rd-5th day of life in the intestine of breast-fed children E. coli and enterococci can be found, the amount of which sharply increases with the change to mixed feeding. After breast feeding is stopped the microflora of the child’s intestine is completely replaced by a microflora typical of adults (E. coli, Clostridium perfringens, Clostridium sporogenes, Streptococcus faecalis. Proteus vulgaris, etc.).

At present it has been established that such a constant inhabitant of the intestine of man as Clostridium perfringens is capable of secreting digestive enzymes. The colibacillus and other species of microbes in the intestine produce the vitamins essential for the human body (B₁, B₂, B₁₂, K). Microbe antagonists (acidophilic, Lactobacillus bulgaricus etc.) are beneficial to the organism as they hinder the development of pathogenic bacteria which, together with infected food and water, may enter the intestine.

The pathogenic serotypes of E. coli which are capable of causing severe diseases (colienteritis) mostly in children have been found to be present in the human intestine together with the on pathogenic species.

Anaerobic bacteria which do not produce spores, the so-called bacteroids, inhabit mainly the lower part of the large intestine in human. They are found during acute appendicitis, postpartum infection, pulmonary abscesses, septicaemia of different aetiology, postoperative infectious complications in the peritoneal cavity, inflammatory processes of the gastrointestinal tract, respiratory tract, and on the skin

Metchnikoff considered some species of intestinal bacteria to be harmful, causing chronic intoxications. He suggested the method (if combating them by introducing lactic acid bacilli (Lactohacillus bulgaricus) bearing antagonistic properties into the intestine. Besides, Metchnikoff recommended a diet of vegetables and fruit, rich in sugar, and considered it advisable to build one’s life according to the principles of orthobiosis (normal work, healthy relaxation, hygienic conditions, and prophylaxis of diseases).

Enteroviruses live in large quantities in the intestine. They may be found for a long time in healthy persons without causing diseases. In unfavourable conditions associated with some species of bacteria they cause the most varied clinical forms of disease.

Microflora of the respiratory tract. People breathe in a large number of dust particles and adsorbed micro-organisms. Experimentally, it has been established that the amount of microbes in inspired air is 200-500 times greater than in expired air Most of them are trapped in the nasal cavity and only a small amount enters the bronchi, The pulmonary alveoli and the terminal branches of bronchi are usually sterile. The upper respiratory tract (nasopharynx, pharynx) contains relatively constant species (Staphylococcus epidermidis, streptococci, diphtheroids, Gaffkya tetragena, etc.).

When the defense mechanisms of the body are weakened as a result of cooling, starvation, vitamin deficiency, or traumas, the constant inhabitants of the respiratory tract become capable of causing different diseases (acute catarrhs of the respiratory tract, tonsillitis, pneumonia, bronchitis, etc.).

The nasal cavity contains a small amount of microbes. The mucous membrane of the nose produces mucin and lysozyme which have a bactericidal action. However, in spite of this, the nasal cavity has a relatively constant microflora (haemolytic or nasal micrococcus, diphtheroids, non-haemolytic staphylococci, haemolytic staphylococci, saprophytic Gram-negative diplococci, capsular Gram-negative bacteria, haemoglobinophilic bacteria of influenza, Proteus, etc). In the respiratory tract, besides the bacterial microflora. many viruses, in particular adenoviruses. can remain viable for long periods without causing pathological processes.

Microflora of the vagina. In the first 2 days after birth the baby’s vagina is sterile. Sometimes it contains a small amount of Gram-positive bacteria and cocci. After 2-5 days of life the coccal microflora becomes fixed and remains constant until puberty, when it is replaced by Dodderlein’s lactic acid bacilli.

During the menstrual cycle the contents of the vagina become alkaline which is favourable for the development of coccal microflora. During sexual life the microflora of the vagina changes, and many microbes appear which are introduced from outside.

The microflora of the vagina undergoes profound changes during gynaecological diseases (endometritis, metritis, ovaritis, etc.), and after abortions.

The vaginal contents of the healthy woman have a relatively high concentration of sugar and glycogen, and a low content of the diastatic enzyme and proteins. The pH is 4.7 during which all other microbes except for Doderlein’s lactic acid bacilli, cannot develop.

As has been established, the acid medium of the vagina depends on the presence of glycogen which under the influence of vaginal bacteria is transformed into mono- and disaccharides and then into lactic acid. The amount of glycogen depends on the function of the ovaries and the condition of the whole body.

Vaginal bacteria have antagonistic properties; because of this, normal microflora should be protected and should not be exposed to the harmful effect of medicines (antibiotics, sulphonamide preparations, rivanol, osarsol. potassium permanganate, etc.) to which Doderlein’s lactic acid bacilli are more sensitive than the bacteria against which these substances are employed.

Microflora of the urinary tract. In men in the anterior part of the urethra there are Staphylococcus epidermidis. diphtheroids and Gram-negative non-pathogenic bacteria. Mycobacterium smegmatis and mycoplasmas are found on the external parts of the genitalia, and also in the urine of men and women.

The female urethra is usually sterile, in some cases it contains a small amount of non-pathogenic cocci.

The bacteria of the mucous membranes of the eyes include Staphylococcus epidermidis, Corynebacterium xerosis, mycoplasmas, etc. When the organism is weakened or when there are visual disturbances and vitamin deficiency, the normal inhabitants of the mucous membranes may become relatively pathogenic and may cause different diseases of the mucous membrane, such as conjunctivitis, blepharitis and other suppurative processes.

The normal microflora is not constant but depends on the age, nutrition and general condition of the macro-organism. The microflora of the human body undergoes profound changes, especially during various diseases.

Disturbances in the species composition of the normal microflora occurring under the influence of infectious and somatic diseases, and long-term and irrational use of antibiotics bring about the state of dysbacteriosis. This is characterized by disturbances in the assimilation of products of digestion, by changes in the enzymatic processes, and by the cleavage of ready-made physiological secretions. The territorial deviations of microflora cause a whole series of complications: intestinal dyspepsia, toxinfections, suppurative processes, catarrhs of the respiratory tract, pneumonia, candidiasis, etc. In dysbacteriosis the number of lactic acid bacteria is diminished, the number of anaerobes increased, Gram-positive bacteria change to Gram-negative and Gram-negative to Gram-positive, etc.

The question arises whether animal life is possible without microbes, It was already known in the last century that micro-organisms were very rarely found in birds and animals of the arctic regions. There were cases with absolutely no microflora found in the body of some birds. Pasteur made an attempt to raise amicrobic animals, but with the level of techniques of that time the problem could not be solved.

A new branch of biology, gnotobiology, is now developing. It studies the microbe-free organisms. Amicrobic chicks, rats, mice, guinea pigs, sucking pigs. and other animals have been reared.

Amicrobic animals, or gnotobiotes, are subdivided into several groups, monobiotes. (absolutely microbe-free animals), dibiotes (animals infected with one microbial species), polyobiotes (animals harbouring more than one species of microbes in their body).

Germ-free (gnotobiotic) animals are good experimental models for investigating the interactions of animals and microorganisms. To determine the role of the normal microbiota, animals can be delivered by aseptic Caesarean section (the surgical removal of the fetus from the uterus via the abdomen) so they will not be contaminated by the normal microbiota of the vagina and birth canal during vaginal delivery. Germ-free animals can been be raised in the absence of microorganisms by being kept in a sterile environment. They are fed sterile food and water and given sterile air to breathe. Comparing animals possessing normal associated microbiota with germ-free animals permits the exploration of the complex relationships between microorganisms and host animals.

Germ-free animals develop abnormalities of the gastrointestinal tract. They are more susceptible to disease than animals with normal associated microbiota. Germ-free animals are more susceptible to bacterial infection. Organisms such as Bacillus subtilis and Micrococcus luteus, which are harmless to other animals, cause disease in germ-free animals. More exotic pathogenic microorganisms such as Vibrio cholerae and Shigella dysenteriae are far more readily able to establish infections where there are no normal microbiota. They do not have to compete for survival within the intestinal tract. At the same time, though, germ-free animals are resistant to Entamoeba histolytica, the causative organism of amebic dysentery. This is because this protozoan requires normal intestinal bacteria as a food source. Likewise, tooth decay is no problem to germ-free animals, even those on high sugar diets, because they do not have lactic acid bacteria — the bacteria that cause tooth decay — in their oral cavities.

Scientists focused their attention at gnotobiotes because it was necessary to study deeply the role of normal microflora in the mechanisms of infectious pathology and immunity. As compared to the commonly encountered animals, gnotobiotes have a larger caecum, underdeveloped lymphoid tissue, internal organs of lesser weight, smaller blood volume, a reduced content of water in the tissue and of antibodies in blood serum.

Gnotobiology provides the means for revealing more precisely the role of normal microflora in the synthesis of vitamins and amino acids, in the production of congenital and acquired immunity, and the relationship of bacteria and viruses. Much importance is attached to gnotobiology in the study of space and the life conditions of man and animals during space flight.

 

Microbiological investigation of soil. For this purpose it is necessary to select most typical area not more then 25 m². The samples are taken from different places of the are field along the diagonal, the angles and the center 10 — 20 cms deep. The weight of each sample must be 100 – 200 g. The total weight of the soil 0,5 – 1 kg.

After careful mixing take an average sample of weight 100 – 200 g. Put the samples of soil in the sterile banks, mark and deliver to the laboratory. The soil specimens for plating are grinded in sterile mortar, make serial dilutions in an isotonic solution of sodium chloride 1: 10, 1:100, 1: 1000 etc. Plate 0,1 – 1 ml of specimens into special media for aerobic and anaerobic microbes. After incubation at optimal temperature count the  colonies on the plates.

 

 

Collection of soil

 

 

Examination of the general microbe number

 

Microbiological investigation of water. The sanitary – bacteriological investigation of water includes determination of total number of microbes in 1 ml of water, determination of a coli-index or coli-titer, and detection of pathogenic microbes, their toxins and Е. соli bacteriophages.

There are quantitative parameters of faecal pollution: a coli-titre and coli-index. Coli-titre is an index, which characterise  an amount of water in millilitres which contain  one E.coli. Coli-index characterises a number of E. coli in one litre of water.

The important part of investigation is taking of water samples. This procedure can do only by special persons (sanitary doctor or his assistant).

The samples of water for investigation are taken in 0,5 L sterile bottles.

 

Water collection

 

Water cocks, pumps, tubes previously must be sterilize by a flame using a cotton plug moistened before with alcohol. Then the water on for 10 min to wash off the bacteria which are on their surface inside. From the open reservoirs water for investigation is taken with the bathometer. This instrument is metal frame with a sterile bottle inside. It gives the possibility to take the water samples from any depth. Usually water is taken from the depth of 15 cms. The bottles with samples of water should have labels, which should mark the source, place  of sample,  time (day, hour), surname of the person who has taken the sampleassay, and also purpose of research.

Water is delivered to the laboratory immediately after its receival. The transportation should be done within 2 hours.  When doctor examines chlorinated water, it is taken in sterile bottles with 2 mL of 1,5 %  of solutions of Sodium hyposulphitum (Na₂S₂Оз • 5Н₂0). Such quantity of Sodium hyposulfite binds chlorine at its concentration up to 2 mg/L  in 500 ml. In summer water transports in ice, and in winter in special hot-water bottle (temperature  must be 1–5 °C). After delivery of samples to the laboratory the investigation begins. It  includes determination of total number of microbes and determination of a coli-titre and coli-index.

Quantitative Analysis. Bacteria cannot be accurately counted by microscopic examination unless
there are at least 100 million (10⁸) cells per mililiter Natural bodies of water, however, rarely contain more than 10⁵ cells per millilitre. The method employed is therefore the plate count. A measured volume of water is serially diluted (see below), following which 1 mL from each dilution tube is plated iutrient agar and the resulting colonies counted. Since only cells able to form colonies are counted, the method is also known as the “viable count”.

A typical example of serial dilution would be the following. One millilitre of the water sample is aseptically transferred by pipette to 9 mL of sterile water. The mixture is thoroughly shaken, yielding a 1:10 dilution (For obvious reasons, this is also known as the “10^–1” dilution). The process is repeated serially until a dilution is reached that contains between 30 and 300 colony-forming cells per millilitre, at which point several 1-mL samples are plated in a nutrient medium. Since the original sample may have contained up to 1 million (10⁶) viable bacteria, it is necessary to dilute all the way to 10^–5, plate 1-mL samples from each dilution tube, and then count the colonies only on those plates containing 30-300 colonies. The reasons for these numerical limits are that with over 300 colonies the plate becomes too crowded to permit each cell to form a visible colony, whereas with below 30 colonies the percent counting error becomes too great. (The statistical error of sampling can be calculated as follows’ The standard deviation of the count equals the  square root of N, where N equals the average of many samples. Ninety-five percent of all samples will give counts within 2 standard deviations of the average. For example, if the average count is 36, then 95% of all samples will lie between 24 and 48 [36 ± 12]. In other words, within 95% confidence limits a sample count of 36 has an error of plus or minus 33%.) Assume that the above procedure has been carried out with the results shown in Table 3. The 10^–3 dilution has a suitable number of colonies, the others being either too high or too low for accuracy. The original water sample is calculated to have contained 72,000 (72 x 10³) viable cells per millilitre.

Qualitative Analysis. The methods of plating and enrichment culture are used to obtain a picture of the aquatic bacterial population. Although such methods are satisfactory for general biologic studies, they are inadequate for the purpose of sanitary water analysis; this involves the detection of intestinal bacteria in water, since their presence indicates sewage pollution and the consequent danger of the spread of enteric diseases. Since any enteric bacteria would be greatly outnumbered by other types present in the water samples, a selective technique is necessary in order to detect them. Two widely used procedures for sanitary water analysis are as follows:

Table.

Example of a viable count

 

              *Each count is the average of 3 replicate plates

 

1.Tube method. Dilutions of a water sample are inoculated into tubes of a medium which is  elective for coliform bacteria and in which all coliform bacteria but few noncoliform bacteria will form acid and gas. Such media include MacConkey’s medium, which contains bile salts as inhibitors of noncoliform bacteria; lactose-containing media; and glutamatecontaining media. Cultures showing both acid and gas may then be subjected to further tests to confirm the presence of Escherichia coli or closely related enteric gram-negative rods. Such tests include streaking cultures on a lactose-peptone agar containing eosin and methylene blue (EMB agar), on which E coli forms characteristic blue-black colonies with a metallic sheen; subculturing at 44 °C; and a series of diagnostic biochemical tests

2. Membrane filtration method. A large measured volume of water is filtered through a sterilized membrane of a type that retains bacteria on its surface while permitting the rapid passage of smaller particles and water (fig.1). The membrane is then transferred to the surface of an agar plate containing a selective differential medium for coliform bacteria (fig. 2). Upon incubation, coliform bacteria give rise to typical colonies on the surface of the membrane. The advantages of this method are speed (the complete test takes less than 24 hours) and quantitation, the number of coliform cells being determined for a given volume of water.

 

 

 

Figure. Water samples (100 ml)  are passed through bacteriological filters (0,2 to 0,45 mm pore size) to trap bacteria The filters with trapped bacteria are placed on a medium containing lactose as a carbon source, an inhibitor to suppress growth of noncoliforms and indicator substances to facilitate differentiation of coliforms. Coliform bacteria form distinct colonies on Endo medium

 

 

Figure. Colonies of Е. coli on membranous  filters.

 

The drinking water should not have more than 100 microbes in 1 ml. The microbic number in water of wells and open reservoirs can be up 1000.

During determination of a coli-index and coli-titre of water it is necessary to take into consideration the ability of Е. coli  of the man and animal to grow at 43 °C

 

Microbiological investigation of the air. The sanitary – hygienic investigation of the microflora of the air includes determination both the total number of microbes in 1 m³ of the air and revealing of pathogenic staphylococci and streptococci. For taking the samples sedimentation and aspiration methods are used.

Plate method (sedimentation method). The Petry’s dishes with meat-peptone agar or another special nutrient media for staphylococci and streptococci, for example blood agar, yolk-salt agar are used. They stay in open form at various height from a floor. It is recommend to take one sample on every 20 m² of a premises. Term of exposition depends on prospective quantity of microbes in the air. With a plenty of microorganisms a cup is opened for 5 – 10 minutes, with a little – for 20 — 40 minutes.

Place the dishes into 37 °C incubator for 24 hrs and then incubate for 48 hrs at room temperature (18-20 °C). Study colonies, count them,  and  isolate  pure culture of different microbes.

According to Omeliansky data on a surface of medium by 100 cm² sedimentate in 5 minutes as so many microbes, as they present in 10 L of air. For example, on the dish surface with MPA after 5 minute exposure 32 colonies have grown. It is necessary to calculate amount of microbes which are present in 1 m³ of the air, applying the Omeliansky’s formula. The plate has 78,5 cm² (S = pr² =3,14 • 5² = 78,5 см²). Thus,  it is possible to determine, what quantity of microbes (х) would grow at the given exposure on a surface of medium in 100 см²,

x = (32 • 100) : 78,5 = 40

This quantity of microbes contains in 10 L of the air, and in 1 m³ (1000 л) there will be  –  (40 • 1000) : 10 = 4000. 

There is a special table for determination of total number of microbes in 1 m³ of the air (Table.).

Table

Account of bacteria number in 1 m³ of the air at a 10-minute exposure

 

For determination of  microbial dissemination degree quantity of the colonies on the dish surface which have been counted should be multiplied with one of multiplier.

If on  a Petry’s dish (78,5 cm²) at a 10-minute exposure 40 colonies have grown, the  quantity of microbes in 1 m³ of the air will be equal 40 • 60 = 2400.

Aspiration method. It  is based on a shock action of an air jet about a surface of a medium. Krotov’s apparatus is used for this purpose. It give us the possibility to let pass 50 –100 L of air with a speed of 25 L per minute through clinoid chink in the special glass above the open dish MPA. The rotation of Petry’s dish (1 rotation/sec) provides uniform dispersion of microorganisms on all surface of a medium. Then dish is incubated in a thermostat at 37 °C for 18-24 hrs.

For example, 250 colonies are revealed  on the surface of dish after 2-minutes exposure with a 25 l/min speed. Thus the number of microbes (x) in 1 l of the air is: x = (250 • 1000) : 50 = 5000.

There are temporary standards       of a sanitary – hygienic state of the air: in operating room the   total number of microbes prior to the beginning of the operation must be no more than 500 in 1 m³, after the operation – 1000.

In preoperative and dressing rooms limiting number of microbes prior before the beginning of work – 750 microbes in 1 m³, after work  – 1500. In birth wards the total number of microbes is about 2000 in 1 m³ of the air, and staphylococci and streptococci  are not higher then 24 in 1 m³, and in newborn rooms  – about 44 in 1 m³.

 

SUPPLEMENT

http://interactive.usask.ca/Ski/agriculture/soils/soilliv/soilliv_micfl.html

http://www.google.com/search?hl=en&q=soil+microflora

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=647419&dopt=Abstract

http://jac.oxfordjournals.org/cgi/content/abstract/46/suppl_1/41

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3103209&dopt=Abstract

www.gsbs.utmb.edu/microbook/ch006.htm

www.pedresearch.org/cgi/content/full/54/5/739

 

References:

1.     Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 29-34.

2.     Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P. 23-27.

3.     Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P. 176-179.

5. Essential of medical microbiology /W. A. Volk.. et al.– 5 ed., 1995.