Theme 26

 

 

Theme 26. Sanitary microbiology. Sanitary microbiology of chemist shop.

Theme 27. Microbiological control of medicines in pharmaceutical manufacturing and pharmaceutical companies. Phytopathogenic microorganisms.

 

 

Plant pathology (also phytopathology) is the scientific study of plant diseases caused by pathogens (infectious organisms) and environmental conditions (physiological factors). Organisms that cause infectious disease include fungi, oomycetes, bacteria, viruses,viroids

, virus-like organisms, phytoplasmas, protozoa, nematodes and parasitic plants.

 

Plant associated bacteria may be beneficial or detrimental. All plant surfaces have microbes on them (termed epiphytes), and some microbes live inside plants (termed endophytes). Some are residents and some are transient. Bacteria are among the microbes that successively colonize plants as they mature. Individual bacterial cells cannot be seen without the use of a microscope, however, large populations of bacteria become visible as aggregates in liquid, as biofilms on plants, as viscous suspensions plugging plant vessels, or colonies on petri dishes in the laboratory. For beneficial purposes or as pathogens, populations of 106 CFU (colony-forming units/milliliter) or higher are normally required for bacteria to function as biological control agents or cause infectious disease.

 

Physiology

Most phytopathogenic bacteria are aerobic (live in the presence of oxygen) and some are facultative anaerobes which can grow with or without oxygen. Some bacteria have thick, rigid cell walls which willretain dye from a cellstaining method developed by Christian Gram, while other bacteria will not accept thisstain. This method ofstaining resultsin the bacteria being classed as Gram-positive or Gram-negative and is an important factor in identification and classification. Gram-positive bacteria appear purple and Gram-negative bacteria appear pink under magnification. Bacteria are also  distinguished by the different kinds of enzymesthey either can or cannot use for nourishment and the nutrient media on which they can grow.

 

Bacteria as plant pathogens can cause severe damaging diseases, ranging from spots, mosaic patterns or pustules on leaves (Figure 1) and fruits, or smelly tuber rots to plant death. Some cause hormone-based distortion of leaves and shoots called fasciation, or crown gall, a proliferation of plant cells producing a swelling at the intersection of stem and soil (Figure 2) and on roots.

 

 

Figure 1. Bacterial pustules on soybean by Agrobacterium tumefacienson.

 

Figure 2. Gall caused caused byXanthomonas campestris glycines. (Courtesy B. W. Kennedy) a young blueberry plant. (Courtesy R. S. yther)

 

 

 

Most of the plant pathogenic bacteria are either Gram-positive, classified within the PhylumActinobacteria, or Gram-negative, in the Phylum Proteobacteria. 

 

 

Dissemination

Dissemination of plant pathogenic bacteria is easy, but fortunately does not always result in disease. Dissemination commonly occurs by windblown soil and sand particles that cause plant wounding, particularly during or after rains or storms (Figure). Wounding is essential for entry by many plant pathogens. Aerosols generated by diurnal temperature fluctuations enable dissemination, if temperature and humidity are aligned (Hirano and Upper 1989). Some plant diseases require certain temperature conditions e.g. Pseudomonas syringae (synonym: P. savastanoi) pv. phaseolicola causes disease below 22°C (72 °F) and Xanthomonas campestris(syn: X. axonopodis) pv. phaseoli, above 22°C on dry bean (Phaseolus vulgaris). Both diseases can occur simultaneously under growth conditions in which day and night temperature differentials enable disease progression in susceptible plants. Infested (surface contamination) or infected seed or any plant part can be sources of bacterial inoculum. Machinery, clothing, packing material and water can also disseminate pathogens, as can insects and birds. Continual monoculture in an area will usually enable increases in inoculum, making it easier for pathogens to be disseminated.

 

 

 

 

 

 

Host-Pathogen Interactions

Infection of plants by bacteria can occur in multiple ways. Infection is generally considered to be passive, i.e. accidental, although a few cases of plant chemoattractants have been reported. Bacteria can be sucked into a plant through natural plant openings such as stomata, hydathodes or lenticels. They can enter through abrasions or wounds on leaves, stems or roots or through placement by specific feeding insects. The nutrient conditions in plants may be such as to favor multiplication in different plant parts e.g. flowers or roots. Wind-driven rain carrying inoculum can be highly effective. Artificially, bacteria are most commonly introduced into plants by wounding, by pressure-driven aerosols mimicking wind-driven rains, vacuum infiltration, or by seed immersion into inoculum.

 

 

Symptomatology

Symptomatology of bacterial diseases is extremely varied, but usually characteristic for a particular pathogen. Symptoms can range from mosaics, resembling viral infections, to large plant abnormalities, such as galls or distorted plant parts. Hormone disruption can produce characteristic abnormal growths on roots, stems, and floral structures (phyllody) and sometimes abnormal flower colors (virescence). The most common symptoms are spots on leaves (Figure 3) or fruit (Figure 4), blights or deadening of tissue on leaves, stems or tree trunks, and rots (Figure5) of any part of the plant, usually roots or tubers. Wilts can also occur, due to plugging of vascular tissue (Figure 6). Symptoms may vary with photoperiod, plant variety, temperature and humidity, and infective dose. In some cases, symptoms may disappear or become inconsequential with further growth of the plant. For example, Holcus spot of corn caused by Pseudomonas syringae pv. syringae is arrested at the onset of hot dry weather.

 

Bacterial spots: the most common symptom of bacterial disease isleafspots. Spots appear on leaves, blossoms, fruits and stems. If the spots appear and advance rapidly the disease is considered a blight. Spots on leaves of dicotyledonous plants often have a rotten or fishy order, are watersoaked and are initially  confined between the leaf veins and will appearangular. In some cases bacterial ooze will be present; thisis diagnostic for bacterial infections. Sometimes a chlorotic halo will surround the bacterial lesion of an infected leaf. Spots may coalesce causing large areas of necrotic tissue. Bacterialspots will appear as streaks orstripes on monocotyledonous plants. Almost all bacterial leafspots and blights are caused by the genera Pseudomonas and Xanthomonas.

Cankers: primarily Pseudomonas and Xanthomonas cause canker disease ofstone fruitAngular leafspots5 and pome fruit trees, and canker disease of citrus respectively. Cankersymptoms can appear on Trunks,stems, twigs and branches. The most conspicuoussymptom of a bacterial canker disease in stone and pome fruit treesisthe development of cankers and gum exudation (gummosis).Cankers can  be slightly sunken, dark brown and much longer than  broad. The cortical tissue of the canker can be orangebrown to dark brown. Gum is produced in most cankers and some branches and twigs. Cankersthat do not produce gum may have a sour odor and be soft,sunken  and moist. Cankersthat girdle trunks and branches can  result in leafstress and eventual dieback ofthe portion of the tree distal to the canker.

Bacterial Galls: bacterial galls can be produced by the genus Agrobacterium and certain species of Arthrobacter, Pseudomonas, Rhizobacter and Rhodococcus. Agrobacterium tumefaciens, A.rubi and A. vitis alone are responsible for gallsin over 390 plant genera worldwide. Galls of these genera have been referred to as crown gall, crown knot, root knot and root gall. Species of these bacteria are thought to be present in most agriculture soil. A wound in  the host isrequired forthe pathogen to gain entry into the host tissue. Gall tissue is composed of disorganized, randomly proliferating cells that multiply in the intercellular (between the cells)spacesin the vicinity ofthe wound. In the presence of the pathogen rapid and continuous cell division  (hyperplasia and hypertrophy) of the plant tissue persists. Gall damage can be benign to deadly.Crown gallfirst appears assmall, whitish,soft round overgrowths typically  on the plants crown or at the main root. The color of galls (tumors) caused by A. tumefaciens can be orange-brown and Gummosis due to bacterial infection – Pseudomona ssp.

Bacterial Vascular Wilts: Vascular wilts caused  by bacteria primarily affect herbaceous plantssuch as vegetables, field crops, ornamentals and some tropical plants. The causal pathogen enters, multipliesin, and movesthrough the xylem vessels of the host plant and interferes with the translocation of nutrients and water by producing gum. The pathogen will often destroy parts of the cell wall of the xylem vesselsresulting in  pockets of bacteria, gums and cellular debris. The symptoms of bacterial wilt disease include wilting and death of the aboveground parts ofthe plant. In some cases bacterial ooze seeps out through stomata or cracks onto the surface of infected leaves. Usually this ooze dose not occur until the infected plant tissue is dead.

Bacterial Soft Rots: Primarily the bacteria that cause soft rotsin living plant tissue include Erwinia spp., Pseudomonasspp., Bacillusspp. and Clostridium spp. Many soft rots are caused by nonphytopathogenic bacteria which are saprophytesthat grow in tissue that has been killed by pathogenic or environmental causes. Softrots attack a large number of hosts and are best known for causing disease in fleshy plantstructures both above and below ground. These bacteria are almost always present where susceptible plants understress are in the field or in storage. Softrot pathogens enter the host through wounds. After entering the host tissue these bacteria produce enzymesthat break down the middle lamella causing separation ofthe cells at the site of the infection. The cells die and disintegrate. Rotting tissue becomes watery and soft and bacteria will form a slimy foulsmelling ooze that will ooze

Bacterial scabs: bacterialscabs primarily infect belowground parts of plantssuch as potatoes. Common scab of potato is caused by Streptomycesscabies which cause localized scabby lesions on  the outersurface of the tuber. Typically corky tissue will form below and around the lesion. Rot pathogens can gain entrance into the host tissue through these lesions and further degrade the host.

Diagnosis

Diagnosis of non-fastidious bacterial diseases depends on characteristic symptomatology, isolation of the presumed infectious agent, and physiological and/or molecular tests (Plant Disease Diagnosis). In heavily infected plants, bacterial populations in leaves or lesions may reach 10⁸ or 10⁹ CFU/gram of plant tissue, and actually visibly ooze from leaves or stems (Figure 18). A simple way to determine if a disease is caused by a bacterium is to cut a typical lesion or discolored area near its boundary with healthy tissue and suspend it in a droplet of water on a microscope slide. If a mass of moving small rods or ‘dots’ is seen at 400-1000x magnification flowing from the cut tissue under a microscope, you are observing bacterial streaming (Figure 19) which is an indicator of a bacterial disease. However, not all bacterial infections show streaming, or it may not be visualized without special microscope attachments. Serological tests, usually enzyme-linked, and physiological assays are available commercially for a few common and economically important bacteria. Molecular tests such as the polymerase chain reaction (PCR), based on specific genomic sequences, are becoming more readily available and used. Diagnostic tests are still evolving (Schaad et al. 2001), so that few are standardized and validated by multiple users, including governments.

Most plant pathogens are capable of inducing a hypersensitive reaction (HR) in plant species that are non-hosts or indicator plants (Klement et al. 1964). The HR is a plant defense mechanism elicited by the presence of a pathogen in non-host tissue. The tissue becomes sensitized to the pathogen, resulting in a rapid death of local plant cells (Figure 20), and entrapment of the pathogen. This, in effect, limits the spread of infection. One may use an HR test to determine if a colony isolated from infected plant tissue is a pathogen by introducing it, in a pure culture water suspension at 10⁸ CFU/ml, into a non-host leaf panel. Tobacco (Nicotiana tabacum) is frequently used in HR tests because its large leaf panels are easily infiltrated, but Four O’clock (Mirabilis jalapa) may be used for some Gram-positive bacteria. Collapse of host tissue in the infiltration area within 48 hours indicates the bacterium is likely a pathogen for another host.

Confirmation that a pathogen causes disease symptoms requires a host and performance of a pathogenicity test. This strategy can be time-consuming (days, weeks, or months). A pure culture of bacteria recovered from diseased tissue is artificially inoculated into the same or related cultivar or another susceptible host species, in an effort to reproduce the same disease symptoms. The bacteria should then be reisolated and compared with the inoculant culture.

With some practice, most bacterial diseases can be easily diagnosed. However, the variation that can occur with different strains may require more sophisticated testing.

Useful Plant Pathogens and Relatives

A few bacterial plant pathogens or their relatives have been widely used in agriculture and food production (Table 2). The thickening agent, xanthan gum, is an extra-cellular polysaccharide derived from the plant pathogen Xanthomonas campestris pv. campestris and is found in an enormous variety of products (Sutherland 1993). Transformation or genetic engineering of plants is best carried out by disarmed vectors (plasmids) of Agrobacterium tumefaciens. The elimination of a gene from a nonpathogenic Pseudomonas syringae that codes for ice formation at relatively high temperatures made history (Lindow 1987) in an ice-minus derivative that prevents frost damage when applied to plants. Other properties await discovery and exploitation.

 

http://horizon.documentation.ird.fr/exl-doc/pleins_textes/divers09-11/40441.pdf

 

 

ANALYSIS AND CONTROL OF PHARMACEUTICAL ENVIRONMENTS TO MINIMIZE MICROBIAL SURVIVAL

 

One of the most important areas in pharmaceutical process control is the development of systems to control the numbers, survival, and proliferation of microorganisms during manufacturing of nonsterile and sterile pharmaceutical products. The facility where products are manufactured is basically a closed environment where people and materials will move in and out to carry out different processes.

Microorganisms, as previously mentioned, have a great catabolic capacity to derive energy from any type of organic or inorganic compounds. Therefore, having microorganisms in a product can cause spoilage of the formula by breaking down active ingredients and excipients. This might compromise the potency and efficacy of the drug. Furthermore, the presence of high numbers of microorganisms and pathogens represents a serious health threat to consumers because products will be ingested, injected, or applied to human skin. Pharmaceutical products are commonly used after a pathological condition (e.g., disease) is diagnosed. The disease can be based upon microbial infection or metabolic disorders.

Therefore, minimizing the numbers or preventing the introduction of significant numbers of microorganisms into pharmaceutical facilities and processes becomes the most important aspect of process control during pharmaceutical manufacturing.  What are the critical areas where microorganisms can be introduced?

First, some of the raw materials utilized for the development of pharmaceutical formulations are based upoatural products that contain a high microbial load. The production processes for these raw materials do not eliminate all microorganisms. Therefore, they are not sterile. Testing must be performed to determine the quality of these materials. The absence of E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhimurium is required before raw materials can be used in pharmaceutical products. However, some of the manufacturing processes are designed to significantly reduce the number of microorganisms. Different types of bacteria commonly found in pharmaceutical raw materials are Lactobacillus spp.,Pseudomonas spp., Bacillus spp., Escherichia spp., Streptoccocus spp., Clostridium spp., Agrobacterium spp., etc. and molds such as Cladosporiums pp. and Fusarium spp.

 Therefore, a dry room provides a more hostile condition for microbes to grow than a humid room. A general practice in pharmaceutical environments is to apply ultraviolet light (UV) to reduce microbial contamination by air. Some of the microbial species commonly found in air samples in pharmaceutical environments are bacteria such as Bacillus spp., Staphylococcus spp., Corynebacterium spp. Common mold species are Aspergillus spp. and Penicillium spp.

A next critical area is the personnel in the plant and testing laboratories. Microorganisms are part of the normal flora of the human skin and body. Therefore, operators and laboratory analysts are the major sources of contamination during manufacturing and testing. Some of the species living in the human skin are Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, Propionibacterium spp., Propionibacterium acnes, Micrococcus spp., etc. The normal flora for the human oral cavity is comprised of Streptococcus salivarius,Streptococcus mutans, etc. Molds can also be possible contaminants. Common molds from human flora areTrichophyton spp., Epidermophyton spp., Microsporon spp., etc. To protect critical areas from human microbial flora, personnel wear gowns, hair covers, hoods, shoe covers, laboratory coats, face masks, gloves, boots, etc.

A third area of concern is water. Water is the most common raw material in pharmaceutical manufacturing. Drinking water is physically and chemically treated to reduce microbial numbers and pathogenic microorganisms. Water for pharmaceutical processes is further treated to minimize microbial numbers, endotoxin substances, and organic and inorganic com pounds. The less organic compounds there are in the water, the fewer microorganisms will be found. Bacterial species such Pseudomonas spp.,

Alcaligenes spp., Stenotrophomonas spp., Burkholderia cepacia, Burkholderia picketti, Serratia spp., and Flavobacterium spp. are commonly found in water samples. Other types of bacteria can also be present but when found, they indicate fecal sources of contamination. These bacteria are E. coli, Enterobacter spp., Klebsiella spp., Salmonella spp., Shigella spp., Clostridium perfringes, and Enterococcus spp. Recent studies using 16S ribosomal analysis, PCR amplification, and denaturing gradient gel electrophoresis (DGGE) testing demonstrated the presence of the following culturable bacterial species:Bradyrhizobium spp.,Xanthomonas spp., and Stenotrophomonas spp. However, the predominant bacterial type in the water system could not be detected on culture media.

Environmental monitoring of all critical areas also relies on standard microbiological assays. When microorganisms contaminate pharmaceutical products, standard methods are performed to quantify, detect, and identify the numbers and types of micro- organisms present in a given pharmaceutical batch. Standard, compendial methods are based upon the enrichment, incubation, and isolation of microorganisms from pharmaceutical samples.

1. INTRODUCTION

This sentence will discuss the microbiological analysis of nonsterile pharmaceutical products with emphasis in the microbiological test requirements and test methods. When a nonsterile pharmaceutical product is manufactured, quality control evaluation includes the microbiological testing of raw materials, excipients, active ingredients, bulk, and finished products. However, because of their nature, nonsterile samples contain high numbers of microbes and objectionable microorganisms that might represent a serious health threat to consumers. High number of microorganisms can also change the chemical composition of a given pharmaceutical formulation by spoilage, aecting the stability and integrity of the product and package. Furthermore, since these products are not sterile, a microbial bioburden is allowed based upon the product specifications. This means that although there are microorganisms present in the sample, their quantity and types will determine the safety of that particular pharmaceutical product and ecacy of the manufacturing process. Therefore the microbiological testing of nonsterile pharmaceuticals is defined as microbial limits.

Nonsterile pharmaceuticals are manufactured under aseptic conditions, but the processes used during production are not monitored on a regular basis. Furthermore, the criteria for manufacturing nonsterile pharmaceu- ticals are completely different when compared to sterile products. To date, there are no regulatory or compendial guidelines. However, according to the code of federal regulations (CFR), companies must have appropriate written procedures, designed to prevent the presence of objection able organisms from drug products not required to be sterile. This includes standard operating procedures (SOPs) for manufacturing and quality control analysis of each nonsterile product. Written procedures for manufacturing, packaging, and quality control analysis allow reproducibility, continuity, accuracy, and process control.

For instance, in sterile manufacturing, water, air, and environmental monitoring are performed on a routine basis preventing sterility failures and system breakdown. However, nonsterile manufacturing does not monitor these areas, if they monitor at all, as frequent as sterile processes. Therefore to control the presence, viability, and proliferation of microorganisms, effective environmental control, equipment and personnel sanitation, aseptic techniques, and good manufacturing practices (GMP) are needed.

The three major pharmacopoeias, U.S. (USP), European (EP), and Japanese (JP), have divided microbial limit testing into two different tests: the quantitative test and qualitative test. The quantitative test ascertains the numbers of microorganisms, bacteria, yeast, and mold present in a given pharmaceutical sample. The qualitative test determines the presence of specific pathogen indicators, e.g.,Salmonella spp., Staphylococcus aureus, Escherichia coli, P. aeruginosa, and the Enterobacteriaceae family which might cause disease to consumers or indicate the presence of other pathogenic bacteria. These indicators are representative microbial species of different types of bacterial populations. For instance,Salmonella spp. andE. coli are gram-negative rods, capable of lactose fermentation, commonly found in fecal sources.Salmonella spp. are virulent pathogens associated to intestinal disorders, while E. coli in general is not a virulent pathogen. However, some strains of  E. coli are known to be producers of toxins associated to gastro- intestinal diseases. P. aeruginosa is a gram-negative nonfermentative rod, which is typically associated to opportunistic infections. S. aureus is a grampositive cocci commonly associated to skin, gastrointestinal, and toxic shock syndrome conditions. The Enterobacteriaceae family comprises genera such as Escherichia, Salmonella, Shigella, Citrobacter, Enterobacter, Klebsiella, Proteus, etc. Most of the members of this family, other thanSalmonella spp. andShigella spp., are opportunistic pathogens. They are widely distributed in the environment.

2. MICROBIAL CONTAMINATION OF NONSTERILE PRODUCTS

This investigation must be fast and accurate so rapid corrective actions can be taken to prevent further contamination of production samples, huge financial losses, and release of contaminated product that can cause disease to consumers.

What are the sources of microbial contamination during the production of nonsterile pharmaceuticals? The great majority of the microbial contamination for nonsterile products has been reported to be due to the presence of microorganisms in raw materials or water or from poor practices during product manufacturing. For instance, manufacturing under nonsterile conditions requires operators to follow specific GMP practices such as raw material testing, equipment sanitization, and wearing of gloves, masks, hats, and laboratory uniforms. To provide continuity and reliability during the performance of all processes, written instructions and procedures are devel- oped for personnel use. Training of manufacturing and laboratory personnel is an important aspect of GMP compliance. Proper documentation of all training is necessary.

A comparison of published scientific studies showed that bacteria from the Enterobacteriaceae family, Pseudomonas spp., and B. cepacia are the most frequently found microorganisms in samples of pharmaceutical products from all over the world. Other nonopportunistic gram-positive bacteria also found are Staphylococcus spp., Bacillus spp.,Clostridium spp., and Streptococcus spp. Of the four USP, EP, and JP bacterial indicators, S. aureus, P. aeruginosa, and E. Coli were found in samples of toothpastes, topical products, shampoos, oral solutions, and disinfectants. On the basis of published scientific studies and government reports, gram-negative bacteria are found

to be the most common microbial contaminant ionsterile pharmaceutical samples regardless of geographical location or time. This indicates that the lack of process control in pharmaceutical environments represents the major factor for nonsterile product contamination.

What is the clinical significance of the presence of microorganisms in nonsterile pharmaceutical formulations? Of the four USP bacterial indicators,Salmonella spp. and some virulent strains of E. coli andS. aureus can cause disease when administered to healthy persons by a natural route. More generally, the USP bacterial indicators and other common pharmaceutical contaminants may cause disease in immunocompromised people or in other classes of susceptible persons. These classes include patients with se- vere preexisting disease, immunocompromised people, and newborn infants. For products intended for immunocompromised patients or infants, the limits must be lower than for people with functional immune systems. This is because the presence of any objectionable microorganism can be fatal for these patients.

 

3. RECOMMENDED MICROBIAL SPECIFICATIONS AND LIMITS

 

What are the threshold limits for the development of microbial specifications for objectionable microorganisms in pharmaceutical products? How many microorganisms are acceptable in a sample? What types of microorganisms are acceptable in a given pharmaceutical raw material and finished product? Are microorganisms, by the numbers and types, present in a sample dangerous to consumers and will they also affect the integrity of the product? There is no comprehensive list of microorganisms, which are called objectionable. Opportunistic pathogens cause disease in children with an infective dosage of 100 colony forming units (CFU), while for adults, 106 CFU are needed to colonize the gut. However, the U.S., European, and Japanese pharmacopoeias recommend different guidelines for the development of microbiological attributes for nonsterile pharmaceutical products. For instance, the USP suggests that some product categories such as plant-, animal-, and mineral-based formulations must be tested for Salmonella species. When products are designed to be administered orally, E. coli should also be tested. With topical pharmaceutical formulations, S. aureus and P. aeruginosa  must also be part of the routine microbiological testing. Vaginal, rectal, and urethral formulations are to be tested for yeast and mold.

The EP recommends more detailed guidelines on the quality of nonsterile pharmaceutical preparations. For the purpose of this chapter, category 2 includes all nonsterile formulations. For topical, transdermal patches, and respiratory tract drugs, a total viable count of not more than 100 CFU/g or mL is recommended. Absence of enterobacteria and other gram negatives, P. aeruginosa, and S. aureus is also recommended.

For category 3 formulations such as taken by oral and rectal route, recommendations specify a total viable count of not more than 1000 CFU/g or mL and not more than 100 CFU yeast and mold/g or mL. When these formulations are based upon raw materials of mineral, animal, or plant origin, the limits for total counts must be no more than 10,000 CFU/g or mL. Furthermore, not more than 100 enterobacteria and other gram-negative bacteria and absence of Salmonella spp.,S. aureus, and E. coli are also recommended. For herbal remedies formulated on one or more vegetable drugs, total viable counts should range from 105 to 107 CFU/g or mL for bacteria and from 104 to 105 CFU/g or mL for mold and yeast. If the formulation is added to boiling water before use, not more than 102 CFU/g or mL ofE. coli are recommended. However, if boiling water is not added,E. coli and Salmonella spp. must be absent.

 

4. TEST REQUIREMENTS

What are the tests required by the different pharmacopoeias for the analysis of nonsterile pharmaceuticals? What kind of criteria do we use to evaluate the effcacy of the methods for detecting microbial contamination ionsterile products?

According to the European (EP), Japanese (JP), and U.S. (USP) pharmacopoeias, for a nonsterile pharmaceutical product, microbial limit testing is performed in a stepwise manner; first, the sample is tested to determine the numbers of microorganisms. This will indicate how many bacteria, yeast, and molds are present in a sample. This is called microbial bioburden. Second, for qualitative analysis, the sample is incubated in broth for at least 24 hr to enhance the isolation of some pathogenic microorganisms. The reason for incubating the samples for at least 24 hr is due to the fact that pathogenic microorganisms are present in lower numbers thaonpathogenic microbes. An enrichment step and growth on selective media will enhance the isolation of pathogenic microorganisms such as Salmonella spp. and E. coli.

Before sample testing is performed, the methods must be shown to be capable of detecting and isolating bacteria, yeast, and mold. This part of the procedure is called the preparatory testing. The preparatory testing involves the inoculation of different types of microorganisms into the samples to demonstrate the accuracy, efficiency, reproducibility, and sensitivity of a given method for detecting microbial contamination. Because of the nonsterile nature of the products, the developing criteria for testing can be completely different for products with different applications. Prior to production, all raw materials are tested and qualified to be of a quality that will minimize the introduction of a significant number of microorganisms to the manufacturing process and finished product. For instance, an oral pharmaceutical product developed for transplant patients will have a completely different microbial limit approach than an oral dosage formulation for gas relief. Since the patients receiving the transplant drug may be immunocompromised, it might be safer to have zero counts of bacteria, yeast, and mold. The pathogen indicator specification can be expanded to include absence of any gram-negative rods. However, for the gas relief formulation targeting a healthy population, a limit of less then 100 colony forming units and absence of four pathogen indicators and gram-negative rods might be a reasonable specification. Therefore to develop the microbiological specifications, we must account again for the intended use of the product, nature of product, target population, manufacturing process, and route of administration.

 

 

5. TEST METHOD VALIDATION

5.1. Quantitative Test

To determine the accuracy and sensitivity of the test methods used for microbial limit testing, according to the USP, 10 g or mL samples of the test material are inoculated with separate viable cultures of S. aureus,Salmonella spp., E. coli, and P. aeruginosa. Some laboratories also use cultures of Candida albicans and Aspergillus niger to validate the quantitative recovery of yeast and mold. The EP recommends inoculating the samples withS. aureus, E. coli, B. subtilis, C. albicans, and A. niger. Same types of microorganisms are used in the JP with the exception of  A. niger. Although compendial recommendations are not specific regarding the number of samples required for method validation, at least three different production batches are generally used. That number will provide important information on the sensitivity, reproducibility, and accuracy of the validation data. When a validated formula has been modified or replaced, further validation work must be performed. Some companies also perform method validation on a yearly basis.

The procedure comprises the addition of no less than a 10 À3 dilution of a 24-hr broth culture of the recommended microorganisms to different dilutions of the test material in diluents such as phosphate buffer, buffered sodium chloride peptone solution, Letheen broth (LB), soybean casein digest broth (SCDB), or lactose broth (LacB). The recommended sample size is 10 g or 10 mL of test material. However, when production batches do not have a significant amount of sample, volumes of less than 10 g or mL can also be used. A positive control solution containing the microorganisms and the diluent without the test article is simultaneously analyzed. For instance, a 1:10 dilution of product suspension and control solution is inoculated with a given microbial culture, thoroughly mixed, and poured or spread plated on some of the most common bacterial growth media such as soybean casein digest agar (SCDA), microbial content test agar (MCTA), or Letheen agar (LA). Mold and yeast samples are plated on media such as Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), or mycological agar (MA). Incubation times for bacterial plates range from 2 to 5 days at 32–35ºC depending upon the company’s specifications. Mold and yeast plates are normally incubated for 5–7 days at 22–25ºC.

At least three replicas of the experiment must be performed and each should show that the average numbers of CFU recovered from the test article are not less than 70% of the inoculum control.

Evidently, a higher dilution of the product allows the recovery of all microorganisms. The testing conditions are then set for routine quality control analysis.

After filtration, the membrane is washed three or more times with a bufer solution to remove any residual antimicrobial substances. The membrane is then placed on agar media, which is incubated for a given period of time. When recovery values fall within the numbers mentioned above, the test ar- ticle is considered to be validated by membrane filtration.

In cases when any of the above strategies are not capable of recovering the microorganisms from the test article, it can be assumed that the strong antimicrobial nature of the formulation will destroy any microorganism present. However, proper documentation of the validation work showing the inefcient neutralization of different methods must be maintained and filed.

As an alternative to the plate count and membrane filtration methods, the USP, JP, and EP recommend the most probable number method (MPN) when no other method is available. However, this method is rarely used by industry. The accuracy and precision of the MPN is less than the plate count and membrane filtration. This method consists in the inoculation of different dilutions of the product suspensions into a suitable medium for bacterial enumeration. The samples are then incubated for 5 days at 30–35º C. After incubation, each dilution tube is observed for the detection of microbial growth by turbidity. The MPN of  bacteria per gram or milliliter is determined from specific tables. However, the MPN method does not provide reliable results for the enumeration of yeast and mold.

The final interpretation of the quantitative results for the EP and JP is based upon the sum of the bacterial count and the fungal count. This sum of the two values is called the total viable aerobic count. For the USP, results are reported separately as total aerobic microbial count and total yeast/fungal counts.

 

5.2. Qualitative Test

Once the quantitative recovery of microorganisms has been validated, the next step is to inoculate the test articles with specific microbial species that might indicate the presence of objectionable microorganisms. These microbial species are called indicators. The USP and JP recommend using the following bacterial species for the validation of pathogen screening: S. aureus, Salmonella spp., E. coli, and P. aeruginosa, while the EP includes the same species along with Enterobacter spp. and Clostridium spp. Although these are the species recommended for validation purposes, as previously discussed, there are reports of microbial contamination and products recalls due to other types of pathogenic or opportunistic microorganisms. Bacteria such as Acinetobacter spp., Pseudomonas putida, Pseudomonas fluorescens, Enterobacter spp., and Klebsiella spp. are frequently found in some samples. This indicates that the pathogen-screening test must not be limited to the recommended indicators but must include other pathogens that might generate serious health threats to consumers and compromise product integrity.

For the validation of the pathogen screening part of the USP, JP, and EP microbial limit test, a 10 À3 dilution of a 24-hr culture of the indicators  previously described or any other pathogenic species are inoculated into a dilution of the test article in SCDB, LB, and LacB with or without neutral izers. Again, sample dilution can range from 1:10 to 1:1000. If a 1:10 dilution does not recover the spiked microorganism, then further dilutions are tested, e.g., 1:100 and 1:1000, to determine the right dilution factor. Furthermore, as in the quantitative step, addition of neutralizers to the media might enhance the recovery of the microorganisms when antimicrobial ingredients are present.

After incubation, the samples are streaked onto different types of se- lective agar media. Incubation times range from 24 to 96 hr at 35–37ºC.

For Salmonella spp., the USP and JP require a preenrichment step in lactose broth followed by transfer to fluid selenite–cysteine medium (FSCM) and fluid tetrathionate medium (FTM). However, the EP requires an enrichment step prior to the lactose enrichment by using bufered sodium chloride peptone solution. After enrichment in FSCM and FTM, all procedures recommend transferring an aliquot of the enrichments on brilliant green (BGA), bismuth sulfite (BSA), and xylose lysine deoxycholate (XLD) agar.

For E. coli, the USP and JP protocols require streaking the lactose broth enrichments onto MacConkey agar medium (Mac). After incubation, if brick-red colonies of gram-negative rods surrounded by a reddish precipitation zone are not found, the samples are negative forE. coli

For Enterobacteriaceae, a preenrichment in lactose broth for 5 hr is the standard procedure. After preenrichment, subculturing in Entobacteriaceae enrichment broth (18–48 hr at 35–37ºC) followed by streaking plates of violet red bile glucose agar (VRBG) (18–24 hr at 35–37ºC) complete the procedure. Absence of growth indicates absence of gram-negative bacteria.

 

MICROBIOLOGICAL TESTING OF HERBAL

AND NUTRITIONAL SUPPLEMENTS

Nutritional supplements and herbal medicines are also tested to determine the microbiological quality of the raw materials and formulations. Because of the continuous health-related claims of these products, regulatory agencies are currently recommending the application of GMP to their manufacturing and quality control. This is done to control the quality, efficacy, and safety of these products.

The test methods are based upon the same requirements and methods described for nonsterile pharmaceutical products. However, these tests are not mandatory since the chapter is part of the informational sections of the USP. The only difference between the nonsterile test and the supplements is that yeast and mold are required to be part of the preparatory test (vali dation test), while nonsterile pharmaceuticals do not require these two microorganisms to be part of it.

 

CONCLUSION

Validation of microbiological testing for nonsterile pharmaceuticals provides a reliable way to determine the potential risk of high microbial bioburden and objectionable microorganisms in finished products and raw materials. Because a bioburden is allowed ionsterile pharmaceutical products, their microbiological risk is based upon the nature of the product, intended use, and route of application. Monitoring of critical areas such as facilities, equipment, raw materials, air, and water must be part of a testing plan to determine the efficacy of process control to minimize microbial contamination and the presence of objectionable microorganisms. A good microbiological program for nonsterile pharmaceuticals relies on cGMP practices to provide safe, stable, and efficacious products.

Contamination of  Pharmaceuticals with microorganisms irrespective of being harmful or objectionable or nonpathogenic, can bring about changes in their physical characteristics, including the breaking of emulsions, the thinning of creams, Fermentation of symps, and appearance of turbidity or deposit, besides producing possible off ordors and color changes.

 

Experimental

Collection of Samples

Non sterile oral Pharmaceutical products including cough syrups, multivitamin syrups, analgesics of different manufacturers were collected from various pharmacies

Test for Escheriehia coli Preparation of Enriched culture:

1.0 ml of each product (syrups, suspensions. drops) was added to 50 ml of sterile Nutrient broth in flasks. Flasks were incubated at 37°C for 24 hours. At the end of

incubation period flasks showing growth were subjected to primary test.

Primary Test:

1.0 ml of enriched culture of different samples were added to 5 nil of Sterile Macconkies broth in test tubes and were incubated at 37°C for 48 hours. At the end of  incubation period tubes were checked for acid and gas production. A secondary test was then carried out for tubes showing formation of add and gas.

Secondary Test:

0.1 ml of the contents of the tubes showing acid and gas was transferred to each of two sets of the tubes containing (a) 5 ml of Mac Conkey’s broth and (b) 5 ml of pepton water- The tubes were incubated at 44°C ± 1 for 74 hours. At the end of incubation period, the first set of tubes(a) were examined for acid and gas formation while second set of tubes (b) for the presence of Indole.

Indole Test:

For indole test 0.5 ml of Kovac’s reagent (Merck) was added into each of the tube. Development of a red colour in the reagent layer indicated the presence of indole. The presence of acid, gas and of Indole in the secondary test indicated the presence of E-coli.

Control Test:

A control test was carried out by repeating the primary and secondary tests adding one nil of enriched culture of E-coli.

Test for Salmonella Species:

Preparation of enriched culture

1.0 ml of each product was added separately to 100 ml of Nutrient broth in flasks. The flasks were incubated at 37°C for 24 hours. At the end of incubation period flasks showing growth were subjected to primary test.

Primary Test:

1.0 ml of the enriched culture was added to each of 2 sets of tubes containing

(a) 10 ml of sterile broth and

(b) 10 ml of tetrathionate broth, and incubated at 37°C for 48 hours.

At the end of the incubation period tubes showing growth were streaked separately on sterile brilliant green agar plates. The plates were incubated at 37°C for 24 hours. At the end of incubation period the colonies developed on these plates were compared with the official standard description (Bergy’s manual 1974).

Secondary Test:

A single colony from the plates was transferred to tripple sugar iron agar (TSI) slope. The TSI slope was incubated at 37°C for 24 hours. The formation of acid and gas in the stab culture (with or without) concomitant blackening and the absence of acidity from the surface growth indicated the presence of Salmonella species.

Control Test for Salmonella Species:

A control test was carried out by repeating the primary and secondary tests using one ml of enriched culture of Salmonella typhi, prepared from a 24 hours culture in

nutrient broth.

Test for pseudomonas Species:

1.0 ml of each sample was transferred to 100 ml of Cetrimide broth in flasks. The flasks were incubated at 32°C for 72 hours. Each flask was then subcultured on plates containing a layer of Certimide agar and incubated at 32°C for 48 hours. At the end of incubation period the resulting growths were examined by Gram’s stain and Oxidase test. A positive Oxidase test by Gram –ve bacilli indicated the presence of  Pseudomonas species.

Oxidase Test:

For Oxidase test few colonies from the Centrimide agar plates were transferred to a piece of filter paper and to this two to three drops of freshly prepared 1% w/w solution of N N N N Tetra methyl-p-Phenylenediammonium dichloride was added. Development of a purple colour with in 5-10 seconds indicated a positive Oxidase test.

Control Test:

A control test was carried out by repeating the test using one ml of enriched culture of Pseudomonas.

 

Sterility is not a requirement in official compondia for oral Pharmaceutical dosage forms. However contamination may occur during manufacturing, packaging and handling by the consumer. This causes concern, since some dosage forms if stored in favourable environment, can serve as substrates for microorganisms. Further more the contaminated drugs can mediate infection in man and hence harmful organisms should be absent from non-sterile Pharmaceutical preparations.

As indicated by the results the samples of analgesics, antipyretics, antidiarrhoeal,

anthelminhc, anabolic drops; antihistaminic, antiemetic, antibiotics, antimalarial, antacids and carminative tested in the present study did not show any growth though they are supposed to be non-sterile. It can be inferred that probably an excessive quantity of preservative is used in the preparation which prevented the growth of microorganisms. The organisms which are detected in the remaining samples are not pathogenic but they are “objectionable” as they can bring about the destruction of active ingredients and thus can interfere with the function of the therapeutic product.

 

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.