Micro week 3- bacterial pathogens 2

growth-dependent diagnostic methods used for the identification of clinical isolates by color changes in various diagnostic media II

(b) A conventional diagnostic test for Enteric bacteria in Triple Sugar Iron (TSI) agar. The medium is inoculated both on the surface of the slant and by stabbing into the solid agar. The medium contains a small amount of Glucose and a large amount of Lactose and Sucrose. Organisms able to ferment only the Glucose cause acid formation only at the bottom, whereas Lactose- or Sucrose-fermenting organisms cause acid formation throughout the slant. Gas formation is indicated by the breaking up of the agar at the bottom. Hydrogen sulfide formation (either from protein degradation or from reduction of thiosulfate in the medium is indicated by a blackening due to reaction of H2S with Fe2+ in the medium.

Antibiotic susceptibility testing cont

(d) Discs containing known amounts of different ATB are placed on the bacteria- inoculated agar surface. (e) After incubation (24, 48hs), inhibition zones are observed and measured. From these data, the susceptibility category of the organism is determined by reference to an interpretive chart of zone sizes.

growth-dependent diagnostic methods used for the identification of clinical isolates by color changes in various diagnostic media IV

(d) Media kits used for the rapid identification of clinical isolates. The principle is the same as before , but the whole arrangement has been miniaturized so that a number of test can be run at the same time. Four separate strips, each with a separate culture, are shown.

growth-dependent diagnostic methods used for the identification of clinical isolates by color changes in various diagnostic media V

(e) Another arrangement of a miniaturized test kit. This one defines sugar utilization in nonfermentative organisms.

What are the four different EIA methodologies that are frequently used?

- Direct EIA: Detection of antigen - Indirect EIA : Detection of antibody - Sandwich EIA : Detection of antibody - Combination EIA : Combination of direct EIA and sandwich EIA

delayed-type hypersensitivity reaction (DTH)

-A number of pathogens induce a delayed-type hypersensitivity reaction (DTH) response mediated by Th1 cells. -For these pathogens, skin testing may be useful for determining exposure. -The prototypic delayed-type hypersensitivity reaction is an artifact of modern medicine the tuberculin test, which it is used to determine whether an individual has previously been in contact with M. tuberculosis. Small amounts of tuberculin (a complex mixture of peptides and carbohydrates derived from M. tuberculosis) are injected intradermally. -In people who have been exposed to the bacterium, either by infection or by immunization with the BCG vaccine (an attenuated form of M. tuberculosis) a local T-cell mediated inflammatory reaction evolves over 24-72h. The response is caused by TH1 cells.

ATB susceptibility determined by Etest

-ATB susceptibility determined by the Etest (AB Biodisk, Solna, Sweden) for different ATB -From 8 o'clock, PTc, piperacilin/tazobactam; AT, aztreonam; CT, cefotaxime; CI, ciprofloxacin; GM, gentamicin; IP, imipenem. -Each strip is calibrated in terms of the minimum inhibitory concentration (MIC) in μg/ml starting with the lowest concentration from the center of the plate. -The lowest concentration of ATB that inhibits bacterial growth is the MIC value for that particular agent -e.g. the MIC for cafotaxime (CT) is 16 μg/ml. this organism is resistant to imipenem (IP); MIC > 31μg/ml

Disc diffusion test

-Agar media are inoculated by evenly spreading a defined density of a suspension of a pure culture on the agar surface (lawn). Filter paper disc containing a defined quantity (μg/disc) of an antimicrobial agent are then placed on the inoculated agar. -After a specific period of incubation, the diameter of the inhibition zone around each disc is measured. inhibition zone diameters are then interpreted into susceptibility categories based on zone size -Standards for the efficacy of different antimicrobial agents against different bacterial pathogens are provided by FDA or the Clinical and Laboratory Standards Institute. -Many of the pathogens for which susceptibility testing is necessary are healthcare-associated pathogens or nosocomial pathogens, those acquired in hospitals an other health care settings. -Hospital infection-control microbiologists generate and examine susceptibility data to generate periodic reports called antibiograms.

Immunofluorescence

-Antibodies can be chemically modified with fluorescent dyes to help detect antigens on intact cells. -Two common fluorescent dyes are Rhodamine B (red) and fluorescein isothiocyanate (yellow-green) -Fluorescent methods: 1) Direct method : The antibody targeted against the surface antigen is covalently linked to the fluorescent dye 2) Indirect method : The presence of a nonfluorescent antibody on the surface of a cell is detected by use of a fluorescent Ab directed against the nonfluorescent antibody. Both methods are widely used

Cerebrospinal fluid (CSF) & collection of CSF

-Cerebrospinal Fluid (CSF): bacterial meningitis is a serious disease associated with high morbidity and mortality if the etiologic diagnosis is delayed. -The Central Nervous System (CNS) consists of the brain and spinal cord. -Cerebrospinal fluid (CSF) is a clear, colorless liquid that surrounds and protects the CNS. It bathes the brain and spine in nutrients and eliminates waste products. -Because some common pathogens are labile (e.g. Neisseria meningitidis, Streptococcus pneumoniae) specimens should be processed immediately after they are collected. -Under NO circumstance should the specimen be refrigerated or placed directly into an incubator -The patient's skin is disinfected before lumbar puncture, and the cerebrospinal fluid (CSF) is collected (5-10ml) into sterile screw-capped tubes. -When the specimen is received in the microbiology lab, it is concentrated by centrifugation, and the sediment is used to inoculate bacteriologic media and prepare a Gram stain.

enriched media: Thayer-Martin agar

-Chocolate agar is modified to be selective for Neisseria gonorrhoeae and Neisseria meningitidis by the addition of antibiotics (V-C-N inhibitor) including: 1. Colistin: to inhibit most Gram negative bacteria other than Neisseria. 2. Vancomycin: to inhibit most Gram positive bacteria . 3. Nystatin or Anisomycin: to inhibit yeast -Modified Thayer-Martin agar includes Trimethoprim to inhibit Proteus.

Chocolate agar with bacitracin

CHOC with bacitracin is a selective medium used to improve the primary isolation of H. influenzae from specimens containing a mixed flora of bacteria and/or fungi.

examination methods

-Clinical specimens or suspension of microorganisms can be placed on a glass slide and examined under the microscope (e.g. direct examination of a wet mount of fungal elements, parasites) and cellular material. -Direct examination are the simplest methods for preparing samples for microscopic examination: 1. The sample can be suspended in water or saline (wet mount) 2. Mixed with alkali to dissolve background material (potassium hydroxide [KOH] method) or 3. Mixed with a combination of alkali and a contrasting dye (e.g. Lactophenol cotton blue, iodine, etc.) 4. A variation is the India ink method, in which the ink darkens the background rather than the cell (this method is used to detect capsules surrounding organisms - the dye is excluded by the capsule, creating a clear halo around the cell).

Antimicrobial drug susceptibility testing

-For many pathogens, appropriate and effective antimicrobial treatment is based on current experience and practices, in other cases decision about appropriate antimicrobial therapy must be made on case-by-case bases. -Include those pathogens for which antimicrobial drug resistance is common e.g. Gram- Negative Enteric bacteria. -Those that cause life-threatening disease e.g. meningitis cause by Neisseria meningitidis -Those that require bacteriocidal rather than bacteriostatic drugs to prevent disease progression and tissue damage e.g. organisms that cause bacterial endocarditis, where total and rapid killing of the pathogen is critical for patient survival e.g. Streptococcus viridans, Staphylococcus aureus.

isolation of pathogens from clinical specimens

-If clinically relevant organisms are to be isolated and identified, the specimens must be obtained and handled properly to ensure that the pathogen survives: -specimens should be obtained from the actual site of infection -sample must be taken aseptically to avoid contaminations with irrelevant microorganisms. -sample size must be large enough to ensure an inoculum sufficient for growth. -metabolic requirements for the organism must be maintained during sampling, storage, and transport under aerobic and anoxic conditions to ensure the survival of potential anaerobic pathogens.

Immunofluorescence - applications

-If the pathogen contains surface antigens reactive with the antibody, the pathogen cells fluoresce Fluorescent antibodies can be applied directly to infected host tissues, allowing for rapid diagnosis -Fluorescent antibody assays are also used in the diagnosis of noninfectious diseases (e.g., malignant cells). -To separate mixtures of cells into relatively pure populations -To define the numbers of individual cell types in complex mixtures

growth-dependent diagnostic methods

-Isolation of Pathogens from Clinical Specimens -Growth-Dependent Identification Methods -Antimicrobial Drug Susceptibility Testing -Safety in the Microbiology Laboratory

Immunoassays for Infectious Diseases

-Many immunoassays utilize antibodies specific for pathogens or their products for "in vitro" test designed to detect individual infectious agents. -It is possible to identify an infection by measuring the patient's antibody titer (quantity) against antigen(s) produced by the pathogen. -Agglutination and EIA are commonly used tests -Skin testing is another method for determining exposure to a pathogen

growth-dependent identification methods

-Many microorganisms recovered from clinical samples can be identified using growth-dependent assays. -Growth on Selective and Differential Media: • Based on its growth characteristic on primary isolation media a pathogen is typically sub-cultured onto specialized media design to measure one of many different biochemical reactions. • Specialized media (selective, differential) are often available as kits containing several distinct tests that can be inoculated simultaneously and rapidly assessed • Differential media incorporate biochemical tests to measure the presence or absence of enzymes involved in catabolism of specific substrate(s) -Eosin-Methylene Blue (EMB) agar is a widely used selective and differential medium for the isolation and differentiation of Enteric bacteria, Methylene blue is a selective dye because it inhibits the growth of Gram-positive bacteria, and thus only Gram-negative organisms can grow. -Fermentations of sugars are measured by incorporating pH indicator dyes that change color on acidification -Production of H2 or CO2 during sugar fermentation is assayed by observing gas production either in gas collection vials or in agar.

Nucleic acid amplification

-Polymerase chain reaction (PCR): presence of amplified gene segment confirms presence of pathogen. -Reverse transcriptase PCR (RT-PCR): uses pathogen-specific RNA to make cDNA. -Quantitative real-time PCR (qPCR): uses fluorescently labeled PCR products. Results are almost immediate.

culture of anaerobic microorganisms

-Some obligate anaerobic bacteria are common causes of infection, and their identification requires special isolation and culture methods. -In general, media for anaerobes do not differ greatly from those used for aerobes except that they are: 1. Usually richer in organic constituents 2. Contain reducing agents e.g. cysteine or thioglycolate to remove O2 3. Contain a redox indicator (resazurin) to indicate that conditions are anoxic. -The isolation, growth, and identification of obligate anaerobes can be complicated by: • Specimen contamination. • Challenge of maintaining anoxic conditions during collection, transport, and culture. • For anoxic incubation, agar plates are placed in a sealed jar, which is made anoxic either by replacing the atmosphere in the jar with an oxygen-free gas mixture (N2+CO2) or by removing O2 from the enclosed vessel by some chemical means.

Urine tract culture

-Urinary tract infections are common, especially in women. -Interpretation of microbial findings can be problematic since the disease-causing agents are often members of the normal flora (e.g. E. coli) -In most cases, the urinary tract becomes infected by organisms that ascend into the bladder from the urethra. -dipstick tests can be used to identify infections by change in colors.

blood cultures

-approximately 20 ml of blood should be collected from an adult for each blood culture. -blood cultures are the only immediate way of isolating and identifying the causative agent of septicemia -The most common pathogens found in blood include: 1. Gram-positive: Staphylococcus and Enterococcus as well as 2. Gram-negative: Pseudomona aeruginosa and Enteric bacteria such as: Enterobacter, E. coli, and Klebsiella pneumoniae.

Antibiogram

-define the susceptibility of clinically isolated microorganisms to the ATB in current use. -are used to monitor control of known pathogens, to track the emergence of new pathogens, and to identify the emergence of ATB resistance, all at the local level.

blood culture procedure

-draw around 20 ml of blood aseptically (careful disinfection of the patient's skin is important) from a vein and inject it into two blood culture bottles (10ml each) containing an anticoagulant and a general-purpose culture medium. -One bottle is incubating in air (aerobically) and the other under anoxic conditions, and both are kept at 370C for up to 5 days. -Automated blood culture systems detect growth by monitoring gas consumption (in oxic conditions) or CO2 production in anoxic conditions as often as every 10min. Some systems also measure turbidity. -Most clinically significant bacteria are recovered within 1 to 2 days but detectable growth of fastidious organisms (mycobacteria or fungi) may take 3-5 days or longer. -Bottles that contain growth are examined by Gram stained and then inoculated onto enrichment and differential media for further isolation and identification.

Immunoblot (Western blot)

-electrophoresis of proteins, followed by transfer to a membrane and detection by addition of specific antibodies. -immunoblot methods detect antibodies to specific antigens or the antigens themselves.

What are the most common urinary tract pathogens?

-enteric bacterial, with E. coli accounting for about 90% of the cases. -other urinary tract pathogens include: Klebsiella, Enterobacter, Proteus, Pseudomonas, Staphylococcus, and Enterococcus.

growth media and culture

-most microbes of clinical importance can be grown, isolated, and identified with specialized growth media. -clinical samples are first grown in: 1) General-purpose media - support the growth of most aerobic and facultative aerobic organisms (e.g., blood agar and chocolate agar). Organisms isolated from such media are often subculture on more specialized media, 2) Enriched media - contain specific growth factors that enhance growth of certain fastidious pathogens such as N. gonorrhoeae (e.g Thayer-Martin agar) 3) Selective media - allow for some organisms to grow while inhibiting the growth of others due to the presence of inhibitory agents (e.g. Oxford Agar media) 4) Differential media - are specialized media that allow identification of organisms based on their growth, color, and appearance on the medium (e.g. Eosin- methylene blue)

Rapid tests

-reagents are absorbed to support material -body fluid is applied to the support matrix (matrix contains a soluble antigen conjugated to a colored molecule - chromophore, matrix also contains a line of fixed antigen, antibodies bind to antigens). -color forms when concentration of chromophore gets high enough.

sealed jar for incubating cultures under anoxic conditions

-the catalyst and hydrogen generator packed produce and maintain a reducing (anoxic) environment. -H2 is generated chemically in the jar, in the presence of a palladium catalyst, the H2 combines with the free O2 in the vessel, forming H2O and removing the contaminated O2.

Agglutination

-the visible clumping of a particulate antigen when mixed with antibodies specific for the particulate antigens. -agglutination tests are simple to perform, specific, inexpensive, rapid, and sensitive. -standardized agglutination tests are used to identify blood group antigens and many pathogens and pathogen products.

Enzyme immunoassay (EIA) or Enzyme-Linked Immunosorbent Assay (ELISA)

-very sensitive immunological assay -widely used in clinical diagnostic and research applications -EIAs employ covalently bonded enzymes attached to antibody molecules -rapid tests are similar to EIAs -both allow detection of antigen-antibody complexes.

Urinary tract pathogens can be cultured using?

1) General purpose enriched media for initial isolation such as blood agar; and/or Selective media such as: MacConkey agar or Eosin-Methylene blue agar, which allows the initial differentiation of Lactose fermenters (E. coli) from non-Lactose fermenters (Shigella, Salmonella, Pseudomona). 2) contains bile salts and the dye crystal violet, which inhibit the growth of gram-positive bacteria and select for gam-negative bacteria. It also contains the carbohydrate lactose, which allows differentiation of gram-negative bacteria based on their ability to ferment lactose. Organisms which ferment lactose produce acid end-products which react with the pH indicator neutral red and produce a pink color.

urinalysis dipstick test

A control strip (CS) is shown underneath the test strip (TS). From left to right, the strip measures abnormal levels of: 1) glucose, 2) bilirubin, 3) ketones, 4) specific density, 5) blood, 6) pH, 7) proteins, 8) urobilinogen, 9) nitrite, 10) leukocytes (esterase) in a urine sample. -Abnormal readings for esterase (10) (trace positive far right) and nitrite (9) (strong positive, second from right) indicate bacteriuria; presence of large amounts of enteric bacteria. -Subsequent culture of this sample indicated the presence of E. coli.

Multidrug-Resistant Pseudomonas

Can be deadly for patients in critical care. An estimated 51,000 healthcare-associated P. aeruginosa infections occur in the United States each year. More than 6,000 (13%) of these are multidrug-resistant, with roughly 400 deaths per year attributed to these infections.

What is the standard procedure for assessing antimicrobial activity?

Disc diffusion test

Chocolate agar with GC base and growth supplement

It is a medium that supports the special growth requirements (hemin and NAD) needed for the isolation of fastidious organisms, such as H. influenzae, when incubated at 35-37°C in a 5% CO2 atmosphere

Methods for determining the susceptibility of an organism to ATB

For the disc diffusion test: (a) Isolated pure colonies are homogenized in a tube with an appropriate liquid medium to achieve a specified density compare to a turbidity standard. (b) A sterile cofon swab is dipped into the bacterial suspension and excess fluid removed by pressing the swab against the side of the tube. (c) The swab is streaked evenly over the surface of an appropriate agar medium.

isolation of pathogens from clinical specimens

If a health care provider suspects a disease is caused by an infectious agent: -Samples of tissues or fluids are collected for microbiological, immunological, and molecular biological analyses. -Samples may include blood, urine, feces, sputum, cerebrospinal fluid, or pus from a wound. -Swabs may be used to obtain samples from suspected infected areas such as skin, nares, or throat. *The swab is used to inoculate the surface of an agar plate or a tube of liquid culture medium. *In some cases, a small piece of tissue (biopsy) may be obtained for culture.

Growth-independent diagnostic methods

Immunoassays for Infectious Disease Agglutination Immunofluorescence Enzyme Immunoassays, Rapid Tests, and Immunoblots Nucleic Acid Amplification

Chocolate agar with Tryptic Soy Agar (TSA) and growth supplements

It is a medium that supports the special growth requirements (hemin and NAD) needed for the isolation of fastidious organisms, such as H. influenzae, when incubated at 35-37°C in a 5% CO2 atmosphere.

Thayer-Martin agar

It is used for the selective isolation of N. gonorrhoeae and Neisseria meningitidis. Thayer-Martin media is a chocolate agar supplemented with Vancomycin, Nystatin, Colistin to inhibit the normal flora, including nonpathogenic Neisseria. The Modified Thayer-Martin agar includes Trimethoprim to inhibit Proteus.

observation of colony characteristics on various media

Tentative identification of an isolate is often achieved by observing colony characteristics on various media -This is then followed by more detailed tests to make a positive identification . Some pathogens can be readily identified by microscopic examination of tissue samples - Example: Neisseria gonorrhoeae, clinically refer as gonococcus.

lab identification of microbial pathogens

The diagnostic methods used for identification of infectious pathogens include growth-dependent microbiology assays, immunoassays, and molecular biology assays. -immunoassays can be used to measure patient immune response, indicating pathogen exposure, or can be used to directly identify the pathogen in host tissue or culture.

ATB susceptibility determined by broth dilution method

The organism is Pseudomona aeruginosa Each row has a different ATB. The microtiter plate enables automation of these tests. The end point is the well with the lowest concentration of ATB that shows no visible bacterial growth. The highest concentration of ATB is in the well at the left; serial two-fold dilutions are made in the wells to the right. For example in rows 1 and 2, the end point is the third well. In row 3 the ATB is ineffective at the concentration tested, since there is bacterial growth in all the wells. In row 4, the end point is in the first well. What about the end point in rows 5, 6,7 and what about 9, 10, and 11 and 12 ????

Immunoassay

biochemical test that uses the reaction of an antibody to its antigen to measure the amount of a substance in a liquid

sensitivity

defines the lowest numbers of a pathogen or the lowest amount of a pathogen product that can be detected. High sensitivity prevents false-negative reactions.

Clinical microbiologist

detects, identifies, and characterizes the microorganisms that cause infectious diseases from a variety of samples collected from sick hosts. -the microbiologist should be prepared to instruct the health care provider (MD, DO, PA, nurse, etc.) about what specimen should be collected if a particular diagnosis is suspected and the health care provider must supply the microbiologist with information about the clinical diagnostic so the right tests are selected.

Diagnostic approaches

diagnostic approaches in clinical microbiology include evaluation of samples by: growth-dependent techniques, molecular techniques, immunoassays, etc.

Direct observation

direct observation of pathogens acquired from clinical specimens is a very important tool for many infectious diseases.

Selective and differential media

for example: Eosin-Methylene Blue (EMB) has dyes that inhibits the growth of Gram+ organisms. It has also bile salts, which is toxic for Gram negative bacteria other than coliforms. -EMB is the selective and differential medium for coliforms. -In addition EMB is a differential medium because it distinguishes factor fermenters such as E. coli (green) from non-lactose fermenting gram- organisms such as Pseudomonas, Salmonella, Shigella, Proteus vulgaris etc.

differential stains

gram stain, acid-fast stain, endospore stain

septicemia or sepsis

is a Blood infection by a virulent organism that enters the blood from a focus of infection, multiplies, and travels to various body tissues to initiate new infectious. -It can cause severe systemic symptoms including fever and chills, followed by prostration. -Severe cases may result in septic shock, a life-threatening systemic condition characterized by severe reduction in blood pressure and multiple organ failures, including heart, kidneys and lung.

endospore stain

is a differential stain used to visualize bacterial endospores (using malachite green or toluidine blue stain). -endospores are formed by a few genera of bacteria, such as bacillus. By forming spores, bacteria can survive in hostile conditions. Spores are resistant to heat, desiccation, chemicals, and radiation.

Etest

is a non-diffusion-based technique that employs a preformed and predefined gradientof an antimicrobial agent immobilized on a plastic strip. When applied to the surface of an inoculated agar plate, the gradient transfer from the strip to the agar and reminds stable for a period that covers the wide range of critical times associated with the growth characteristics of different microorganisms. -After overnight incubation or longer, an elliptical zone of inhibition centered along the axis of the strip develops. -The MIC value (μg/ml) can be read at the point where the ellipse edge intersects the precalibrated Etest strip, providing a precise MIC. This value can then be interpreted using current standards.

bacteremia and fungemia

is the presence of bacteria and fungi, respectively in the blood. *It is extremely uncommon in healthy individuals, normally occurring transiently in response to invasive procedures such as tooth brushing, dental surgery or trauma.

N. gonorrhoeae

is usually found as a Gram-Negative diplococci, NO SIMILAR microorganisms are observed among the normal flora of the urogenital track. -Thus a Gram-negative stain of urethral, vaginal or cervical smear of diplococci is diagnostic for gonorrhea. -In acute gonorrhea, microscopic examination of purulent discharges usually reveals Gram-Negative diplococci in neutrophils. -One of several selective media used for primary isolation of Neisseria gonorrhoeae is Modified Thayer-Martin (MTM) agar, this media incorporates the ATB: Vancomycin, Nystatin, Colistin and Trimethoprim to suppress the growth of normal flora. -they have no effect on Neisseria gonorrhoeae. -a non-selective enriched media for the isolation of N. gonorrhoeae contains heat-lysed blood "chocolate agar", the heated blood interacts with the media components, absorbing compounds that are normally toxic for the microorganism.

acid-fast stain

like the Ziehl-Neelsen used to stain mycobacteria and other acid-fast microorganisms, the background is counter-stained with methylene blue. The stain is: carbol fuchsin.

gram stain

most widely used, forms the basis for the phenotypic classification of bacteria. Yeast can also be stained with this method (Gram +).

Minimum inhibitory concentration (MIC)

procedure for antibiotic susceptibility testing employs an antibiotic dilution assay in agar, in culture tubes or in the wells of a microtiter plate. -Wells containing serial dilutions of antibiotics are inoculated with a standard amount of a test organism, growth in the presence of each ATB is then observed by measuring turbidity -Look for inhibited growth -the MIC is the concentration of the higher dilution (lowest concentration) of ATB that completely inhibits growth.

specificity

the ability of the test to recognize a single pathogen, optimal specificity implies that the test is specific for a single pathogen and will not identify any other. High specificity prevents false-positive results.

Diagnostic microbiology

the idea is: to grow, isolate, and identify most pathogenic bacteria within 48 hours of sampling. In general the usefulness of any diagnostic test depends on the test's specificity and sensitivity.