Medical Microbiology Lectures - Dr. Issam Jumaa

 ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ )2( ‫الفصل الدراسي الثاني علم البكتيريا الطبية‬ CONTENTS No. Lecture title Page No. 1. Neisseria...

 ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ )2( ‫الفصل الدراسي الثاني علم البكتيريا الطبية‬ CONTENTS No. Lecture title Page No. 1. Neisseria 2 Enteric Gram-negative rods: 2. 7 Escherichia coli 3. Klebsiella 12 4. Pseudomonads and Acinetobacter 18 5. Shigella and Salmonella 22 6. Yersinia 28 7. Vibrio 31 8. Campylobacter 36 9. Helicobacter pylori 38 10. Haemophilus 41 11. Bordetella and Brucella 45 12. Chlamydia 49 13. Spirochetes 52 14. Mycobacterium 55 15. Mycoplasma and Rickettsia 59 ‫الدرجة‬ ‫ الى‬- ‫المحاضرات من‬ ‫الجزء‬ ‫اسم المادة‬ ‫المرحلة‬ 33 5 -1 ‫االول‬ 34 10 - 6 ‫الثاني‬ ‫علم البكتيريا الطبية‬ ‫الثانية‬ )2 ( 33 15 -11 ‫الثالث‬ 1  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 1. Neisseria - Gram-negative cocci, occur in pairs (diplococci). Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are exclusively pathogenic for humans and typically are found associated with or inside polymorphonuclear cells (PMNs). Morphology and Identification A. Typical Organisms: Neisseria is (aerobic, Gram-negative, nonmotile diplococcus, approximately 0.8 µm in diameter. Individual cocci are kidney bean shaped; when the organisms occur in pairs, the flat or concave sides are adjacent. B. Culture: -grow on sheep blood agar, chocolate agar, and selective agar media (eg, modified Thayer-Martin agar, Martin-Lewis agar and New York City medium). N. meningitis grows on sheep blood agar as well as selective media. N. gonorrhoeae requires enriched chocolate agar and/or selective media for optimal growth. The selective media contain: -vancomycin (suppression of Gram-positive bacteria). - colistin (suppression of Gram-negative bacteria). - and other inhibitory substances to suppress the growth of many of the commensal microorganisms from these clinical sites. (N. gonorrhoeae, N. meningitides, and N. lactamica are colistin-resistant, and are therefore able to grow on these selective media). C. Growth Characteristics: The neisseriae grow best under aerobic conditions; however, some Neisseria species (eg, N. gonorrhoeae) are capable of growing under anaerobic conditions as well. The neisseriae produce acid but not gas by oxidation of various carbohydrates (not by fermentation!); the oxidase test is hence a key test for identifying neisseriae. Furthermore, all Neisseria species, with the exception of N. elongata, are catalase positive. Neisseria species are grow best on media containing complex organic substances, such as heated blood, hemin, and animal proteins, and in an atmosphere containing 5% CO2. These organisms are also rapidly killed by drying, prolonged exposure to sunlight, moist heat, and many disinfectants. They produce autolytic enzymes that result in rapid swelling and lysis in vitro at 25°C and at an alkaline pH. 2  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ NEISSERIA GONORRHOEAE Gonococci oxidize only glucose and differ antigenically from the other neisseria. Antigenic Structure N. gonorrhoeae is antigenically heterogeneous and capable of changing its surface structures in vitro—and presumably in vivo—to avoid host defenses. Surface structures include the following. A. Pili (Fimbriae): Pili are the hairlike appendages. They enhance attachment to host cells and resistance to phagocytosis. They are made up of stacked pilin proteins B. Por: Por protein extends through the gonococcal cell membrane. It forms pores in the surface through which some nutrients enter the cell. Por proteins may impact intracellular killing of gonococci within neutrophils by preventing phagosome– lysosome fusion. C. Opa Proteins: adhesion of gonococci within colonies and in attachment of gonococci to host cell receptors. D. Rmp (Protein III): is a reduction-modifiable protein (Rmp) and changes its apparent MW when in a reduced state. It associates with Por in the formation of pores in the cell surface. E. Lipooligosaccharide: In contrast to the enteric Gram-negative rods, gonococcal lipopolysaccharide (LPS) does not have long O-antigen side chains and is called a lipooligosaccharide (LOS). Toxicity in gonococcal infections is largely attributable to the endotoxic effects of LOS. F. Other Proteins: Lip (H8) is a surface exposed protein that is heat modifiable like Opa. The Fbp (ferric-binding protein). Pathogenesis, Pathology, and Clinical Findings Gonococci that form opaque colonies are isolated from men with symptomatic urethritis and from uterine cervical cultures at midcycle. Gonococci that form transparent colonies are frequently isolated from men with asymptomatic urethral infection, from menstruating women, and from patients with invasive forms of gonorrhea, including salpingitis and disseminated infection. Gonococci attack mucous membranes of the genitourinary tract, eye, rectum, and throat, producing 3  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ acute suppuration that may lead to tissue invasion; this is followed by chronic inflammation and fibrosis. Men usually have urethritis, with yellow, creamy pus and painful urination. Gonococcal bacteremia leads to skin lesions (especially hemorrhagic papules and pustules) on the hands, forearms, feet, and legs and to tenosynovitis and suppurative arthritis, usually of the knees, ankles, and wrists. Gonococci can be cultured from blood or joint fluid of only 30% of patients with gonococcal arthritis. Gonococcal endocarditis is an uncommon but severe infection. Gonococcal ophthalmia neonatorum, an infection of the eye in newborns, is acquired during passage through an infected birth canal. Diagnostic Laboratory Tests A. Specimens: Pus and secretions are taken from the urethra, cervix, rectum, conjunctiva, throat, or synovial fluid for culture and smear. Blood culture is necessary in systemic illness. B. Smears: Gram-stained smears of urethral or endocervical exudates typically reveal many diplococci within PMNs, therefore providing a presumptive diagnosis. C. Culture Immediately after collection, pus or mucus is streaked on enriched selective medium (eg, modified Thayer-Martin medium [MTM]) and incubated in an atmosphere containing 5% CO2 at 37°C. To avoid overgrowth by contaminants, selective media contain antimicrobial drugs (eg, vancomycin, colistin, nystatin, and trimethoprim). If immediate incubation is not possible, the specimen should be placed in a CO2 - containing transport-culture system. Forty-eight hours after culture, identified presumptive identification can be achieved by the organisms’ appearance on a Gram-stained smear and by a positive oxidase test. The definitive species level of the sub-cultured bacteria may be determined by their ability to produce acid from certain carbohydrates by oxidation; the only carbohydrate used by N. gonorrhoeae is glucose NEISSERIA MENINGITIDIS Antigenic Structure Capsular polysaccharides: The six most important serogroups associated with disease in humans, worldwide, are A, B, C, X, Y, and W-135. Incorporation of human sialic acid derivatives such as NANA into the meningococcal capsules allows the organism to be overlooked by the host immune system (often referred to as “molecular mimicry”). 4  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ The outer membrane of N. meningitidis consists of proteins and LPS that play major roles in organism virulence. There are two porin proteins (Por A and Por B), interact with host cells. The opacity proteins (Opa) are comparable to Opa of the gonococci and play a role in attachment. Meningococci are piliated and these structures initiate binding to nasopharyngeal epithelial cells and other host cells such as endothelium and erythrocytes. The lipid A disaccharide of meningococcal LPS is responsible for many of the toxic effects found in meningococcal disease. The highest levels of endotoxin. Pathogenesis, Pathology, and Clinical Findings: The nasopharynx is the portal of entry. There, the organisms attach to epithelial cells with the aid of pili; they may form part of the transient microbiota without producing symptoms and/or disease. Invasive meningococcal diasease (IMD) occurs in only a small number of individuals who acquired the organism and are transient carriers; infants and adolescents have the highest incidence of IMD in developed countries. From the nasopharynx, organisms may reach the bloodstream, producing meningococcal bacteremia; the initial symptoms during this stage of the actual infection may be similar to those of an upper respiratory tract, “flu-like” infection, but IMD quickly ensues. IMD typically presents as meningitis, sepsis (ie, meningococcemia), or as a combination of both. Meningitis is the most common complication of menigococcal bacteremia. It usually begins suddenly with an intense headache, vomiting, photophobia, confusion, and stiff neck; it may progress to coma within a few hours. Fulminant meningococcemia is more severe, presenting with a high fever and a hemorrhagic rash; the patient may also develop disseminated intravascular coagulation and ultimate circulatory collapse with bilateral hemorrhagic necrosis of the adrenal glands with subsequent adrenal failure (Waterhouse-Friderichsen syndrome). In meningitis, the meninges are acutely inflamed, with thrombosis of blood vessels and exudation of polymorphonuclear leukocytes, so that the surface of the brain is covered with a thick purulent exudate. The exact mechanisms that transform an asymptomatic colonization of the nasopharynx into meningococcal bacteremia, subsequently leading to meningococcemia and meningitis, are not very well understood. 5  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Diagnostic Laboratory Tests A. Specimens: The typical specimens for isolation of N. meningitides include blood for culture and cerebrospinal fluid (CSF) for smear and culture. Puncture material or biopsies from petechiae may be taken for smear and culture. Nasopharyngeal swab cultures are suitable for carrier surveys. B. Smears: Gram-stained smears of the sediment of centrifuged spinal fluid or of petechial aspirate often show typical neisseriae within polymorphonuclear leukocytes or extracellularly. C. Culture: Although neisseriae are inhibited by certain toxic factors present in media and polyanethole sulfonate (anticoagulant) present in commercial blood culture broths, this seems to be of a lesser problem for the ability to recover N. meningitis from blood cultures, compared to N. gonorrhoeae. CSF specimens are plated on sheep blood agar and chocolate agar and then incubated at 37°C in an atmosphere of 5% CO 2. A MTM agar favors the growth of neisseriae, inhibits many other bacteria, and is used for nasopharyngeal cultures. Colonies of N. meningitidis are gray, convex, and glistening, with entire edges; a positive oxidase test together with a Gram-stain showing Gram-negative diplococci provides presumptive organism identification. Spinal fluid and blood generally typically yield pure cultures that can be further identified by carbohydrate oxidative reactions and subsequent agglutination with type-specific or polyvalent serum. D. Serology: Antibodies to meningococcal polysaccharides can be measured by latex agglutination or hemagglutination tests or by their bactericidal activity. 6  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 2. Enteric Gram-negative rods: Escherichia coli - The general characteristics of Enterobacteriaceae Gram-negative bacilli. Found as commensals in the intestinal tract of mammals. They are also referred to as coliforms or enteric bacteria. Aerobic and facultative anaerobic growth. Optimal growth normally at 37 C˚. Grow readily on simple media. Ferment wide range of carbohydrates. According to the Lactose fermantation they are classified into: ❖ Lactose fermenter Fermentation of lactose to produce pink colonies on MacConkey’s agar is characteristic of Escherichia, Enterobacter and Klebsiella. ❖ Non-lactose fermenter Salmonella, Shigella, Serratia, Proteus and Yersinia do not ferment lactose and form pale ‫ شاحب‬colonies on MacConkey’s agar. ❖ Late Lactose fermenters, Shigella sonnei. Oxidase-negative. Some are motile (Motile except Shigella and Klebsiella ). Bile tolerant and grow readily on bile-salt containing media, e.g. MacConkey’s agar. Some of them produce urease. (which splits urea with release of ammonia). Some of them produce Hydrogene sulphide. Some of them decarboxylase amino-acids. Some of them derive the indole ring from the amino acid tryptophan. None-spore forming. None acid fast. Ferment glucose with acid production Reduce nitrates into nitrites Non-capsulated except Klebsiella Non-fastidious Enterobacteriaceae possess a variety of antigens: - lipopolysaccharide somatic antigen(‘O’), - flagellar antigen(‘H’) - capsular polysaccharide (‘K’) antigens. 7  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Escherichia coli Morphology E. coli is Gram-negative (-ve) rod-shaped bacteria. It is 1-3 x 0.4-0.7 µm in size and 0.6 to 0.7 µm in volume. It is arranged singly or in pairs. It is motile due to peritrichous flagella (see classification of flagella ). Some strains are non-motile. ‫هذا استثناء نادر‬ Some strains may be fimbriated. The fimbriae are of type 1 (hemagglutinating & mannose- sensitive) and are present in both motile and non-motile strains. Some strains of E. coli isolated from extra-intestinal infections have a polysaccharide capsule. They are non-sporing. They have a thin cell wall with only 1 or 2 layers of peptidoglycan. They are facultative anaerobes. Growth occurs over a wide range of temperatures from 15-45°C. Antigenic Structure and Pathogenicity ✓ Specific fimbriae (adhesins) facilitate adherence to mucosal surfaces and colonization of the intestinal and urinary tracts. ✓ E. coli possesses 4 antigens; H, O, K and F. A. Flagellar or (H) Antigen Heat and alcohol labile protein Present on the flagella Genus specific Present as monophasic 75 ‘H’ antigens have been recognized B. Somatic or (O) Antigen Heat stable, resistant to boiling. Occur on the surface of the outer membrane An integral part of the cell wall 173 ‘O’ antigens have been recognized C. Capsular or (K) Antigen Heat labile 8  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Acidic polysaccharide (polysaccharides that contain carboxyl groups, phosphate groups and/or sulfuric ester groups) antigen presents in the envelope Boiling removes the K antigen Inhibit phagocytosis 103 ‘K’ antigens have been recognized D. Fimbrial or (F) Antigen Heat labile proteins Present in the fimbriae K88, K99 antigens ✓ The heat stable lipopolysaccharide (endotoxin) “in the cell wall is liberated when Gram- negative bacteria lyse”, resulting in production of inflammatory mediators and complement activation “ plasma proteins that can be activated directly by pathogens or indirectly by pathogen-bound antibody”. This results in endotoxic shock and intravascular coagulopathy. Different protein toxins (exotoxins) produced by E. coli. Verocytotoxin-producing E. coli (VTEC), also known as (Shiga toxin-producing E. coli )(STEC) particularly the O157:H7 serotype, are an important cause of diarrhoea and hemolytic uremic syndrome (HUS). Enteropathogenic (EPEC): cause of infantile diarrhoea, non-invasive. Enterotoxigenic (ETEC): travelers’ diarrhoea, non-invasive. Enteroinvasive (EIEC): causes dysentery-like illness. Enteroaggregative (EAEC): watery diarrhoea without fever. ‫تم حذف التفاصيل الخاصة بكل نوع‬ Pathogenicity of E. coli Most infections (with the exception of neonatal meningitis and gastroenteritis) are endogenous; that is, the E. coli that are part of the patient’s normal microbial flora are able to establish infection when the patient’s defenses are compromised (e.g., through trauma or immune suppression). This organism is associated with a variety of diseases, including gastroenteritis and extra-intestinal infections such as UTIs, meningitis, and sepsis. Clinical Feature of E. coli 1. Gastroenteritis 2. Urinary Tract Infection 3. Sepsis 4. Meningitis 9  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Laboratory Diagnosis E. coli on Blood Agar 1. Colonies are big, circular, gray, and moist. 2. Non-hemolytic colonies (gamma-hemolysis) (Above Figure) OR Beta (β)hemolytic (Below Figure) colonies are formed. 3. Many pathogenic strains are haemolytic on blood agar. E. coli on MacConkey Agar 1. Colonies are circular, moist, smooth, and of entire margin. 2. Colonies appear flat and pink. 3. They are lactose fermenting colonies. 10  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ E. coli on Eosin Methylene Blue (EMB) Agar 1. Green Metallic sheen colonies are formed. ‫تم االعتماد على األوساط شائعة االستعمال‬ E. coli Biochemical Characters, - Glucose, Lactose, Mannitol, Maltose fermented with Acid and Gas. - Indole (+ve) - Methyl Red (+ve) - Voges Proskauer (-ve) - Citrate (-ve) - Urease not produced. - H2S (-ve) - Motility test (+ve) 11  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 3-Klebsiella The genus was originally divided into 3 main species based on biochemical reactions. Today, 7 species with demonstrated similarities in DNA homology are known. These are (1) Klebsiella pneumoniae, (2) Klebsiella ozaenae, (3) Klebsiella rhinoscleromatis, (4) Klebsiella oxytoca, (5) Klebsiella planticola, (6) Klebsiella terrigena, and (7) Klebsiella ornithinolytica Klebsiella pneumoniae General characteristics: K. pneumoniae is typically colonizes human mucosal surfaces of the oropharynx and gastrointestinal (GI) tract. It is recorded to be associated with pneumonia in the alcoholic and diabetic patient population. K. pneumoniae is also a well-known cause of community-acquired pneumonia. It is mostly commonly isolated Gram-negative, non-motile bacteria possesses a polysaccharide capsule, which protects against phagocytosis and antibiotics and makes the colonies moist and mucoid. has a distinctive “yeasty” odor. Antigenic Structure Members of the genus Klebsiella form large capsules consisting of polysaccharides (K antigens) covering the somatic (O or H) antigens and can be identified by capsular swelling tests with specific antisera. Cultural and biochemical characteristics Klebsiella species exhibit mucoid growth, large polysaccharide capsules Table 3.1 and Figure 3.1, and lack of motility, and they usually give positive test results for lysine decarboxylase and citrate. Klebsiella, species usually give positive Voges-Proskauer reactions Table 3.2. Table 3.1 Cultural Characteristics of Klebsiella pneumoniae on some laboratory media Cultural Nutrient Agar MacConkey Agar Blood Agar EMB Agar Characteristics Medium (NAM) medium Medium medium Shape Circular Circular Circular Circular Size 2-3 mm 2-3 mm 2-3 mm 2-3 mm Elevation Dome-shaped Convex Dome-shaped Convex Surface Mucoid Mucoid Mucoid Mucoid Color Greyish white Pink – Red Greyish white Pink – Purple Structure Translucent–Opaque Opaque Translucent–Opaque Translucent– Opaque Hemolysis ----- ----- γ-Hemolysis (Non- ----- hemolytic) 12  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Figure 3.1 Klebsiella pneumoniae Growing on MacConkey, Nutrient, Blood and Eiosin Methylene Blue agar plates respectively. Table 3.2 Biochemical tests of Klebsiella pneumoniae Characteristics Klebsiella pneumoniae Capsule +ve Catalase +ve Citrate +ve Gelatin Hydrolysis -ve Gram Staining -ve H2 S -ve Indole -ve Motility -ve MR (Methyl Red) -ve Nitrate Reduction +ve Oxidase -ve Pigment -ve Shape Rod Spore -ve TSIA (Triple Sugar Iron Agar) A/A Urease +ve VP (Voges Proskauer) +ve 13  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Pathogenesis and Clinical Findings Klebsiella species are present in the nasopharynx and feces of about 5% of normal individuals. The most commonly isolates species are K. pneumoniae and K. oxytoca. While K. pneumoniae may be isolated more frequently than K. oxytoca by clinical laboratories, both species are important human pathogens. K. pneumoniae can produce a lobar pneumonia, the production of “currant jelly” sputum. Klebsiella species also cause urinary tract infections, wound and soft tissue infections, and bacteremia/sepsis. K. pneumoniae has emerged as a cause of community-acquired pyogenic liver abscess. Klebsiella species responsible for hospital-acquired infections. Klebsiella granulomatis (formerly Calymmatobacterium granulomatis) causes a chronic genital ulcerative disease, and is thought to be a sexually transmitted disease. Proteus, Morganella and Providencia - Normal flora of the GI tract (except Providencia). - Non-lactose ferment - All motile, with Proteus swarming (Figure 3.2) motility with peritrichous flagella, non- spore forming, - Phenylalanine Deaminase Test Positive (PA+Phenylalanine Agar) - Lysine deamination + (LIA (Lysine Iron Agar) Lysine Iron Agar (LIA) is used to differentiate enteric bacilli based on their ability to decarboxylate or deaminate lysine and produce hydrogen sulfide (H2S). LIA also is used in combination with Triple Sugar Iron Agar to identify members of Salmonella and Shigella R/A) - Urease production was positive for most members and it's strongly + for Proteus - TSI variable for every genus - Indole test positive except P. mirabilis is -ve 14  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Proteus species - P. mirabilis and P. vulgaris are widely recognized human pathogens. - The spot-indole test is useful for differentiation between the two most common Proteus species: is P. vulgaris indole positive, whereas P. mirabilis is negative. - Isolated from urine, wounds, and ear and rarely from bacteremia - Both produce swarming (Rauss phenomenon) colonies on non-selective media and have a distinctive “burned chocolate” odor - Both are strongly urease positive - Both are phenylalanine deaminase positive The swarming of Proteus can be inhibited by: Figure 3.2 Swarming phenominon of Proteus species Increasing the concentration of agar from 1-2% to 6%. Incorporation of sodium azide, boric acid, or chloral hydrate in the medium. The addition of growth inhibitors like sulphonamides to the medium. Addition of Teepol (a surface-active agent), which is present in Teepol Lactose agar Table 3.3: Culture Characteristics of Proteus vulgaris Cultural Nutrient Agar MacConkey Blood Agar EMB Agar Characteristics Medium (NAM) Agar medium Medium medium Shape Irregular (due to Circular Irregular (due to Circular swarming) swarming) Size 1-2 mm 2-3 mm 1-2 mm 2-3 mm Elevation Effuse Low Convex Effuse Effuse Surface Glistening Smooth Glistening Glistening Color Greyish white Colorless or Pale Greyish white Colorless colored Structure Translucent Transparent Translucent – Transparent Opaque Hemolysis ----- ----- γ-Hemolysis (Non- ----- hemolytic) 15  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ https://www.slideserve.com/yardley-carver/shigella-proteus https://www.med.muni.cz/mikroblg/atlas/atlas/bacteriology/proteus/atlas_en.html https://microbe-canvas.com/Bacteria/gram-negative-rods/facultative-anaerobic-3/catalase-positive-3/oxidase- negative/colistin-resistant/proteus-vulgaris.html Antigenic Structure The Proteus possess thermostable, somatic (O), and thermolabile flagellar (H) antigens upon which, several serotypes have been recognized. Pathogenicity and Pathogenesis The two species to most commonly produce infections in humans are P. mirabilis and P. vulgaris. Both species produce urease, resulting in rapid hydrolysis of urea with liberation of ammonia. Thus, in urinary tract infections with Proteus species, the urine becomes alkaline, promoting stone formation and making acidification virtually impossible. The rapid motility of Proteus may also contribute to its invasion of the urinary tract. P. mirabilis causes urinary tract infections and occasionally other infections, such as bloodstream infection (frequently secondary due to a UTI) and respiratory tract infections. P. vulgaris is probably more frequently implicated in wound and soft tissue infections than UTIs. Biochemical tests and identification Basic Characteristics Properties (Proteus mirabilis) Capsule Negative (-ve) Catalase Positive (+ve) Citrate Positive (+ve) 16  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Basic Characteristics Properties (Proteus mirabilis) Flagella Positive (+ve) Gas from Glucose Positive (+ve) Gelatin Hydrolysis Positive (+ve) Gram Staining Negative (-ve) H2S Positive (+ve) Indole Negative (-ve) Motility Positive (+ve) MR (Methyl Red) Positive (+ve) Nitrate Reduction Positive (+ve) Oxidase Negative (-ve) Pigment Negative (-ve) Shape Rods Spore Negative (-ve) Urease Positive (+ve) VP (Voges Proskauer) Negative (-ve) Fermentation of Glucose Positive (+ve) Lactose Negative (-ve) Enzymatic Reactions Acetate Utilization Negative (-ve) Esculin Hydrolysis Negative (-ve) Lipase Positive (+ve) Lysine decarboxylases Negative (-ve) Phenylalanine Deaminase Positive (+ve) Tryptophan Deaminase Negative (-ve) 17  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 4. Pseudomonads and Acinetobacter Pseudomonads The pseudomonads are Gram-negative, motile, aerobic rods, some of which produce water-soluble pigments. The pseudomonads occur widely in soil, water, plants, and animals. P aeruginosa is frequently present in small numbers in the normal intestinal flora and on the skin of humans, and is the major pathogen of the group. Other pseudomonads infrequently cause disease. In the general population P. aeruginosa is carried by very few people but this can rise to over 30% after a stay in hospital. The invasive potential of this organism means that it causes disease in a wide range of hospital patients. It is a particular problem to the neutropenic patient where it can cause fulminant septicaemia and death. Patients undergoing artificial ventilation for extended periods in intensive therapy units may become colonized with P. aeruginosa and secondary lower respiratory tract infection may follow. Extensive burns become colonized and septicaemia develops in a proportion of patients. Multidose optical solutions can be contaminated by P. aeruginosa which, when used, can produce a rapidly progressive corneal infection which ends in ocular perforation. Pseudomonas aeruginosa is an important pathogen for patients with cystic fibrosis where colonization with this organism is inevitable. Skin infection may arise in healthy subjects exposed to high infective doses such as deep sea divers and users of contaminated hydrotherapy pools and Jacuzzi. Pseudomonas aeruginosa. It is widely distributed in nature and is commonly present in moist environments in hospitals. It can colonize normal humans; in whom it is a saprophyte. It causes disease in humans with abnormal host defenses, especially in individuals with neutropenia. Classification: There are more than 100 species in the genus Pseudomonas. There are two primary pathogens, P. pseudomallei and P. mallei. Morphology and Identification: A. Typical Organisms P. aeruginosa is motile (except P. mallei) and rod shaped, measuring about 0.6 ×2 μm. It is Gram-negative and occurs as single bacteria, in pairs, and occasionally in short chains. 18  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ B. Culture P aeruginosa is an obligate aerobe that grows readily on many types of culture media, sometimes producing a sweet or grape-like or corn taco–like odor. Some strains hemolyze blood. P aeruginosa forms smooth round colonies with a fluorescent greenish color pyoverdin which gives a greenish color to the agar. It often produces the non-fluorescent bluish pigment pyocyanin, which diffuses into the agar. Other Pseudomonas species do not produce pyocyanin, Some strains produce the dark red pigment pyorubin or the black pigment pyomelanin. C. Growth Characteristics P aeruginosa grows well at 37–42°C; its growth at 42°C helps differentiate it from other Pseudomonas species that produce fluorescent pigments. It is oxidase positive. It does not ferment carbohydrates, but many strains oxidize glucose. Antigenic Structure and Toxins: Pili: Adhere to epithelial cells Exopolysaccharide: Anti-phagocytic property/ inhibit pulmonary clearance. Lipopolysaccharide: Endotoxic effect Enzymes Elastases: Digests protein (elastin, collagen, IgG) Proteases Hemolysins Phospholipases C (heat labile): Degrade cytoplasmic membrane components Exotoxin A: Cytotoxic by blocking protein synthesis. Endotoxin: like that of other gram-negative bacteria, causes the symptoms of sepsis and septic shock. Pathogenesis: Pseudomonas aeruginosa is primarily an opportunistic pathogen that causes infections in hospitalized patients (e.g., those extensive burns), with in whom the skin host defenses are destroyed; in those with chronic respiratory disease (e.g., cystic fibrosis), in whom the normal clearance mechanisms are impaired; in those who are immunosuppressed; Urinary tract infection- chronic, complicated Urinary tract infection and associated with indwelling catheter. Wound infection of burn sites, pressure sores and ulcers. Septicaemia- “Ecthyma gangrenosum” skin lesion (haemorrhagic skin necrosis) 19  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Otitis externa- Malignant external ear infection in poorly treated diabetic patients. Pneumonia- Infection of the lung in patients with cystic fibrosis. Eye infection- Secondary to trauma or surgery. Laboratory diagnosis: Isolation Bacteria of the genus Pseudomonas grow readily on simple media such as nutrient or blood agar, and will also grow on the less inhibitory selective media such as MacConkey. Specimen: pus, urine, sputum, blood, eye swabs, surface swabs Smear: Gram-negative rods. Pseudomonas pseudomallei is usually isolated from sputum, blood or pus from abscesses. Culture: Obligate aerobe, grows readily on all routine media over wide range of temperature (5-42 OC). Bluish-green pigmented large colonies with characteristic “fruity” odor on culture media. Media can be made selective for Pseudomonas by the incorporation of one or more of the antibiotics or disinfectants to which it is naturally resistant such as irgasin, cetrimide or nalidixic acid. Colonies of P. aeruginosa are morphologically diverse and dwarf, rough, mucoid, rugose, coliform- like colonies and the more commonly encountered large convex, flat, oval colonies are described. A culture of P. aeruginosa has a characteristic musty odour. The colonies of P. pseudomallei and P. mallei are slower to appear and are typically wrinkled with a faint pinkish colour developing after about five days. P. aeruginosa are lactose and fructose oxidation, arginine dehydrolase, gelatinase and lysine decarboxylase. In Centrimide agar: Pseudomonas aeruginosa colonies (greenish-blue in color) are medium sized and characterized by an irregular growth In blood agar: Colonies of Pseudomonas aeruginosa surrounded by a wide zone of beta-hemolysis. Cultivation 48 hours in an aerobic atmosphere, 37°C. 20  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Pseudomonas aeruginosa may produce the characteristic blue-green pigment or none at all Biochemical reactions: Oxidase positive Catalase-positive Citrate-positive Indole-negative Produce acid from carbohydrate by oxidation, not by fermentation. ADH: Arginine dehydrolase, ODC: Ornithine decarboxylase Acinetobacter Acinetobacter species are aerobic, Gram-negative bacteria that are widely distributed in soil and water and can occasionally be cultured from skin, mucous membranes, secretions, and the hospital environment. A baumannii is the species most commonly isolated. Acinetobacter lwoffii and other species are isolated occasionally. A. Morphology and Identification: Acinetobacters are usually coccobacillary or coccal in appearance; they resemble neisseriae on smears, because diplococcal forms predominate in body fluids and on solid media. Rod-shaped forms also occur, and occasionally the bacteria appear to be Gram-positive. B. Culture: Acinetobacter grows well on most types of media used to culture specimens from patients. Acinetobacter recovered from patients with meningitis, bacteremia, female genital, sputum, skin, pleural fluid, and urine, usually. 21  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 5- Shigella and Salmonella Genus: Shigella Familly: Enterobacteriaceae Tribe: Escherichia Genus: Salmonella Discovered by Kiyoshi Shiga in 1898. It is the causative agent of human shigellosis. Classification: There are more than 40 serotypes. The classification of shigellae relies on biochemical and antigenic characteristics (O antigens). The pathogenic species are Shigella sonnei, Shigella flexneri, S. dysenteriae, and Shigella boydii. Important Properties: Shigellae are - short Gram-negative rods. - non–lactose-fermenting. - Resistant to bile salts - Divided into four groups: A, B, C, and D according to (O) antigen. Shigella can be distinguished from salmonellae by three criteria: They produce no gas from the fermentation of glucose They do not produce H2S They are non-motile. Virulence Factors: 1. K. capsular antigen 2. O. antigen (HL) 3. Shiga toxin: with cytotoxic and neurotoxic activity. 22  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Pathogenesis of Shigella: Shigella causes bacillary dysentery , Low infective dose < 200 bacilli ( can be transmitted easily unlike salmonella (More serious and virulent than salmonella) Incubation period = 1-3 days Upon ingestion, the bacteria pass through the gastrointestinal tract until they reach the small intestine. There they begin to multiply until they reach the large intestine. In the large intestine, the bacteria cause cell injury and the beginning stages of Shigellosis via two main mechanisms: direct invasion of epithelial cells in the large intestine and production of enterotoxin 1 and enterotoxin 2. High fever, chill, abdominal cramp and pain accompanied by tenesmus, bloody stool with mucus & WBC and HUS are involved. 23  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Laboratory Diagnosis: Specimens: include fresh stool, mucus flecks, and rectal swabs for culture. Large numbers of fecal leukocytes and some red blood cells often are seen microscopically. Culture: The materials are streaked on differential media (eg, MacConkey or EMB agar) and on selective media (Hektoen enteric agar or xylose-lysine-deoxycholate agar), which suppress other Enterobacteriaceae and Gram-positive organisms. Colorless (lactose-negative) colonies are inoculated into TSI agar. Organisms that fail to produce H2S, that produce acid but not gas in the butt and an alkaline slant in TSI agar medium. Salmonella Shigella (SS) Agar: Shigella Clear, colorless, transparent. XLD- Agar: Shigella flexneri Red Colonies TSI-Agar: Salmonella Alkaline slant/acidic butt (K/A); - H2S and Gas- Genus: Salmonella Introduction:. The organisms are named after the American veterinary pathologist Daniel Elmer Salmon in 1885. Currently, there are three recognized species: S. enterica, S. bongori and S. subterranean. Salmonella is found worldwide in cold- and warm-blooded animals (including humans), and in the environment. They cause illnesses such as typhoid fever, paratyphoid fever, and foodborne illness. Classification: The members of the genus Salmonella were originally classified on the basis of epidemiology; host range; biochemical reactions; and structures of the O, H, and Vi (when present) antigens. Salmonella spp. have both H and O antigens. There are over 60 different O antigens, and individual strains may possess several O and H antigens; the latter can exist in variant forms, termed ‘phases’. Salmonella serotype Typhi also has a capsular polysaccharide antigen referred to as ‘Vi’ (for virulence), which is related to invasiveness Over 2500 serotypes are distinguished, most of which belong to the species S. enterica. However, many of these have been given binomial names (e.g. Salmonella typhimurium and Salmonella enteritidis), although they are not separate species. In clinical practice, laboratories identify microorganisms according to their binomial name. 24  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Important Properties: Salmonellae are motile rods that characteristically ferment glucose and mannose without producing gas but do not ferment lactose or sucrose. Most salmonellae produce H2S. They are often pathogenic for humans or animals when ingested. Virulence Factors: 1. Type III secretion systems: which facilitate secretion of virulence factors of Salmonella into host cells. 2. Endotoxin: Endotoxin is responsible for many of the systemic manifestations of the disease caused by Salmonella spp. 3. Fimbriae: The species-specific fimbriae mediate binding of Salmonella to M (microfold) cells present in Peyer patches of the terminal part of the small intestine. These M cells typically transport foreign antigens, such as bacteria to the underlying macrophages for clearance. 4.Acid tolerance response gene: The acid tolerance response (ATR) gene protects Salmonella spp. from stomach acids and the acidic pH of the phagosome, thereby facilitating survival of bacteria in phagosomes 5. Enzymes: Catalase and superoxide dismutase are the enzymes that protect the bacteria from intracellular killing in macrophages. Pathogenesis of Salmonella: The three types of Salmonella infections (enterocolitis, enteric fevers, and septicemia ‫ )وجود البكتريا بمجرى الدم وتكاثرها‬have different pathogenic features. 25  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ (1) Enterocolitis: is characterized by an invasion of the epithelial and sub-epithelial tissue of the small and large intestines. (2) In typhoid and other enteric fevers, infection begins in the small intestine, but few gastrointestinal symptoms occur. (3) Septicemia accounts for only about 5−10% of Salmonella infections and occurs in one of two settings: a patient with an underlying chronic disease, such as sickle cell anemia or cancer, or a child with enterocolitis. 26  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Laboratory Diagnosis: In enterocolitis: the organism is most easily isolated from a stool sample in selective media e.g. XLD (Xylose lysine deoxycholate agar), DCA (deoxycholate citrate agar), salmonella-shigella (SS) agar, and enrichment media, e.g. selenite broth; identification of Salmonella spp. by biochemical agglutination tests. Phage typing can be used for typing individual strain. Salmonella Shigella (SS) Agar: salmonella colorless, transparent, with a black center if H2S is produced XLD- Agar: Salmonella Typhi red Colonies, black centers. TSI-Agar: Salmonella Alkaline slant/acidic butt (K/A); + H2S and Gas +. In the enteric fevers: a blood culture is the procedure most likely to reveal the organism during the first weeks of illness. Stool cultures may also be positive, especially in chronic carriers in whom the organism is secreted in the bile into the intestinal tract. Urine culture results may be positive after the second week. Serologic Methods: I. Agglutination test II. Tube dilution agglutination test (Widal test): Serum agglutinins rise sharply during the second and third weeks of S serotype Typhi infection. 27  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 6-Yersinia Yersinia pestis is short, pleomorphic, Gram-negative rods that often exhibit bipolar staining (A bipolar stain is a particular staining pattern those colors only the two opposite poles of the microorganism in question, leaving the rest of the bacterium unstained or a lighter color) with special stains such as Wright, Giemsa, Wayson, or methylene blue, appear as single cells or as pairs or short chains in clinical material. They are catalase positive and microaerophilic or facultatively anaerobic. Cultural and Biochemical Characteristics It is non-motile, grows as a facultative anaerobe on many bacteriologic media, and can be readily isolated when sterile specimens such as blood or lymph node aspirates are plated onto sheep blood agar. Growth is more rapid when agar plates are incubated at 28°C. In cultures on sheep blood agar incubated at 37°C, colonies may be smaller when compared to colonies from agar plates incubated at 28°C. Colonies of Y. pestis are typically gray to white, sometimes opaque, and are 1–1.5 mm in diameter with irregular edges; the organism does not produce hemolysis. Antigenic Structure All yersiniae possess - lipopolysaccharides that have endotoxic activity when released. - Y. pestis and Y. enterocolitica also produce antigens and toxins that act as virulence factors. - They have type III secretion systems that consist of a membrane-spanning complex that allows the bacteria to inject proteins directly into cytoplasm of the host cells. - The virulent yersiniae produce V and W antigens. 28  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Clinical Findings The clinical manifestations of plague depend on the route of exposure, and three forms of the disease have been described: - bubonic plague,‫( الطاعون الدبلي‬the most common, incubation period of 2–7 days, sudden onset of high fever and development of painful lymphadenopathy, tender lymph nodes (buboes) in the neck, groin, or axillae). - pneumonic plague, commonly with greatly enlarged - septicemic plague. (Occur spontaneously or as a complication of untreated bubonic plague, Y. pestis multiplies intravascularly, can be seen in blood smears) - Patients typically present with a sudden onset of high fever, chills, and weakness, progressing rapidly to septic shock with associated disseminated intravascular coagulation, hypotension (septic shock), - altered mental status, and renal and cardiac failure. - Bleeding into skin and organs can also occur. Vomiting and diarrhea may develop during the early stages of septicemic plague. Terminally, - signs of pneumonia and meningitis can appear. Pathogenesis and Pathology When a flea ‫ برغوث‬feeds on a rodent ‫ قوارض‬infected with Y. pestis, the ingested organisms multiply in the gut of the flea and, helped by the coagulase, block its proventriculus ‫ االمعاء من امامي جزء‬so that no food can pass through. Subsequently, the “blocked” and hungry flea bites ferociously ‫بشراسة‬, and the aspirated blood, contaminated with Y. pestis from the flea, is vomited into the bite wound. The inoculated organisms may be phagocytosed by polymorphonuclear cells and macrophages. The Y. pestis organisms are killed by the polymorphonuclear cells but multiply in the macrophages; because the bacteria are multiplying at 37°C, they produce the antiphagocytic protein and subsequently are able to resist phagocytosis. The pathogens rapidly reach the lymphatics, and an intense hemorrhagic inflammation develops in the enlarged lymph nodes, which may undergo necrosis and become fluctuant. 29  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Diagnostic Laboratory Tests A. Specimens Blood is taken for culture and aspirates of enlarged lymph nodes for smear and culture. Acute and convalescent ‫ نقاهة‬sera be examined for antibody levels. In pneumonia, sputum is cultured; in possible meningitis, cerebrospinal fluid is taken for smear and culture. B. Smears Wright, Giemsa, or Wayson stains may be more useful when staining material from a suspected buboes or a positive blood culture result because of the striking bipolar appearance (safety pin shape) of the organism using these stains that is not evident on a direct Gram-stain. More specific direct staining methods include the use of fluorescent antibody stains targeting the capsular F1 antigen. C. Culture All materials are cultured on blood, chocolate, and MacConkey agar plates and in brain–heart infusion broth. Growth on solid media may be slow, requiring more than 48 hours, but blood culture results are often positive in 24 hours. Y. pestis produces non-lactose-fermenting colonies on MacConkey agar, and it grows better at 28°C than at 37°C. The organism is ✓ catalase positive; ✓ indole, oxidase, and urease negative; ✓ Non-motile. ✓ Definite ‫ حتمي‬identification of cultures is best done by immunofluorescence or by lysis by a specific Y. pestis bacteriophage. All cultures are highly infectious and must be handled with extreme caution inside a biological safety cabinet. 30  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 7-Vibrio Vibrios are among the most common bacteria in marine and estuarine waters, worldwide. They are comma-shaped, curved, and sometimes straight facultatively anaerobic, fermentative rods; they are catalase and oxidase positive, and most species are motile by means of monotrichous or multitrichous polar flagella. Vibrios can grow within a broad temperature range (14–40°C), and all species require sodium chloride (NaCl) for growth; hence the term halophilic (“salt loving”). V. cholerae serogroups O1 and O139 cause cholera in humans, and other vibrios, most commonly V. parahaemolyticus and V. vulnificus, are important human pathogens, causing skin and soft tissue infections, sepsis, or gastroenteritis. VIBRIO CHOLERAE The bacterium V. cholerae is the cause of cholera. The epidemiology of cholera closely parallels the recognition of V. cholerae transmission in water and the development of sanitary water systems. Cholera is associated with poor sanitation, as well as direct contact with or consumption of contaminated water and/or food (eg, water used for drinking, cooking, bathing, and crop irrigation). Morphology and Identification A. Typical Organisms Upon first isolation, V. cholerae is a comma-shaped, curved rod 2–4 µm long. It is actively motile by means of a polar flagellum. On prolonged cultivation, organisms may become straight rods that can resemble other Gram-negative enteric bacteria. B. Culture V. cholerae produces convex, smooth, round colonies that are opaque and granular in transmitted light. V. cholerae and most other vibrios grow well at 37°C on routine agar media to recover enteric bacteria (eg, blood agar and MacConkey agar); however, selective agars for Vibrio species, such as thiosulfate-citrate-bile salts-sucrose (TCBS) agar and enrichment broth (eg, alkaline peptone broth), can also be used to recover vibrios, especially from specimens (eg, stool) when a mixture of organisms is expected. All vibrios, including V. cholerae, grow well on TCBS agar; V. cholerae produces yellow colonies (sucrose fermented) on TCBS agar that are readily visible against the dark- green background of the agar. Non-sucrose-fermenting vibrios (eg, most strains of V. parahaemolyticus and V. vulnificus) produce green colonies on TCBS agar. 31  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Characteristically, vibrios grow at a very high pH (8.5–9.5) and are rapidly killed by acid. To ensure optimal recovery of vibrios, stool specimens should be collected early in the course of the diarrheal illness; prompt inoculation onto appropriate agar media is necessary. If processing of specimens may be delayed, the stool specimen should be mixed in a Cary-Blair transport medium and refrigerated. In areas where cholera is endemic, direct cultures of stool on selective media, such as TCBS, and enrichment broth cultures (eg, alkaline peptone water with 1% NaCl, pH 8.5) are appropriate. In the United States and other countries where cholera is rare, routine use of TCBS agar for stool cultures in clinical laboratories is generally not necessary or cost effective; exceptions may be made if recovery of other vibrios (eg, V. parahaemolyticus) is a frequent and/or seasonal occurrence (eg, coastal U.S. regions with regular and frequent consumption of bivalve mollusks and crustaceans). The Medically Important Vibrios C. Growth Characteristics V. cholerae regularly ferments sucrose and mannose but not arabinose. A positive oxidase test result is a key step in the preliminary identification of V. cholerae and other vibrios. While most Vibrio species are halophilic, requiring the presence of NaCl (range from < 0.5–4.5%) to grow, V. cholerae can grow on most agar media without additional salt. 32  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ FIGURE Gram-stain of V. cholerae. Often they are comma shaped or slightly curved (arrows) and 1 × 2 to 4 µm. Original magnification ×1000. FIGURE Colonies of V. cholerae growing on thiosulfate, citrate, bile salts, and sucrose agar. The glistening yellow colonies are 2–3 mm in diameter and are surrounded by a diffuse yellowing of the indicator in the agar up to 1 cm in diameter. The plate is 10 cm in diameter. Antigenic Structure and Biologic Classification Many vibrios share a single heat-labile flagellar H antigen. Antibodies to the H antigen are probably not involved in the protection of susceptible hosts. V. cholerae has O lipopolysaccharides that confer serologic specificity. Based on the O antigen, there are over 200 serogroups; however, only V. cholerae strains of serogroup O1 and O139 cause epidemic and pandemic cholera. Occasionally, non-O1/non-O139 V. cholerae strains have been described as causes of cholera-like diarrheal disease. Antibodies to the O antigens tend to protect laboratory animals against infections with V. cholerae. The V. cholerae serogroup O1 antigen has determinants that make possible further subtyping; these serotypes are Ogawa, Inaba, and Hikojima. Furthermore, two biotypes of epidemic V. cholerae have been defined, classic and El Tor. The El Tor biotype produces a hemolysin, gives positive results on the Voges-Proskauer test, and is resistant to polymyxin B. Molecular techniques can also 33  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ be used to type V. cholerae. Typing is used for epidemiologic studies, and tests generally are done only in reference laboratories. V. cholerae O139 is very similar to V. cholerae O1 El Tor biotype. V. cholerae O139 does not produce the O1 lipopolysaccharide and does not have all the genes necessary to make this antigen. V. cholerae O139 and other non-O1 V. cholerae strains, as well as V. vulnificus produce acidic polysaccharide capsules; however, V. cholerae O1 does not make a capsule. Vibrio cholerae Enterotoxin V. cholerae produce a heat-labile enterotoxin with a molecular weight (MW) of about 84,000, consisting of subunits A (MW, 28,000) and B (see Chapter 9). Ganglioside GM1 serves as the mucosal receptor for subunit B, which promotes entry of subunit A into the cell. Activation of subunit A 1 yields increased levels of intracellular cyclic adenosine monophosphate (cAMP) and results in prolonged hypersecretion of water and electrolytes. There is increased sodium-dependent chloride secretion, and absorption of sodium and chloride by the microvilli is inhibited. Electrolyte-rich diarrhea occurs with as much as 20–30 L/day, resulting in dehydration, shock, acidosis, and death. The genes for V. cholerae enterotoxin are located on the bacterial chromosome. Cholera enterotoxin is antigenically related to LT of Escherichia coli and can stimulate the production of neutralizing antibodies. However, the precise role of antitoxic and antibacterial antibodies in protection against cholera is not clear. Diagnostic Laboratory Tests A. Specimens As stated above, stool specimens should be collected early in the course of the diarrheal illness and inoculated within 2–4 hours of collection onto appropriate agar media, to ensure optimal recovery of vibrios. If processing of specimens may be delayed, the stool specimen should be mixed in a Cary- Blair transport medium and refrigerated. B. Smears Direct detection of V. cholerae on smears made from stool samples is not distinctive of the organism, and therefore not routinely recommended. Dark-field or phase-contrast microscopy can be used to detect V. cholerae O1 directly from stool samples or the enrichment broth. Observation of “shooting 34  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ star” motility is suggestive of V. cholerae O1; if the motility is extinguished after mixing the sample with a polyvalent O1 antiserum, the organism is confirmed as V. cholerae O1. However, if there is no motility or the type of motility does not change after applying the antiserum, the organism is not V. cholerae O1. C. Culture Vibrios, including V. cholerae, grow well on most agar media (including MacConkey and blood agar) used in clinical laboratories. Some strains of V. cholerae may however be inhibited on MacConkey agar. Growth is rapid in alkaline peptone broth or water, containing 1% NaCl with a pH of 8.5, or on TCBS agar; typical colonies can be picked in 18 hours of growth. For enrichment, a few drops of stool can be incubated for 6–8 hours in taurocholate peptone broth (pH, 8.0–9.0); organisms from this culture can then be stained or subcultured onto other appropriate agar media. Accurate identification of vibrios, including V. cholerae, using commercial systems and kit assays is quite variable. MALDI-TOF MS is a promising newer methodology for identification of vibrios, and studies have shown rapid and reproducibly accurate identification for V. parahaemolyticus. 35  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 8- CAMPYLOBACTER Introduction: Campylobacters cause both diarrheal and systemic diseases, and are among the most widespread causes of infection, worldwide. Campylobacter infections of wild and domesticated animals, which are also the natural reservoirs for these organisms, are also widespread. C. jejuni is the prototype organism in the group and is a very common cause of diarrhea in humans. Other campylobacters, less commonly isolated from humans, include C. fetus, C. coli, and C. upsaliensis. Scientific Content: Morphology and Identification A. Typical Organisms C. jejuni and other campylobacters are curved, comma-, or S-shaped, Gram-negative, non-spore- forming rods; they have also been described as having “sea gull wing” shapes. Campylobacters are motile, with a single polar flagellum at one or both ends, but some organisms may lack flagella all together. B. Culture Campylobacter species, including C. jejuni, multiply at a slower rate when compared to other Gram- negative, enteric bacteria; therefore, selective media, containing various antibiotics (eg, Campy- Blood agar and Skirrow’s media) are needed for isolation of campylobacters from stool specimens. Campylobacter species require a microaerobic atmosphere, containing reduced O2 (5–7%) and increased 10% CO2 for incubation and optimal growth. A relatively simple way to produce the incubation atmosphere is to place the plates in an anaerobe incubation jar without the catalyst and to produce the gas with a commercially available gas- generating pack or by gas exchange. Furthermore, most campylobacters grow best at 42°C, although growth can be seen on agar media with incubation between 36°C and 42°C. Incubation of primary plates for isolation of C. jejuni should always be at 42°C. Several selective agar media are in widespread use for isolation of campylobacters; Skirrow’s medium contains vancomycin, polymyxin B, and trimethoprim to inhibit growth of other bacteria, but this medium may be less sensitive than other commercial products that contain charcoal, other inhibitory compounds, as well as cephalosporin antibiotics. 36  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ These selective media are suitable for isolation of C. jejuni and C. coli at 42°C. However, C. upsaliensis, while growing at 42°C, is not recovered on selective media, and C. fetus shows variable growth at 42°C, and may not be recovered at that temperature. The colonies of Campylobacter species may have different appearances; generally, the colonies tend to be colorless or gray. They may be watery and spreading or round and convex, and both colony types may appear on one agar plate. Hemolysis on blood-containing agar media is not observed. Gram-stain of C. jejuni showing “comma”- or “gull wing”-shaped Gram- negative bacilli (arrows). Campylobacters stain faintly and can be difficult to visualize. C. Growth Characteristics Because of the selective media and incubation conditions for growth, an abbreviated set of tests is usually all that is necessary for further identification of campylobacters. C. jejuni as well as C. coli are positive for both oxidase and catalase. Campylobacters do not oxidize or ferment carbohydrates. Gramstained smears show typical morphology. Nitrate reduction, hydrogen sulfide production, hippurate hydrolysis tests, and antimicrobial susceptibilities can be used for further identification of species. A positive hippurate hydrolysis test distinguishes C. jejuni from the other Campylobacter species. Antigenic Structure and Toxins The campylobacters have lipopolysaccharides with endotoxic activity. Cytopathic extracellular toxins and enterotoxins have been found, but the significance of the toxins in human disease is not well defined. Diagnostic Laboratory Tests A. Specimens Diarrheal stool is the preferred specimen when attempting to isolate campylobacters in patients with gastrointestinal illness. Rectal swabs may also be acceptable specimens. C. jejuni and C. fetus may occasionally be recovered from blood cultures usually from immunocompromised or elderly patients. B. Smears Gram-stained smears of stool may show the typical “gull wing”–shaped rods. Dark-field or phase-contrast microscopy may show the typical darting motility of the organisms. C. Culture Culture on the selective media as described earlier is the definitive test to diagnose C. jejuni enteritis. If C. fetus or another species of Campylobacter is suspected, an agar medium without cephalosporins should be used and incubation at 36–37°C is necessary. 37  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 9- HELICOBACTER PYLORI Members of the genus Helicobacter are usually spiral, curved, or fusiform rod-shaped Gram-negative bacteria. Helicobacter species have been isolated from the gastrointestinal and hepatobiliary tract of many different mammalian hosts, including humans, dogs, cats, pigs, cattle, and other domestic and wild animals. The various helicobacters can be divides into two groups: Helicobacter species that primarily colonize the stomach (gastric helicobacters), and those that colonize the intestines (enterohepatic helicobacters). Humans are the primary host-reservoir for H. pylori, which is a spiral- shaped, Gram-negative, catalase- and oxidase-positive, and urease positive rod. H. pylori is associated with antral gastritis, duodenal (peptic) ulcer disease, gastric ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. Morphology and Identification A. Typical Organisms Helicobacter species, including H. pylori, have many characteristics in common with campylobacters. Helicobacter species are motile and have single and/or multiple monopolar flagella that are typically sheathed and can vary greatly in their flagellum morphology. B. Culture While H. pylori can be readily isolated from gastric biopsy specimens, culture sensitivity may be limited by several factors, including delayed specimen transport and processing, prior antimicrobial therapy, or contamination with other mucosal bacteria. Special transport media (eg, Stuart’s transport medium) should be used to main the organisms’ viability when transport to the laboratory is anticipated to exceed 2 hours. H. pylori usually grows within 3–6 days when incubated at 37°C in a microaerophilic and humid atmosphere; however, incubation of up to 14 days may be necessary before resulting the culture as negative. To achieve a higher yield for recovery of the organism, the biopsy specimen may be homogenized prior to streaking onto the agar plate. The agar media for primary isolation include enriched agar media supplemented with blood and/or blood products (eg, Chocolate agar) or antibiotic-containing media such as Skirrow’s medium, in order to suppress overgrowth by other competing bacterial flora. The colonies have varying appearance on blood agar ranging from gray to translucent and are 1–2 mm in diameter. 38  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ C. Growth Characteristics H. pylori is oxidase positive and catalase positive, and has a characteristic Gram-stain morphology; the organism is motile, and is a strong producer of urease. Pathogenesis and Pathology H. pylori is able to survive in the acidic environment of the stomach and ultimately establish lifelong colonization of the gastric mucosa in the absence of antimicrobial treatment. While H. pylori grows optimally at a pH of 6.0–7.0, it would be killed or not grow at the pH within the gastric lumen (pH 1–3). Several factors contribute to the organism’s ability to overcome the acidic environment of the stomach, contributing to colonization, inflammation, changes in gastric acid production, and tissue destruction. Gastric mucus is relatively impermeable to acid and has a strong buffering capacity. On the lumen side of the mucus, the pH is low (1.0–3.0); on the epithelial side, the pH is about 5.0–7.0. After entering the stomach, H. pylori utilizes its urease activity to neutralize the gastric acid; intracellular urease activity as well as urease located on the bacterial cell surface allow for the breakdown of urea into ammonia and CO 2; NH3 is converted to ammonium (NH4+) and extruded from the bacterial cell leading to neutralization of the gastric acid. Diagnostic Laboratory Tests A. Specimens Gastric biopsy specimens can be used for histologic examination or minced in saline and used for culture. Blood is collected for determination of serum antibodies. Stool samples may be collected for H. pylori antigen detection. Diagnostic testing methods are summarized in Table 1 B. Smears The diagnosis of gastritis and H. pylori infection can be made histologically; this approach is generally more sensitive than culture. A gastroscopy procedure with biopsy is required. Routine stains (eg, hematoxylin & eosin stain) demonstrate acute/chronic gastritis, and Giemsa or special stains (eg, silver stains or immunohistochemical stains) can show the curved or spiral-shaped organisms. C. Culture Since H. pylori organisms adhere to the gastric mucosa, the bacteria cannot be recovered from stool specimens like other gastrointestinal pathogens. As described above, culture is usually performed when patients are not responding to treatment, and there is a need to perform antimicrobial susceptibility testing. Tissue for culture is obtained by endoscopy and biopsy of the gastric mucosa. 39  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ TABLE 1 Diagnostic Testing Methods for H. pylori D. Antibodies Several assays have been developed to detect serum antibodies specific for H. pylori. While testing for IgG serum antibodies against H. pylori is useful to confirm the exposure to the organism, either for epidemiologic purposes or for the evaluation of a symptomatic patient, the antibody titers do not typically correlate with the severity of the disease. Furthermore, IgM antibodies disappear rapidly during the initial course of an acute infection, and are of little diagnostic value. The relevance of IgA testing remains controversial, and both IgA and IgG serum antibodies persist even if the H. pylori infection is eradicated. The role of antibody testing in differentiating active H. pylori infection from past infection and/or completion of therapy is therefore limited. 40  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 10-Haemophilus General Characteristics The genus Haemophilus contains significant genetic diversity. Members of the genus are small, nonmotile, pleomorphic gram-negative bacilli. The cells are typically coccobacillary or short rods. Species of the genus Haemophilus require protoporphyrin IX (a metabolic intermediate of the hemin biosynthetic pathway), referred to as X factor and V factor, nicotine adenine dinucleotide (NAD), or nicotine adenine dinucleotide phosphate (NADP) for in vitro growth. Haemophilus spp. are facultative anaerobes enhanced in a 5% to 7% CO 2-enriched atmosphere. The morphologic and physiologic features of individual species are presented in the discussion of laboratory diagnosis. Epidemiology As presented in Table 1, except for Haemophilus ducreyi, Haemophilus spp. normally inhabit the upper respiratory tract of humans. Asymptomatic colonization with Haemophilus influenzae type b is rare. Although H. ducreyi is only found in humans, the organism is not part of our normal microbiota, and its presence in clinical specimens indicates infection. TABLES-1 Organism Habitat (Reservoir) Mode of Transmission Normal microbiota: upper Person-toperson: respiratory Haemophilus influenzae respiratory tract dropletsEndogenous strains Not part of normal human Person-toperson: sexual Haemophilus ducreyi microbiota; only found in contact humans during infection Other Haemophilus spp. Normal microbiota: upper Haemophilus parainfluenzae Endogenous strains respiratory tract Haemophilus parahaemolyticus 41  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Pathogenesis and Spectrum of Disease Production of a capsule and factors that mediate bacterial attachment to human epithelial cells are the primary virulence factors associated with Haemophilus spp. In general, infections caused by H. influenzae are often systemic and life-threatening, whereas infections caused by nontypeable (do not have a capsule) strains are usually localized. Most serious infections caused by H. influenzae type b are biotypes I and II. Most H. influenzae infections are now caused by nontypeable strains (NTHi). Transmission is often via respiratory secretions. The organism is able to gain access to sterile sites from colonization in the upper respiratory tract. Clinical infections include otitis media (ear infection), sinusitis, bronchitis, pneumonia, and conjunctivitis. Immunodeficiencies and chronic respiratory problems such as chronic obstructive pulmonary disease may predispose an individual to infection with NTHi. Chancroid is the sexually transmitted disease caused by H. ducreyi. The initial symptom is the development of a painful genital ulcer and inguinal lymphadenopathy. TABLES-2 42  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Laboratory Diagnosis Specimens Specimens consist of expectorated sputum and other types of respiratory specimens, pus, blood, and spinal fluid for smears and cultures depending on the source of the infection. Direct Observation To increase the sensitivity of direct Gram stain examination of body fluid specimens, especially CSF, specimens may be centrifuged (2000 rpm for 10 minutes), and the smear is prepared from the pellet deposited in the bottom of the tube. Gram stains of the smears from clinical specimens must be examined carefully. Haemophilus spp. stain a pale pink and may be difficult to detect in the pink background of proteinaceous material often found in clinical specimens. Antigen Detection H. influenzae type b capsular polysaccharide in clinical specimens, such as CSF and urine, can be detected directly using commercially available particle agglutination assays. Molecular Methods Rapid screening procedures are very useful for patient therapy and evaluating outbreaks and have been developed for detection from CSF, plasma, serum, and whole blood. A polymerase chain reaction (PCR) for H. influenzae capsular types a and f has been developed. Incubation Conditions and Duration Most strains of Haemophilus spp. are able to grow aerobically and anaerobically (facultative anaerobes). Growth is stimulated by 5% to 10% carbon dioxide (CO2). It is recommended that cultures be incubated in a candle jar, CO2 pouch, or CO2 incubator. FIGURE 1. Example of Haemophilus influenzae growing on chocolate (CHOC) agar. Notice the tan mucoid colonies characteristic of encapsulated strains 43  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Cultivation / Media of Choice Haemophilus spp. typically grow on chocolate agar as smooth, flat or convex, buff or slightly yellow colonies. Chocolate agar provides hemin (X factor) and NAD (V factor), necessary for the growth of Haemophilus spp. Most strains will not grow on 5% sheep blood agar, which contains protoporphyrin IX but not NAD. Several bacterial species, including Staphylococcus aureus, produce NAD as a metabolic byproduct. Therefore, tiny colonies of Haemophilus spp. may be seen growing on sheep blood agar very close to colonies of bacteria capable of producing V factor; this is known as the satellite phenomenon. FIGURE: 2 Haemophilus influenzae satellite phenomenon Treatment Invasive H. influenzae infection often requires hospitalization. The current recommended treatment of life-threatening illness caused by H. influenzae is cefotaxime or ceftriaxone. Alternative drugs include trimethoprim-sulfamethoxazole, imipenem, and ciprofloxacin. 44  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 11-Bordetella and Brucella Bordetella Bordetella pertussis is mesophilic coccobacillus causes whooping cough (pertussis). It is obligate pathogens of humans colonizing the ciliary epithelial cells of the respiratory tract. B. pertussis is fastidious, slow-growing organism. The cells of B. pertussis are Gram-negative minute coccobacilli ranging in size between 0.2-0.5 µm × 0.5-2.0 µm. The cells are occasionally filamentous that can elongate several µm in length, usually observed in clinical samples. B. pertussis is non-motile with no flagella. The cells are either encapsulated or surrounded by a sheath of slime. The capsule is usually observed in freshly isolated species whereas the slime formation occurs in vitro in the form of biofilm. Both the capsule and slime sheath are composed of polysaccharides. The surface of the cell consists of fine filamentous appendages. The lipopolysaccharide of B. pertussis is different from that of other Gram-negative bacteria with different phosphate composition than the lipid A in other bacteria. The lipopolysaccharide of B. pertussis acts as endotoxins. The organisms are minute gram-negative coccobacilli resembling H influenzae. With toluidine blue stain, bipolar metachromatic granules can be demonstrated. A capsule is present. Culture Primary isolation of B pertussis requires enriched media. Bordet-Gengou medium (potato-blood-glycerol agar) that contains penicillin G, 0.5 g/mL, can be used; however, a charcoal-containing medium. The plates are incubated at 35–37 °C for 3–7 days in a moist environment (eg, a sealed plastic bag). The small, faintly staining gram-negative rods are identified by immunofluorescence staining. B pertussis is non-motile. The organism is a strict aerobe and forms acid but not gas from glucose and lactose. It does not require X and V factors on subculture. Hemolysis of blood-containing medium is associated with virulent B pertussis. The medium of choice for the selective isolation of B. pertussis is the Bordet-Gengou medium with glycerol. The medium is composed of a potato-extract medium without peptone containing 50% blood. 45  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ The charcoal horse blood agar is a better medium for the selective cultivation of B. pertussis as it has a longer shelf-life and is superior in its ability to support B. pertussis growth. Commercial media for B. pertussis include Stainer-Scholte broth and cyclodextrin solid medium. B. pertussis is an obligate aerobe with the most efficient growth at the temperature range of 30-37°C. The metabolism is mostly based on the oxidation of amino acids as these bacteria do not usually utilize carbohydrates. The growth of B. pertussis on artificial media is difficult due to the susceptibility of the bacteria to various compounds like unsaturated fatty acids, colloidal sulfur, and sulfides. The Virulence factors 1- Adhesins such as filamentous hemagglutinin, fimbriae. 2- Pertussis toxin. 3- Adenylate cyclase, 4- Tracheal cytotoxin. Pathogenesis & Epidemiology Bordetella pertussis, a pathogen only for humans, is transmitted by airborne droplets produced during the severe coughing episodes. The organisms attach to the ciliated epithelium of the upper respiratory tract but do not invade the underlying tissue. Decreased cilia activity and subsequent death of the ciliated epithelial cells are important aspects of pathogenesis. Clinical Findings Whooping cough is an acute trachea-bronchitis that begins with mild upper respiratory tract symptoms followed by a severe paroxysmal cough, which lasts from 1 to 4 weeks. The paroxysmal pattern is characterized by a series of hacking coughs, accompanied by production of copious amounts of mucus, that end with an inspiratory “whoop” as air rushes past the narrowed glottis. Laboratory Diagnosis The organism can be isolated from nasopharyngeal swabs taken during the paroxysmal stage. Bordet-Gengou1 medium used for this purpose contains a high percentage of blood (20%–30%) to inactivate inhibitors in the agar. Treatment Azithromycin is the drug of choice. Prevention There are two types of vaccines: an acellular vaccine containing purified proteins from the organism and a killed vaccine containing inactivated B. pertussis organisms. 46  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Brucella Disease Brucella species cause brucellosis (undulant fever‫الحمى المتموجة‬, Malta Fever‫)حمى مالطا‬. Important Properties Brucellae are small, aerobic, Gram-negative “but often stain irregularly” rods without a capsule, In young culture thaey varies from cocci to rods 1.2µm in length, with short cocco-bacillary forms predominating and they are, non-motile, and non-spore-forming. There are three major human pathogens: - Brucella melitensis (goats and sheep). - Brucella abortus (cattle), - Brucella suis (pigs). Growth Characteristics Fresh specimens from animal or human sources are usually inoculated on trypticase-soy agar or blood culture media. Brucella colonies are small, convex, smooth colonies appear on enriched media in 2–5 days. B. abortus requires 5–10% CO2 for growth, whereas the other three species grow in air. Brucellae utilize carbohydrates but produce neither acid nor gas in amounts sufficient for classification. Catalase and oxidase are produced by the species. Hydrogen sulfide is produced by many strains, and nitrates are reduced to nitrites. Virulence factors lipopolysaccharide (LPS), T4SS secretion system and BvrR/BvrS system, which allow interaction with host cell surface Pathogenesis The common routes of infection in humans are the intestinal tract (ingestion of infected milk), mucous membranes (droplets), and skin (contact with infected tissues of animals). Cheese made from unpasteurized goats' milk is a particularly common vehicle. They localize in the reticuloendothelial system, namely, the lymph nodes, liver, spleen, and bone marrow. Many organisms are killed by macrophages, but some survive within these cells, where they are protected from antibody. The host response is granulomatous, with lymphocytes and epithelioid giant cells, which can progress to form focal abscesses. 47  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Clinical Findings After an incubation period of 1 to 3 weeks, nonspecific symptoms such as fever, chills, fatigue, malaise, anorexia, and weight loss occur. The onset can be acute or gradual. The undulating (rising- and-falling) fever pattern that gives the disease its name occurs in a minority of patients Enlarged lymph nodes, liver, and spleen are frequently found. Pancytopenia occurs. Note:- Brucella melitensis infections tend to be more severe and prolonged, whereas those caused by B. abortus are more self-limited. Treatment The treatment of choice is tetracycline plus rifampin. Prevention Prevention of brucellosis involves pasteurization of milk, immunization of animals, and slaughtering of infected animals. There is no human vaccine. 48  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 12-Chlamydia Order: Chlamydiales Family Chlamydiaceae. They are: obligate intracellular bacteria (like viruses) require biochemical resources of eukaryotic host cells to fuel their metabolism for growth and replication. Chlamydia spp. are similar to Gram-negative bacilli in that they have lipopolysaccharide (LPS) as a component of the cell wall. The chlamydial LPS, however, has little endotoxic activity. They have a major outer membrane protein (MOMP) that is very diverse. Chlamydiae have a unique developmental life cycle, ✓ an intracellular, replicative form, the reticulate body (RB), ✓ an extracellular, metabolically inert, infective form, the elementary body (EB). The EB cannot live long periods of time outside of a host cell. The EB transforms into an RB after infecting a host cell. Within vacuoles, the RB divides via binary fission. The vacuole enlarges and becomes an intracytoplasmic inclusion as the number of RB rises. The RB then transform back into EB, which are then discharged from the host cell 48 to 72 hours after infection. There is evidence that, in addition to the replicative cycle associated with acute chlamydial infections, Chlamydia can persist in vitro in an abnormal form. 49  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Differential Characteristics Among Chlamydiae That Cause Human Disease Chlamydia trachomatis General Characteristics C. trachomatis infects humans almost exclusively and is responsible for various clinical syndromes. Based on major outer membrane protein (MOMP) antigenic differences, C. trachomatis is divided into 18 different serovars that are associated with different primary clinical syndromes. Spectrum of Disease Trachoma is manifested by a chronic inflammation of the conjunctiva and remains a major cause of preventable blindness worldwide. Lymphogranuloma venereum (LGV) is a sexually transmitted disease. Oculo-genital Infections C. trachomatis can cause acute inclusion conjunctivitis in adults and newborns. The organism is acquired when contaminated genital secretions get into the eyes via fingers or during passage of the neonate through the birth canal. Perinatal Infections Approximately one fourth to one half of infants born to females infected with C. trachomatis develop inclusion conjunctivitis. Usually, the incubation period is 5 to 12 days after birth, but it may be as long as 6 weeks 50  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ Laboratory Diagnosis 1. Indirect method: Culture: Several different cell lines have been used to isolate C. trachomatis in cell culture, including McCoy, HeLa, and monkey kidney cells; cycloheximide-treated McCoy cells are commonly used. After shaking the clinical specimens with 5-mm glass beads, centrifugation of the specimen onto the cell monolayer (usually growing on a coverslip in the bottom of a vial, commonly called a “shell vial”) facilitates adherence of elementary bodies. After 48 to 72 hours of incubation, monolayers are stained with a fluorescein labeled monoclonal antibody. 2. Direct Detection Methods Cytologic Examination. Cytologic examination of cell scrapings from the conjunctiva of newborns or persons with ocular trachoma can be used to detect C. trachomatis inclusions, usually after Giemsa staining. Antigen Detection and Nucleic Acid Hybridization. To circumvent the shortcomings of cell culture, antigen detection methods are commercially available. 51  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ 13-Spirochetes The spirochetes are long, slender, helically coiled, spiral, or corkscrew shaped bacilli. T. pallidum has an outer sheath. Inside the sheath is the outer membrane, which contains peptidoglycan and maintains the structural integrity of the organisms. Endoflagella (axial filaments) are the flagella- like organelles in the periplasmic space coated by the outer membrane. The endoflagella begin at each end of the organism and wind around it, extending to and overlapping ‫ يتشابك‬at the midpoint. Inside the endoflagella is the inner membrane (cytoplasmic membrane) that provides osmotic stability and covers the protoplasmic cylinder. A series of cytoplasmic tubules (body fibrils) are inside the cell near the inner membrane. Treponemes reproduce by transverse fission. TREPONEMA PALLIDUM AND SYPHILIS Morphology and Identification A. Typical Organisms: T. pallidum are slender spirals measuring about 0.2 μm in width and 5–15 μm in length. The spiral coils are regularly spaced at a distance of 1 μm from one another. The organisms are actively motile, rotating steadily‫ تدور بثبات‬around their endoflagella even after attaching to cells by their tapered ends‫نهايات مستدقة‬. The long axis of the spiral is ordinarily straight but may sometimes bend ‫ ينحني‬so that the organism forms a complete circle for moments at a time, returning then to its normal straight position. The spirals are so thin that they are not readily seen unless immunofluorescent stain or dark-field illumination is used. They do not stain well with aniline dyes, but they can be seen in tissues when stained by a silver impregnation method. 52  ‫ عصام جمعه ناصر‬.‫ د‬.‫م‬.‫أ‬ B. Culture Pathogenic T. pallidum has never been cultured continuously on artificial media, in fertile eggs, or in tissue culture. In proper suspending fluids and in the presence of reducing substances, T. pallidum may remain motile for 3–6 days at 25°C. In whole blood or plasma stored at 4°C, organisms remain viable for at least 24 hours, which is of potential importance in blood transfusions. Antigenic Structure: The outer membrane The peptidoglycan–cytoplasmic membrane complex. Membrane proteins are present that contain covalently bound lipids at their amino terminals. The lipids appear to anchor the proteins to the cytoplasmic or outer membranes. The endoflagella are in the periplasmic space. T. pallidum h