Clinical Microbiology I Lecture Notes (LM 323) 2-10-2018 PDF

CLINICAL MICROBIOLOGY I LM 323 Lecture no. (2) 2 -10 - 2018 Dr. Muhanmad Shakhatreh Traditional Cultivation and Identification of Bacteria Cultivation is the process of growing microorganisms in culture by taking bacteria from the infection site (i.e., the in vivo envi...

CLINICAL MICROBIOLOGY I LM 323 Lecture no. (2) 2 -10 - 2018 Dr. Muhanmad Shakhatreh Traditional Cultivation and Identification of Bacteria Cultivation is the process of growing microorganisms in culture by taking bacteria from the infection site (i.e., the in vivo environment) by some means of specimen collection and growing them in the artificial environment of the laboratory (i.e., the in vitro environment). Various principles and methods are required for bacterial cultivation and identification. Direct laboratory methods such as microscopy provide preliminary information about the bacteria involved in an infection Bacterial growth is usually required for definitive identification and characterization.  PRINCIPLES OF BACTERIAL CULTIVATION Bacterial cultivation has three main purposes: To grow and isolate all bacteria present in a clinical specimen To determine which of the bacteria that grow are most likely causing infection and which are likely contaminants or colonizers To obtain sufficient growth of clinically relevant bacteria to allow identification and characterization BACTERIAL CULTIVATION : Once grown in culture, most bacterial populations are easily observed without microscopy and are present in sufficient quantities to allow laboratory identification procedures to be performed. The successful transition from the in vivo to the in vitro environment requires that the nutritional and environmental growth requirements of bacterial pathogens be met.  BACTERIAL CULTIVATION The in vivo to in vitro transition is not necessarily easy for bacteria. In vivo they are utilizing various complex metabolic and physiologic pathways developed for survival on or within the human host. Then, relatively suddenly, they are exposed to the artificial in vitro environment of the laboratory. The bacteria must adjust to survive and multiply in vitro:. Their survival depends on the availability of essential nutrients and appropriate environmental conditions  Bacterial Isolation Techniques Microbial cultures are mostly mixed: Identification depend on isolating individual colonies Testing requires pure cultures Isolation technique provides an essential microbiological tool Original inoculum containing a mixture of bacteria is spread into 4 quadrants on solid media. To reduce the number of bacteria in each subsequent quadrant. Colonies are masses of offspring from an individual cell therefore streaking attempts to separate individual cells. Discrete colonies form as the individual cells are separated and then multiply to form isolated colonies in the later quadrants  NUTRITIONAL REQUIREMENTS Bacteria have numerous nutritional needs that include different gases, water, various ions, nitrogen, sources for carbon, and energy. General Concepts of Culture Media: In the laboratory: Nutrients are incorporated into culture media on or in which bacteria are grown. If a culture medium meets a bacterial cell’s growth requirements, then that cell will multiply to sufficient numbers to allow visualization by the unaided eye. Bacterial growth after inoculation also requires that the medium be placed in optimal environmental conditions. Because different pathogenic bacteria have different nutritional needs, various types of culture media have been developed for use in diagnostic microbiology. For certain bacteria the needs are relatively complex, and exceptional media components must be used for growth (bacteria with such requirements are said to be fastidious). Alternatively, the nutritional needs of most clinically important bacteria are relatively basic and straightforward (non- fastidious bacteria).  Phases of Growth Media Growth media are used in either of two phases: liquid (broth) or solid (agar). In some instances (e.g., certain blood culture methods), a biphasic medium that contains both a liquid and a solid phase may be used. In broth media, nutrients are dissolved in water, and bacterial growth is indicated by a change in the broth’s appearance from clear to turbid (i.e., cloudy). The turbidity, or cloudiness, of the broth is due to light deflected by bacteria present in the culture The more bacteria growth, the greater the turbidity. At least 1,000,000 bacteria per milliliter of broth are needed for turbidity to be detected with the unaided eye  Bacterial Culture media: Solid media are made by adding a solidifying agent to the nutrients and water. Agarose, the most common solidifying agent, has the unique property of melting at high temperatures (≥95° C) but re-solidifying only after its temperature falls below 50° C. Addition of agar allows a solid medium to be prepared by heating to an extremely high temperature, which is required for sterilization, and cooling to 55° to 60° C for distribution into petri dishes. On further cooling, the agarose-containing medium forms a stable solid gel referred to as agar. The petri dish containing the agar is referred to as the agar plate Different agar media usually are identified according to the major nutritive components of the medium (e.g., Sheep Blood Agar, bile esculin agar, xylose- lysine-desoxycholate agar).  Media culture: With appropriate incubation conditions, each bacterial cell inoculated onto the agar medium surface will proliferate to sufficiently large numbers to be observable with the unaided eye The resulting bacterial population is considered to be derived from a single bacterial cell and is known as a colony. All bacterial cells within a single colony are the same genus and species, having identical genetic and phenotypic characteristics (i.e., are a single clone). Bacterial cultures derived from a single colony or clone are considered “pure” Pure Cultures are required for subsequent procedures used to identify and characterize bacteria. The ability to select pure (individual) colonies is one of the first and most important steps required for bacterial identification and characterization. Bacteria grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.  Media Classifications and Functions Media are categorized according to their function and use. In diagnostic bacteriology there are four general categories of media: Enrichment, supportive, selective, and differential. Enrichment media contain specific nutrients required for the growth of particular bacterial pathogens that may be present alone or with other bacterial species in a patient specimen. This media type is used to enhance the growth of a particular bacterial Pathogen from a mixture of organisms by using nutrient specificity. One example of such a medium is buffered charcoal-yeast extract agar, which provides L-cysteine and other nutrients required for the growth of Legionella pneumophila, the causative agent of legionnaires’ disease Gram-negative bacterium Legionnaires' disease is a severe, often lethal, form of  Culture media: Supportive media contain nutrients that support growth of most nonfastidious organisms without giving any particular organism a growth advantage. Selective media contain one or more agents that are inhibitory to all organisms except those being sought. These media select for the growth of certain bacteria to the disadvantage of others. Inhibitory agents used for this purpose include dyes, bile salts, alcohols, acids, and antibiotics. An example of a selective medium is phenylethyl alcohol agar (PEA), which inhibits the growth of aerobic and facultatively anaerobic gram-negative rods and allows gram positive cocci to grow  Bacterial Culture media: Differential media employ some factor (or factors) that allows colonies of one bacterial species or type to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria growing on the same agar plate. One commonly used differential medium is MacConkey agar, which differentiates between gram-negative bacteria that can and cannot ferment the sugar lactose  Culture media: Many media used in diagnostic bacteriology provide more than one function. For example, MacConkey agar is both differential and selective because it will not allow most Gram-positive bacteria to grow. Another example is sheep blood agar (SBA). This is the most commonly used supportive medium for diagnostic bacteriology because it allows many organisms to grow. Blood agar is considered as nutritive and differential media because it differentiates between organisms based on hemolysis: In many ways this agar is also Beta hemolysis differential because the Alpha hemolysis appearance of colonies produced Gamma hemolysis by certain bacterial species is readily distinguishable Sheep Blood Agar  Isolation of bacteria from specimens To enhance the growth: Isolation, and selection of etiologic agents, specimen inoculum is usually spread over the surface of plates in a standard pattern so that individual bacterial colonies are obtained and semi quantitative analysis can be performed. Individual bacterial colonies are obtained and semi-quantitative analysis can be performed, using streaking technique, the relative numbers of organisms in the original specimen can be estimated based on the growth of colonies past the original area of inoculation. The commonly used streaking technique for isolation of bacteria from clinical specimens Loop is the flipped over or flamed and quadrant two is struck by pulling the loop through quadrant one of a few times and streaking the rest of the area. The nichrome loop is then flamed again and quadrant three is streaking the rest of the area. Finally quadrant four is streaked by pulling the loop over the rest of the agar without further flaming.  Bacterial growth and isolation A commonly used is streaking method: Using this method, the relative numbers of organisms in the original specimen can be estimated based on the growth of colonies past the original area of inoculation. To enhance isolation of bacterial colonies, the loop should be flamed for sterilization between streaking each subsequent quadrant. Streaking plates inoculated with a measured amount of specimen, such as when a calibrated loop is used to quantify colony-forming units (CFUs) in urine cultures, is accomplished by spreading the inoculum evenly over the entire agar surface This facilitates counting colonies by ensuring that individual bacterial cells will be well separated over the agar surface. Calibrated loop is used that delivers a specific amount of urine to the media Isolation of bacteria from specimens Bacterial Isolation Techniques  Bacterial Isolation: Microorganisms occur in huge numbers. Isolation of single species (pure culture) is based on diluting sample out to a point where a single cell will give rise to a single colony Using a sterile loop a portion of an isolated colony is taken and transferred to the surface of a suitable enrichment medium that is then incubated under conditions optimal for the organism. When making transfers for subculture it is beneficial to flame the inoculating loop between streaks to each area on the agar surface. Subculturing refers to transferring microorganisms from one medium to another. For example, bacteria growing in broth may be transferred to an agar plate. This avoids over-inoculation of the subculture media and ensures individual colonies will be obtained (To enhance isolation of bacterial colonies, the loop should be flamed for sterilization between streaking each subsequent quadrant). Once a pure culture is available in a sufficient amount an inoculum for subsequent identification procedures can be prepared  Pure bacterial culture isolation: In streaking method: Microorganisms present in the specimen are successively diluted out as each quadrant is streaked until finally each morphotype is present as a single colony Numbers of organisms present can subsequently be graded as: 4+ (many, heavy growth) if growth is out to the fourth quadrant, 3+(moderate growth) if growth is out to the third quadrant, 2+ (few or light growth) if growth is in the second quadrant, 1+ (rare) if growth is in the first quadrant. This tells the clinician the relative numbers of different organisms present in the specimen; Such semiquantitative information is usually sufficient for the physician to be able to treat the patient. Pure culture isolation (streaking method)  BACTERIAL CULTIVATION: ISOLATION OF BACTERIA FROM SPECIMENS The process of bacterial cultivation involves: The use of optimal artificial media and incubation conditions to isolate and identify the bacterial etiologies of an infection as rapidly and as accurately as possible. ISOLATION OF BACTERIA FROM SPECIMENS Cultivation of bacteria from infections at various body sites is accomplished by inoculating processed specimens directly onto artificial media. Incubation conditions are selected for media ability to support the growth of the bacteria most likely to be involved in the infectious process. Infection: The invasion and multiplication of microorganisms such as bacteria, viruses, and parasites that are not normally present within the Human host body  Evaluation of Colony Morphologies Initial evaluation of colony morphologies on the primary plating media is extremely important. Microbiologists can provide physicians with early preliminary information regarding the patient’s culture results. This information also is important for deciding which subsequent steps to take for definitive organism identification and characterization. Type of Media Supporting Bacterial Growth. Different media are used to recover particular bacterial pathogens so that determining which media support growth is a clue to the type of organism isolated (e.g., growth on MacConkey agar indicates the organism is a Gram negative bacillus). The incubation conditions that support growth may also be a preliminary indicator of which bacteria have been isolated (e.g., aerobic vs. anaerobic bacteria). Relative Quantities of Each Colony Type. Establishing the Bacterial Clinical Significance depends on : The predominance of a bacterial isolate is often used as one of the criteria, Direct smear results Microorganism virulence, The body site from which the culture was obtained  Colony Characteristics Noting key features of a bacterial colony is important for any bacterial identification; success or failure of subsequent identification procedures often depends on the accuracy of these observations. Criteria frequently used to characterize bacterial growth include the following: Colony size (usually measured in millimeters or described in relative terms such as pinpoint, small, medium, large) Colony pigmentation Colony shape (includes form, elevation, and margin of the colony Colony surface appearance (e.g., glistening, opaque, dull, transparent) Changes in agar media resulting from bacterial growth (e.g., hemolytic pattern on blood agar, changes in color of pH indicators, pitting of the agar surface Odor (certain bacteria produce distinct odors that can be helpful in preliminary identification)  Bacterial Colony Characteristics Many of these criteria are somewhat subjective, and the adjectives and descriptive terms used may vary among different laboratories. Regardless of the terminology used, nearly every laboratory’s protocol for bacterial identification begins with some agreed-upon colony description of the commonly encountered pathogens. Although careful determination of colony appearance is important, it is unwise to place total confidence on colony morphology for preliminary identification. Bacteria of one species often exhibit colony characteristics that are nearly indistinguishable from those of many other species. Bacteria of the same species exhibit morphologic diversity For example, certain colony characteristics may be typical of a given species, but different strains of that species may have different morphologies.  Gram stain and subculture: The Gram stain and microscopic evaluation of cultured bacteria are used with colony morphology to decide which identification steps are needed. To avoid confusion, organisms from single colony are stained. In many instances, staining must be performed with each different colony morphology that is observed on the primary plate. In other cases, staining may not be necessary because growth on a particular selective agar provides dependable evidence of the organism’s Gram stain morphology Following characterization of growth on primary plating media all subsequent procedures for definitive identification require the use of pure cultures (i.e., Cultures containing one strain of a single species). If sufficient inoculum for testing can be obtained from the primary media Then a subculture is not necessary except as a precaution: To obtain more of the etiologic agent if needed To ensure that a pure inoculum has been used for subsequent tests (i.e., a purity check).  Classification Systems in the Prokaryotes 1. Macroscopic morphology – colony appearance 2. Microscopic morphology 3. Physiological / biochemical characteristics 4. Chemical analysis 5. Serological analysis 6. Genetic and molecular analysis G + C base composition by assessing molecular guanine and cytosine (GC) content DNA analysis using genetic probes Nucleic acid sequencing and rRNA analysis The systematic classification and categorization of organisms into ordered groups: are called taxonomy: The critical feature for all Useful for: these classification systems is an organism identified by one individual (scientist, clinician, Diagnostic microbiology epidemiologist), is recognized as the same organism by Studies in epidemiology and pathogenicity another individual. The Gram stain, which divides most clinically significant bacteria into two main groups, is the first step in bacterial identification  PRINCIPLES OF IDENTIFICATION Microbiologists use various methods to identify organisms (most clinically relevant bacteria) cultivated from patient specimens. Accurate bacterial identification is very significant because identity is central to diagnostic bacteriology issues, including: - Determining the clinical significance of a particular pathogen (e.g., is the isolate a pathogen or a contaminant?) - Guiding physician care of the patient - Determining whether laboratory testing for detection of antimicrobial resistance is warranted - Determining the type of antimicrobial therapy that is appropriate - Determining whether the antimicrobial susceptibility profiles are unusual or aberrant for a particular bacterial species - Determining whether the infecting organism is a risk for other patients in the hospital, the public, or laboratory workers  The identification of a bacterial isolate requires: Analysis of information gathered from laboratory tests that provide characteristic profiles of bacteria. The tests and the order in which they are used for organism identification are often referred to as an identification scheme. Identification schemes can be classified into one of two categories: (1) Those that are based on genotypic characteristics of bacteria (2) Those that are based on phenotypic characteristics. Certain schemes rely on both genotypic and phenotypic characteristics. Some tests, such as the Gram stain, are an integral part of many schemes used for identifying a wide variety of bacteria, These schemes utilize the bacterial morphology and staining properties of the organism, as well as O2 growth requirements of the species combined with a variety of biochemical tests.  ORGANISM IDENTIFICATION USING GENOTYPIC CRITERIA Genotypic identification methods involve characterization of some portion of a bacterium’s genome Using molecular techniques for DNA or RNA analysis. This usually involves detecting the presence of a gene, or a part thereof, or an RNA product that is specific for a particular organism. The presence of a specific gene or a particular nucleic acid sequence is interpreted as a definitive identification of the organism. The genotypic approach is highly specific and often very sensitive. With the ever-expanding list of molecular techniques being developed, the genetic approach to organism identification will continue to grow and become more integrated into diagnostic microbiology laboratory protocols Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)  16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Microbiology Laboratory PCR-amplified 16S-rRNA gene sequencing can be useful for straightforward classification of purified isolates The 16S rRNA gene, a molecular marker for identification of bacterial species: 16S ribosomal RNA (rRNA) sequencing is a common amplicon sequencing method used to identify and compare bacteria present within a given sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study. The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker used for a number of reasons. These reasons include: (i) Its presence in almost all bacteria, often existing as a multigene family, or operons; (ii) The function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution) (iii) The 16S rRNA gene (1,500 bp) is large enough for informatics purposes Identification based on the 16S rRNA gene is by no means: successful and effective  BACTERIAL IDENTIFICATION USING PHENOTYPIC CRITERIA Phenotypic criteria are based on observable physical or metabolic characteristics of bacteria, that is, identification is through analysis of gene products rather than through the genes themselves. The phenotypic approach is the classic approach to bacterial identification, and most identification strategies are still based on bacterial phenotype. Some characteristics may require subcellular analysis Involving sophisticated instrumentation (e.g., high-performance liquidhromatography [HPLC] to analyze cell wall components. Other characterizations are based on the antigenic makeup of the organisms and involve techniques based on antigen-antibody interactions Most of the phenotypic characterizations used in diagnostic bacteriology are based on tests that establish a bacterial isolate’s morphology and metabolic capabilities. The most commonly used phenotypic criteria include: Microscopic morphology and staining characteristics Macroscopic (colony) morphology Environmental requirements for growth Resistance or susceptibility to antimicrobial agents Nutritional requirements and metabolic capabilities  Principles of Identification Identification schemes or charts for final identification are found: In most instances, identification schemes for final identification are based on the cellular morphologies and staining characteristics of bacteria. Bacterial identification schemes are based on: Bacterial Cellular morphologies Staining characteristics Organism nutritional requirements Organism metabolic capabilities Abbreviated identification flowcharts for commonly encountered bacteria are found This flowchart simply illustrates how information about microorganisms is integrated into subsequent identification schemes that are usually based on the organism’s nutritional requirements and metabolic capabilities In certain cases Microscopic Morphology and staining characteristics alone are enough to identify bacterial species Example: Using the fluorescent- labeled specific antibodies and fluorescent microscopy to identify two bacteria: Legionella pneumophila and Bordetella pertussis Gram-negative pathogen , and the causative agent of pertussis or whooping cough.  Microscopic Morphology and Staining Characteristics Microscopic evaluation of bacterial cellular morphology which is facilitated by the Gram stain or Other enhancing methods provides the most basic and important information on which final identification strategies are based For this reason, a Gram stain of bacterial growth from isolated colonies on various media is usually the first step in any identification scheme. Based on these findings, most clinically relevant bacteria can be divided into four distinct groups: Gram-positive cocci, Gram-negative cocci, Gram-positive bacilli, and Gram-negative bacilli Some bacterial species are morphologically indistinct and are described as “Gram-negative coccobacilli,” “Gram-variable bacilli,” or pleomorphic (i.e., exhibiting various shapes). Still other morphologies include curved and/or rods and spirals. Even without staining, examination of a wet preparation of bacterial colonies under oil immersion (1000× magnification) can provide clues as to possible identity. For example, a wet preparation prepared from a translucent, alpha-hemolytic colony on blood agar may reveal cocci in chains, a strong indication that the bacteria are probably streptococci “Gram-negative example for Gram -ve coccoacilli: Hemophilus influenzae coccobacilli,” Bacterial Identification: Examples of characteristics used as criteria for bacterial identification and classification