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Lab 16: Isolation and Identification of Neisseriae, Mycobacteria, and Obligate Anaerobes

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  • A. THE NEISSERIAE

    DISCUSSION

    The neisseriae are a group of Gram-negative diplococci 0.6-1.5 µm in diameter (see Fig. 1A). Two species of Neisseria, N. gonorrhoeae and N. meningitidis, are considered as true human pathogens. Both of these organisms possess pili and adhesins for adherence to host cells, produce endotoxins, and resist destruction within phagocytes. N. meningitidis also produces a capsule to resist phagocytic engulfment.

    • Scanning electron micrograph Neisseria gonorrhoeae; courtesy of Dennis Kunkel's Microscopy.
    • Scanning electron micrograph of Neisseria meningitidis. © Margaret Ketterer, author. Licensed for use, ASM MicrobeLibrary.
    • Transmission electron micrograph Neisseria gonorrhoeae; courtesy of CDC.

    For further information on virulence factors associated with N. gonorrhoeae and N. meningitidis, see the following Learning Objects in your Lecture Guide:

    Neisseria gonorrhoeae (the gonococcus), causes gonorrhea. It is estimated that there are approximately 2,000,000 cases of gonorrhea per year in the U.S. Infection usually occurs following sexual contact, with the incubation period averaging 2-7 days. In males, the gonococcus typically invades the anterior urethra, usually producing a purulent discharge, pain upon urination, and a frequency of urination. Approximately 5-10% of infected males are asymptomatic but will still be infectious. The infection may spread up the reproductive tract, infecting the prostate, vas deferens, epididymis, and testes, causing painful inflammation and scar tissue formation that can result in sterility.

    In females, 30-50 percent of those initially infected are asymptomatic or show mild symptoms. They are, however, still infectious. Initially, the organism invades the cervix, the urethra, and frequently the rectum. In about 15 percent of the cases, the organism spreads up the reproductive tract and infects the fallopian tubes causing pelvic inflammatory disease (PID). The resulting inflammation and scar tissue formation may result in sterility or abnormal (ectopic or tubal) pregnancies.

    The gonococcus may also cause extragenital infections such as pharyngitis (from oral-genital sex), ophthalmia (from inoculation of the eyes with contaminated fingers), and proctitis (from anal sex). In 1% - 3% of infected women and a lower percentage of infected males, the organism invades the blood and disseminates, causing a rash, septic arthritis, endocarditis, and/or meningitis. Dissemination occurs more frequently in females. Congenital gonorrhea is known as ophthalmia neonatorum and occurs as a result of the eyes of newborns becoming infected as the baby passes through the birth canal.

    Neisseria meningitidis (the meningococcus) is the causative organism of meningococcal (epidemic) meningitis. There are between 2000 and 3000 cases of meningococcal meningitis per year in the U.S. Approximately 50% of the cases occur in children between 1 and 4 years old. N. meningitidis infects the nasopharynx of humans causing a usually mild or subclinical upper respiratory infection. Colonization of the nasopharynx may persist for months. However, in about 15% of these individuals, the organism invades the blood and disseminates, leading septicemia and from the there may cross the blood-brain barrier causing meningitis. A petechial skin rash, caused by endotoxin in the blood, appears in about 75 percent of the septic cases and fatality rates for meningococcal septicemia are as high as 30 percent as a result of the shock cascade. A fulminating form of the disease, called Waterhouse-Frederichsen syndrome, can be fatal within several hours due to massive intravascular coagulation and resulting shock, probably a result of massive endotoxin release. N. meningitidis is especially dangerous in young children.

    Diagnosis of Gonorrhea

    A presumptive diagnosis of gonorrhea is made on the basis of two of the following three criteria:

    1. Typical Gram-negative diplococci inside of polymorphonuclear leukocytes seen in a Gram stain of urethral exudates in men and endocervical secretions in women (a positive GC smear; see below);

    2. Growth of N. gonorrhoeae from the urethra of men or the endocervix of women on a culture medium with typical colony morphology, positive oxidase reaction, and Gram-negative diplococcal morphology in a Gram stain; and/or

    3. Detection of N. gonorrhoeae by nonculture tests such as:

    • antigen tests, e.g., Gonozyme [Abbott]; an enzyme immunoassey (EIA) test for N. gonorrhoeae antigens detected from endocervical and male urethral swab specimens.
    • direct specimen nucleic acid probes, e.g., Pace 2C Assay[GenProbe]); a single-tube assay to screen for the presence of both Chlamydia trachomatis and Neisseria gonorrhoeae from endocervical and male urethral swab specimens.
    • nucleic acid amplification tests, e.g., LCR (ligase chain reaction) [Abbott]; DNA probes based on N. gonorrhoeae pilin gene sequences.

    Antibiotic therapy may be started with a presumptive diagnosis.

    A definitive diagnosis of gonorrhea is made on the basis of:

    1. Isolation of N. gonorrhoeae from the site of exposure by culture, usually on a selective medium, demonstration of typical colony morphology, positive oxidase reaction, and Gram-negative diplococcal morphology in a Gram stain and

    2. Conformation of isolates by biochemical, enzymatic, serologic, or nucleic acid testing, e.g., carbohydrate utilization, rapid enzyme substrate tests, serologic methods such as fluorescent antibody tests, or a DNA probe culture confirmation technique.

    Isolation and Identification of Neisseria gonorrhoeae

    a. The GC smear

    A GC smear (gonococcus smear) is a Gram stain of urethral exudates in men and endocervical secretions in women and can be part of a presumptive diagnosis of gonorrhea. One looks for Gram-negative diplococci with flattened adjacent walls that are seen both inside and outside of polymorphonuclear leukocytes (see Fig. 1).This test is quite sensitive in symptomatic males but only 40-60% sensitive in symptomatic females. In asymptomatic males and females the Gram stain has a lower predictive value.

    b. Isolation of Neisseria gonorrhoeae

    To diagnose genital gonorrhea in males, the sample to be cultured is taken from the urethra. In females, cultures are taken from the cervix and the rectum. In non-genital gonorrhea, the infected site is cultured. The gonococcus requires an enriched medium with increased carbon dioxide tension for growth. They are usually cultured on selective media such as modified Thayer-Martin (MTM) Chocolate agar or Martin-Lewis agar. Plates are then incubated at 35-37°C under increased carbon dioxide tension (5-10% CO2) such as that provided by a candle jar or a carbon dioxide incubator.

    MTM Chocolate agar is selective for Neisseria.

    The medium contains enrichment factors to promote the growth of Neisseria. In addition, it contains antibiotics to inhibit normal body flora: vancomycin to inhibit Gram-positive bacteria; colistin to inhibit Gram-negative bacteria; trimethoprim to suppress Proteus; and nystatin to inhibit yeast. The "chocolate" color is due to the hemoglobin enrichment added to the medium. Plates are then incubated under increased carbon dioxide tension as mentioned above. (Transgrow Medium is a convenient flask containing MTM Chocolate agar and CO2.) N. gonorrhoeae forms small, convex, grayish-white to colorless, mucoid colonies in 48 hours at 35-37°C (see Fig. 4).

    c. Identification of Neisseria gonorrhoeae

    Once isolated, N. gonorrhoeae can be identified by the oxidase test, Gram-staining, carbohydrate utilization reactions, rapid enzyme substrate tests, serologic methods such as fluorescent antibody tests, or a DNA probe culture confirmation technique.

    1. Oxidase test

    All Neisseria are oxidase positive. The oxidase test can be performed as in Lab 13 using a Taxo N® disc. A moistened Taxo N® disc can be placed on a growing culture and a blackening of the colonies surrounding the disc indicates a positive oxidase test (see Fig. 2). All oxidase-positive cultures would be Gram stained to confirm Gram-negative diplococci.

    2. Carbohydrate utilization

    The various species of Neisseria can be differentiated according to carbohydrate utilization patterns. Neisseria species do not produce acid from carbohydrate fermentation but rather by an oxidative pathway. These tests are done using a media with single carbohydrates ( glucose, maltose, sucrose, or lactose) and a pH indicator to detect acid. The medium seen today uses phenol red as a pH indicator. If the sugar is utilized, acid end products cause the phenol red pH indicator to turn yellow. N. gonorrhoeae produces acid from only glucose (see Fig. 5) whereas N. meningitidis produces acid from glucose and maltose (see Fig. 6).

    There are a variety of Neisseria species and other genera with similar morphology that often colonize the body. By testing for lactose and sucrose utilization as well as performing other tests such as rapid enzyme substrate tests, serologic methods, or DNA probes, N. gonorrhoeae and N. meningitidis can be definitively identified. Typical sugar utilization reactions can be seen in Table 1.

    d. Serologic and Nucleic Acid Tests to Identify N. gonorrhoeae

    Serologic tests are also available for rapidly identifying N. gonorrhoeae. These include an ELISA test to detect gonococci in urethral pus or on a cervical swab, as well as a direct serologic test using fluorescent monoclonal antibodies to detect N. gonorrhoeae. Serologic testing will be covered in Labs 17 and 18.

    Nucleic acid probes and nucleic acid amplification tests are also available for identifying N. gonorrhoeae.

    Highlighted Bacterium: Neisseria gonorrhoeae

    Click on this link, read the description of Neisseria gonorrhoeae.

    2. Isolation and Identification of Neisseria meningitidis

    a. Gram stain

    A presumptive diagnosis of meningococcal meningitisis often made by doing a Gram stain of cerebrospinal fluid or petichial skin lesions and looking for Gram-negative diplococci seen both inside and outside of polymorphonuclear leukocytes. This can be followed by serologic tests, nucleic acid probes, or culturing.

    b. Serologic identification

    There are at least 12 different serological groups of N. meningitidis based on their capsular polysaccharides, but over 90 percent of meningococcal meningitis cases are caused by five serologic groups: A, B, C, Y, and W135. Serogroups A and C usually causes the epidemic form of meningitis. Serogroup C is is the most common serogroup in North America whereas B is the most common in Europe and Latin America. Serogroups A and C are common in China, the Middle East, and parts of Africa. Serogroup Y has been increasing in the U.S., Israel, and Sweden. Direct serologic testing to detect meningococcal capsular polysaccharides can be performed on cerebrospinal fluid or on organisms from skin lesions for rapid identification. Direct serologic testing will be discussed in more detail in Lab 17.

    c. Nucleic acid identification

    A polymerase chain reaction test to amplify meningococcal DNA can also be used to detect N. meningitidis in cerebrospinal fluid or blood.

    d. Isolation of Neisseria meningitidis

    To isolate N. meningitidis, cultures are taken from the nasopharynx, blood, cerebrospinal fluid, and skin lesions. Typically cultures are done on an enriched, non-selective medium such as blood agar or chocolate agar grown in 3-7% carbon dioxide. MTM Chocolate agar is also sometimes used for isolation. Medium to large, blue-gray, mucoid, convex, colonies form in 48 hours at 35-37°C.

    e. Identification of Neisseria meningitidis

    Once isolated, N. meningitidis, like N. gonorrhoeae discussed above, is identified by the oxidase test, Gram staining, and carbohydrate utilization reactions. N. meningitidis, like all neisseriae, is oxidase-positive and appears in a Gram stain as Gram-negative diplococci. In carbohydrate utilization tubes, N. meningitidis produces acid from both glucose and maltose (see Fig. 6) but not from lactose and sucrose (see Table-1). The acid end products turn the phenol red pH indicator from red to yellow. N. meningitidis also produces gamma-glutamylaminopeptidase, an enzyme that can be detected by biochemical testing.

    Highlighted Bacterium: Neisseria meningitidis

    Click on this link, read the description of Neisseria meningitidis.

    B. THE MYCOBACTERIA

    DISCUSSION

    The mycobacteria are rod-shaped bacteria generally 0.4 by 3.0 µm in size that are said to be acid-fast. This means that because of their unique cell wall (see Fig. 7), when they are stained by the acid-fast procedure (Appendix C), they will resist decolorization with acid-alcohol and stain red (see Fig. 8, Fig. 12A, and Fig. 12B), the color of the initial stain, carbol fuchsin. With the exception of a very few other acid-fast bacteria such as Nocardia, all other bacteria will be decolorized and stain blue (the color of the counterstain, methylene blue). The acid-fast stain is an important test for the genus Mycobacterium. Fluorescent microscopy staining may also be used to identify Mycobacterium.

    The most common species of Mycobacterium to cause human infections are M. tuberculosis, M. leprae, M. avium complex, M. kansasii, M. fortuitum, M. chelonae, and M. abscessus. M. tuberculosis is the causative organism of tuberculosis, M. leprae causes leprosy, and M. avium complex (MAC) frequently causes systemic infections in people with HIV/AIDS. The other species of Mycobacterium occasionally cause tuberculosis-like infections, especially in the debilitated or immunosuppressed host.

    • Scanning electron micrograph of Mycobacterium tuberculosis; courtesy of CDC

    It is estimated by the World Health Organization that worldwide, there are over 8,800,000 million new cases of tuberculosis each year and nearly 3,000,000 deaths. One-third of the world’s population - approximately 1.9 billion people - are thought to be infected with M. tuberculosis. In the U.S. there are approximately 20,000 new cases of tuberculosis a year. M. tuberculosis is typically acquired by inhalation of aersolized infectious particles. In the vast majority of people, primary infection is asymptomatic or minimally symptomatic. Approximately 5% of people exposed to M. tuberculosis develop active disease within 2 years; another 5% - 10% develop disease sometime later in life. Typical symptoms are malaise, cough, weight loss, and night sweats.

    Primary infection typically occurs after inhalation of the organism and subsequent generation of a peripheral lung lesion. The body responds with what is termed delayed hypersensitivity to form characteristic lesions called granuloma or tubercles (see Fig. 3). The formation of granuloma is actually the result of cell-mediated immune responses attempting to "wall-off" and localize infections that the body cannot effectively remove with macrophages. Although primary infection may be self-limiting, progression of the localized lesion may lead to pneumonia. The organisms may eventually die within the granuloma, or the tissue may undergo caseation, liquification, and cavitation. This can result in bronchogenic spread of the M. tuberculosis. In rare instances, the organism may enter the blood causing disseminated miliary tuberculosis.

    Secondary infection is usually due to a relapse of either self-resolved lesions or a previously treated disease. Approximately 90% of tuberculosis in immunocompetent patients is a result of secondary infection. M. tuberculosis is relatively resistant to many disinfectants and most antibiotics and is also able to resist destruction within phagocytic macrophages.

    For further information on virulence factors associated with M. tuberculosis and M. leprae, see the following Learning Objects in your Lecture Guide:

    Diagnosis of tuberculosis

    The diagnosis of tuberculosis is traditionally based on clinical findings, chest radiographs, a sputum or tissue smear showing acid-fast bacilli or fluorescing mycobacteria after using fluorochrome staining. Confirmation can be made using cultures, DNA probes, and polymerase chain reactions to amplify mycobacterial DNA.

    1. Presumptive tests for tuberculosis

    a. The PPD skin test (or other presumptive skin tests) detects delayed hypersensitivity to purified protein from the cell wall of M. tuberculosis. A positive skin test, assessed at 48-72 hours, indicates that the person has developed a cellular immunity to the organism as a result of either a previous or a current infection. Recommendations for interpretation of positive skin tests are based on the size of induration in relationship to other risk factors the patient may have:

    • Induration of 5 mm or more

      Contacts of persons known to be infected
      Patients with abnormal chest film
      Organ-transplant recipients
      HIV-positive patients
      Other immunosuppressed patients

    • Induration of 10 mm or more
      Residents of prisons, nursing homes, institutions
      Recent immigrants from countries with a high incidence of tuberculosis
      Heatlhcare workers
      Children aged <4 years or infants, children, and adolescents exposed to high-risk adults
      Patients with other high-risk medical factors (eg, diabetes, renal failure, cancer, silicosis)

    • Induration of 15 mm or more
      No risk factors.

    b. Chest X-rays are used to detect confluent granuloma formation in the lungs and possible cavitation, which could be a result of past or present infection with tuberculosis or with some other pulmonary infection that may be mistaken for tuberculosis.

    c. An Acid-fast stain of the sputum may indicate acid-fast bacilli, which is a presumptive test for active tuberculosis. In reporting acid-fast slide results, the slide should be observed for 10-15 minutes before considered negative. Results are reported as positive or negative for acid-fast bacilli. Sometimes the amount of acid-fast bacilli are indicated, with 3-9 per slide reported as rare, 10 or more per slide reported as few, and more than one per oil immersion field reported as numerous.

    • Photomicrograph showing a positive acid-fast stain of Mycobacterium tuberculosis in sputum.
    • Photomicrograph showing a positive acid-fast stain of Mycobacterium tuberculosis in sputum.

    2. Confirmation of Active Tuberculosis

    a. Cultures

    Active tuberculosis is confirmed by culturing the organism. Sputum is usually treated with sodium hydroxide, which is cidal for contaminants but not for M. tuberculosis. The liquified sputum is then neutralized, centrifuged, and the sediment is inoculated onto special enrichment media such as Lowenstein-Jensen agar slants, Middlebrook agar, or 7H 10 Oleic acid agar plates. Felsen Quadrant plates with agar containing different antimicrobial agents are also inoculated to determine drug sensitivity.

    Plates are incubated in a carbon dioxide atmosphere. It takes from 3-8 weeks for colonies to form. In recent years, more rapid culture methods for detection of M. tuberculosis have been developed. One involves adding radioisotopic substrates to a broth growth medium in order to detect the release of carbon dioxide during mycobacterial growth. Another uses fluorometric technology to detect oxygen consumption during growth. These techniques usually detect mycobacterial growth in less than two weeks.

    b. Nucleic acid tests

    Polymerase chain reactions to amplify mycobacterial DNA, DNA probes, and probes to detect ribosomal RNA from M. tuberculosis are also being used with increased frequency to confirm tuberculosis.

    The above procedures represents a very simplified outline for the diagnosis of tuberculosis. Culturing of M. tuberculosis, atypical mycobacteria, and other clinically significant mycobacteria involves a complicated series of complex procedures carried out only in large, well-equipped labs by experienced personnel.

    Highlighted Bacterium: Mycobacterium tuberculosis

    Click on this link, read the description of Mycobacterium tuberculosis.

    C. THE OBLIGATE ANAEROBES

    DISCUSSION

    Obligate anaerobes are organisms that grow only without oxygen and, in fact, oxygen inhibits or kills them. They obtain energy from anaerobic respiration or fermentation. A variety of obligate anaerobic bacteria, which are usually normal flora of the body, may cause human infections. Obligate anaerobes primarily cause infections of anaerobic wounds, although they may participate in all varieties of infections and involve any tissue or organ. Five organisms or groups of organisms account for about two-thirds of all clinically significant anaerobic infections. These are Bacteroides fragilis, Bacteroides melaninogenicus, Fusobacterium nucleatum, Clostridium perfringens, and the anaerobic cocci.

    • Bacteroides fragilis is the most common cause of anaerobic infections in humans. It is also a predominant organism of the normal human intestinal tract. It mainly causes wound infections. B. fragilis is a Gram-negative bacillus.
    • Bacteroides melaninogenicus is normal flora of the upper respiratory, gastrointestinal, and genitourinary tracts.
    • Fusobacterium species are normal flora of the upper respiratory, gastrointestinal, and genitourinary tracts.
    • Clostridium perfringens, as well as other clostridial species, are normal flora of the intestinal tract of various animals and may cause gas gangrene. C. perfringens is a Gram-positive bacillus. C. tetani causes tetanus and C. botulinum causes botulism.
    • Anaerobic cocci such as Peptostreptococcus, Peptococcus, and Veillonella are also normal flora of the body.

    Â Clostridium difficile causes severe antibiotic-associated colitis and is an opportunistic Gram-positive, endospore-producing bacillus transmitted by the fecal-oral route. C. difficile is a common healthcare-associated infection (HAIs) and is the most frequent cause of health-care-associated diarrhea. C. difficile infection often recurs and can progress to sepsis and death. CDC has estimated that there are about 500,000 C. difficile infections (CDI) in health-care associated patients each year and is linked to 15,000 American deaths each year.

    Â Antibiotic-associated colitis is especially common in older adults. It is thought that C. difficile survives the exposure to the antibiotic by sporulation. After the antibiotic is no longer in the body, the endospores germinate and C. difficile overgrows the intestinal tract and secretes toxin A and toxin B that have a cytotoxic effect on the epithelial cells of the colon. C.difficile has become increasingly resistant to antibiotics in recent years making treatment often difficult. There has been a great deal of success in treating the infection with fecal transplants, still primarily an experimental procedure. Polymerase chain reaction (PCRs) assays, which test for the bacterial gene encoding toxin B, are highly sensitive and specific for the presence of a toxin-producing Clostridium difficile organism. The most successful technique in restricting C. difficile infections has been the restriction of the use of antimicrobial agents.

    Although anaerobic procedures are no more difficult than those used in aerobic bacteriology, strict adherence to proper technique is necessary to ensure recovery of the organism. There must be proper selection of the specimen, proper specimen collection, proper specimen transport to the lab, and provision of a proper anaerobic environment. Common ways of culturing obligate anaerobes in the lab include:

    1. Brewer anaerobic jar with GasPak®

    A GasPak® is a commercially-produced disposable hydrogen and carbon dioxide generator envelope. When water and catalyst are added, hydrogen and carbon dioxide are produced. The hydrogen then combines with oxygen to form water, thus creating an anaerobic atmosphere. The cultures are placed in a Brewer jar, water is added to the GasPak®, the lid of the jar is sealed, and the jar is placed in an incubator.

    2. Media containing reducing agents Media such as Thioglycolate medium (see Fig. 9) and Anaerobic agar contain chemicals which function as reducing agents. The reducing agents absorb oxygen and create a reduced environment required by anaerobes.

    3. Carbon dioxide incubators Carbon dioxide incubators are frequently used to culture anaerobes. After the cultures are placed in the incubator, the air is evacuated and replaced by carbon dioxide gas.

    Isolation and Identification of Clostridium perfringens

    1. Direct microscopic examination of exudates

    Gram stains of purulent exudates from gas gangrene show stout Gram-positive bacilli frequently surrounded by a capsule. Endospores are usually not produced on ordinary culture media or in tissues.

    2. Isolation on Blood agar

    When inoculated onto Blood agar plates and grown anaerobically, C. perfringens produces smooth, glossy colonies which are usually surrounded by a double-zone hemolysis (see Fig. 10). The double-zone hemolysis appears as a narrow zone of beta-type hemolysis (due to theta toxin) near the colony surrounded by a wider zone of incomplete hemolysis (due to alpha toxin).

    3. Identification in Litmus Milk medium

    In anaerobically-grown Litmus Milk cultures, enzymes of C. perfringens will attack the proteins and carbohydrates of the milk producing a "stormy fermentation" with acid production (litmus turns pink), clotting of milk proteins, and gas formation (see Fig. 11). For further information on virulence factors associated with C. perfringens, see the following Learning Objects in your Lecture Guide:

    Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free.

    Contributors and Attributions

    • Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)