Fastidious Gram Negative Coccobacilli There are 3 genera of m.o. that belong to different families, but all share the characteristics of being gram negative fastidious coccobacilli. These m.o. are the followings: 1. Genus: Haemophilus 2. Genus: Bordetella 3. Genus: Brucella
Haemophilus The word Haem means blood, and philus means loving. So, this m.o. Requires blood for its growth. Classification: There are 2 criteria for the classification of these m.o.: 1. Their ability to haemolyse blood. 2. Their requirments for X and/or V factors.
Haemophilus influenzae (Pfeiffer’s bacillus)Morphology: The typical m.o. are short bacilli (coccobacilli), but some times they are long bacilli and even filamentous forms. This characteristic is called pleomorphism. From young cultures (6-8 hr.), the m.o. are coccobacilli and capsulated, while from old cultures (18-24 hr), they are long bacilli with filamentous forms and are non-capsultated.The capsule can be typed by anti-sera in a reaction called “Quellung Reaction” which is capsule swelling test. The capsular antigen can be identified by counter current electrophoresis (CCE) and immunofluorescent (IF) test.
CULTURES:1. Media:The media used for the cultivation of these m.o. have to contain blood from rabbits or horses and not from human or sheep because it contains anti-Haemophilus antibodies. The most important media used are:A. Brain- heart infusion supplemented with blood.B. Chocolate agar.C. Levinthal’s agar which is nutrient agar supplemented with blood.
2. Cultural requirments: H. Influenzae requires: A. Both X and V factors; The X factor is haem or haematin which is a heat stable factor important for the respiratory enzymes of the m.o. including cytochrome oxidase, catalase and peroxidase. The V factor is nicotinamide adenine dinucleotide (NAD) or NADP (P=phosphate). The NAD is coenzyme I and NADP is coenzyme II. These two types of enzymes are heat labile factors and are important for the oxidation-reduction system of the m.o. B. Temperature; these m.o. tolerate temperatures of 25 - 40 C, but the optimum is 37 C. C. CO2; these m.o. Require 10% CO2 for their growth which is provided by candle jar. D. Isovitalex (1%) added to the culture media can enhance the growth of the m.o.
3. Cultural characteristics:On broth medium; the medium does not become turbid (cloudy) when the m.o.is grown in it.On solid media; the colonies are about 0.5 – 1.5 mm in diameter, rounded, convex, and in young cultures they have strong iridescence or some times are described as dew-like colonies, while in old cultures the colonies are rough and not glistening. Also, the growth have a bleach –like or mousy odour. Such odours can be smelled from the mouth of patients when infected with H. influenzae.
4. Isolation of H. influenzae: Bacitracin can be added to the media to make more selective. Satellite phenomenon (Satellitism); around the growth of Staphylococcus aureus (or Strep. pneumoniae or Neisseria gonorrhoeae) the colonies of H. influenzae will be larger. This is because the former growth provides an extra amount of V factor which enhances the size of the colonies of the latter m.o..
Antigenic structure and factors of pathogenicity:1. Capsule: H. influenzae could have a capsule which is of polysaccharides in nature. It is composed of polyribose ribitol phosphate (PRP). According to the serological types of PRP , the m.o. can be typed into 6 serotypes (a – f). The most important and virulent type is H. influenzae type b. Type ( a ) can cause chronic sinusitis, and (e )and (f ) may cause post-operative infections. It has anti-phogocytic function in the absence of anti-capsular antibodies.2. Somatic antigens (P and M); P constitutes much of the bacterial body, while M is a labile surface antigen.3. Endotoxin; its nature and role is unclear.
Pathogenesis of H. Influenzae type b: The m.o. has no exotoxin and the role of somatic antigens is unclear. The non-capsulated m.o. is not pathogenic ( may be part of the normal flora of the mouth and upper respiratory tract). The capsulated H. influenzae type - b is virulent m.o. and can cause : 1. suppurative R.T. Infections as sinusitis, epiglottitis or laryngotracheitis which could be fulminating obstructive and may require prompt tracheostomy. 2. Otitis media; it is a common cause. 3. Meningitis; most common cause in children of age 5 months to 5 years. 4. Secondary invader following influenza which is caused by influenza virus. It can cause bronchitis ,pneumonitis or empyema . 5. Septic arthritis and cellulitis. This infection could start as Upper R.T. Infection and then spread to the middle ear, meninges or joints.
Diagnostic laboratory tests: 1. Specimens: Nasopharyngeal swabs, pus, blood, and spinal fluid. 2. Direct identification; by immunofluorescent (FA), Quellung reaction for capsule swelling test, or commercial kits for identification of H. influenzae antigens in the CSF. 3. Culture (mentioned above).
Immunity: 1. Infants under age 3 months may have antibodies transmitted from the mother. During this time the infection is rare, and then the antibodies are lost. 2.Unimmunized children by the age of 3 - 5 years have anti- PRP antibodies. 3. Immunization of children with H. influenzae type b conjugate vaccine induces anti- PRP antibodies. Resistance to H. influenzae correlates with anti- PRP antibodies, but these are not protective against pneumonia or arthritis.
Treatment: Mortality rate from H. influezae meningitis may reach up to 90%. Many strains of this m.o. are sensitive to ampicillin and 25% of them are beta lactamases producer. Also, most strains are susceptible to chloramphenicol. So, combined use of ampicillin and chloramphenicol in the treatment of meningitis is important to prevent neurological complications and intellectual impairment. Moreover, cefotaxime may also give excellent results . Subdural fluid accumulation after meningitis requires surgical drainage.
Epidemiology, Prevention & Control:1. Haemophilus b conjugate vaccine to children can prevent infections. Also, this vaccine reduces the carrier rates of H. influenzae.2. Prophylactic rifampin to exposed nonimmune children under 4 years of age is recommended.3. Carrier rate of capsulated m.o. is about 2 – 4 %, but for non- capsulated (nontypeable) is 50-80%. The capsulated m.o. is transmitted from person to person by respiratory route.
Genus: Bordetella There are 3 important species of Bordetella: 1. B. pertussis. 2. B. Parapertussis. 3. B. Bronchiseptica. B. pertussis is the causative agent of a highly infectious disease which is called pertussis (whooping cough). B. parapertussis can cause a disease similar to whooping cough, but ofter subclinical of mild. It grows more rapidly and produces larger colonies than B. pertussis. B. bronchisepica is important in canines, produces smaller m.o., and infrequently is responsible for chronuic RTI in humans.
Bordetella pertussis: These m.o. are fastidious gram negative cocco -bacilli (resemble Haemophilus). With toluidine blue stain, bipolar metachromatic granules can be demonstrated. A capsule is present which can be stained by safranin O stain. Culture: Because it is a fastidious m.o., it requires enriched media for its cultivation which are: 1. For primary isolation; an enrichment media is used. This medium is called Bordet-Gengou (potato-blood-glycerol agar). The medium contains 20-30 % sheep blood cells and penicillin -G or methicillin to make more selective.
2. Buffered charcoal yeast extract agar (BCYE); this medium can also be used for the isolation of Legionella pneumophila . 3. Blood Charcoal Agar (BCA) to which an antibiotic may be added as cephalexin to make more selective ( inhibits respiratory flora). 4. Lowe-Regan agar which is similar to BCA, but contains a half strength of charcoal. 5. Charcoal and cephalexin. Cultural requirments: This m.o. Is a strict aerobe, best grow at 35-37 C for 3-7 days, and the media should be kept in moist enviroment ( in sealed plastic bag).
Growth (cultural) characteristics: The colonies appear as rounded, convex, 1-2 mm in diameter, and look like mercury drops or pearl-appearance. The colonies have iridescence and surrounded by narrow zones of haemolysis. Biochemically; the m.o. can ferment glucose with acid, but no gas production. Also, these m.o. do not require neither X nor V factors.
Variations of B. pertussis: This m.o. has 4 phases: Phase - I : when the m.o. is isolated from patients, and cultured on enriched media. The m.o. are capsulated, haemolytic, pertussis toxin-producing , and the colonies are smooth. Phase II & III : Are intermediate phases between I & IV. Phase IV: Opposit to phase I (i.e., all charactrestics are opposit). The shift from phase I to IV is related phenotypic modulation (enviromental).
Antigenic structure and biologically active substances:1. Capsule (K-Ag) which is mainly present in phase I and is a virulent factor.2. Pertussis Toxin which is a major virulent factor and has the following effects:A. It gives a prolonged immunity because it is an immunogenic factor.B. It causes anaphylactic –like reaction due to histamine sensitization.C. It is responsible for the characteristic paroxysmal cough that is followed by deep inspiration .D. It causes hypoglycaemia (enhances insulin secretion).E. It reduces the migratory and phagocytic activity of macrophages and neutrophils.F. It can increase the release of both B and T cells from the bone marrow, spleen and L.N. These cells loose their ability to return back to their lymphoid tissues. However, they remain in the blood causing an absolute lymphocytosis and the W>B>C> count may reach up to 30000 /cmm. Cont/...
3. Filamentous haemagglutinin that mediates adhesion to ciliated epithelial cells. 4. Leucocytes promoting factor that promote lymphocytosis. 5. Lethal toxin that causes local necrosis. 6. Tracheal cytotoxin which affects the activity of epith. and cilia of respiratory tract. 7. Adenylate cyclase complex that can impair phagocytosis.
Pathogenesis and pathology: B. Pertusis can survive only for a short time out side the body. Transmission of the m.o. occurs via the respiratory tract from patients and carriers. There are two stages in the pathogenesis of the m.o.: 1. Colonization Stage: During this stage the m.o. adher to the epithelial cells of the trachea and bronchi , multiply locally and interfer with the ciliary functions. The m.o. do not invade to the blood. 2. Toxaemic Stage: The m.o. start to liberate the toxins and biologically active substances that determine their pathogenicity.
Clinical picture of pertussis: The incubation peroid of pertussis is about 2 weeks.There are two stages in pertussis that are parallel to the two stages of the pathogenicity stages of B. pertussis: 1. Catarrhal stage: This stage coincides with the colonization stage of the causative m.o. This stage takes about 10 days and the patient suffers from flu-like illness with sneezing and mild cough. During this stage the patient is very infectious because spraying huge numbers of m.o. through the respiratory tract.
2. Paroxysmal stage: This stages coincides with toxaemic phase of the pathogenicity of the m.o. During this stage the patients suffer from the characteristic cough (whooping) which may be associated with vomiting and some times aspiration of the vomitus. Also, the patients may have cyanosis and convulsion. Secondary infections may also occur. Obstruction of the bronchioles with mucous blugs may lead to the collapse of lung tissues (ateletasis). Moreover, during this stage the toxic effects of pertussis toxin are manifested as absolute lymphocytosis or hypoglycaemia. Fatal encephalitis may occur in depilitated (weak) children. This disease should be differentiated from other diseases in particular viral or chlamydial infections.
Diagnostic laboratory tests:1. Specimens: A saline nasal wash is the preferred specimen. Nasopharyngeal swabs or cough droplets expelled onto a “ cough plate” held in front of the patient’s mouth during a paroxysm are some times used.2. Direct fluorescent antibody (FA) test to examine nasopharyngeal swab specimens with sensitivity of 50%. Also, it is most useful in identifying B. pertussis after culture on solid media.
3. Cultures: Mentioned previously. The m.o. can be identified by FA or slide agglutination test with specific antisera. 4. Polymerase chain reaction (PCR): It is the most sensitive method for the diagnosis of pertussis (i.e., replaced culture and FA test). 5. Serology: Serological tests are of limited value because specific antibodies ( agglutinating or precipitating) rise does not occur until the third week of illness.
Immunity: Recovery from pertussis or adequate vaccination is followed by immunity. Second infections may occur, but are mild; reinfection occuring years later in adults may be severe. The first defence against infection is the antibody that prevents attachment of B. pertussis to the cilia of the respiratory tract. Stage I m.o. are used to make pertussis vaccine.
Treatment: 1. Antimicrobial drugs: B. pertussis is sensitive to many drugs in vitro. During the catarrhal stage erythromycin promotes the elemination of the m.o. and may have prophylactic value. Treatment during the paroxysmal stage rarely alters the clinical course. 2. Oxygen inhalation and sedation may prevent anoxic damage to the brain.
Prevention: 1. Vaccination (part of DTP): 3 doses in the first year of life, followed by booster of a total of 5 injections. 2. New acellular vaccine of 5 antigens is also available. 3. Prophylactic erythromycin for 5 days is given for unimmunized infants or heavly exposed adults.
Genus: BrucellaThe name “Brucella” was derived from the name of the scientist “Sir David Bruce” who discovered it to be causative agent of undulant fever. This disease is also called “Malta Fever” because it was prevalent in Island of Malta. Undulant fever can be also called “ Brucellosis”.Species of genus Brucella:Brucellae are obligate bacteria of animals and humans and are characteristically located intracellularly.Six species of genus Brucella can infected animals and may be transmitted to man as a zoonotic disease:1. B. abortus : cattle 2. B. melitensis : sheep and goat 3. B. suis : pig 4. B. canis : Dog 5. B. ovis : Sheep 6. B. neotomae : Desert wood rat
Morphology and Identification: Genetically genus Brucella is of a single species which is B. melitensis and all other m.o. are biovars of this species. A. The typical m.o. are predominantly gram negative coccobacilli, and from young cultures they vary from cocci to short bacilli and are capsulted , and stain irregularly. They are aerobic and nonmotile m.o.
B. Culture: Brucellae are adapted to intracellular compartment of cells (fastidious). So, their requirments are complex including a.a., sugars, salts, and vitamines. The best media used for the cultivation of these m.o. are: 1. Trypticase soy agar. 2. Castaneda biphasic medium (broth & agar). This medium is prepared in bottles and has two portions; solid one (bottom), and liquid (top). The m.o. can grow on the surface of the solid portion and in the liquid rendering it turbid. Cont/...
3. Blood agar & blood culture media.4. MacConkey’s agar supplemented with 5% of heated horse or rabbit serum.5. Brucella selective medium (good medium but expensive).6. Thionine tryptose agar.7. Serum dextrose agar.7. Glucose dextrose agar.The growth characteristics:Fresh specimens from animals or human sources are usually inoculated on trypticase-soy agar or blood culture media. B. abortus requires 5-10% CO2 for growth, whereas the other species grow in air. The colonies are small, convex, smooth, translucent, nonhaemolytic, slightly yellowish in young cultures but brownish in old ones, and appear in 2- 5 days (may be delayed up to 3 weeks).
Also, the colonies from young cultures are smooth, mucoid, and the m.o. are capsultaed when stained are vireulent, whereas from old culture the colonies are rough and the m.o. are uncapsulat-ed and are avirulent. In acute brucellosis, the maximum positivity obtained by culture does not exceed 40- 50% in best centres. Biochemically: Brucellae can utilize carbohydrates, but with no acid and gas production (not fermenter), H2S is produced only by B. abortus and B. suis, most species reduce nitrate to nitrite, oxidase positive, and are sensitive to acid (e.g., gastric juice) and heat (e.g., killed by pasteurization). Also, thionine (1/25000) and carbol Fuchsin (1/50000) sensitivity of both B. melitensis and abortus is ( - , + ), while for B. suis is ( + , - ).
Antigenic Structure: 1. Two lipopolysaccharides antigens (A & M) are present in different proportions among the different species. In B. Abortus the A:M ration is 20:1, and in B. Melitensis is 1:20. 2. Superficial L antigen resembles the Vi antigen (virulence) of Salmonella.
Pathogenesis & Pathology: The common routes of infections in humans are: 1. Intestinal tract (ingestion of milk or dairy products in particular white cheese). 2. Mucous membrane (droplets). 3. Skin (contact with infected animals or tissues as meat for workers in slaughter houses, butchers,veterinarians, or house wives). 4. Conjunctiva (rare; airborne). 5. Breast milk (lactating woman). The m.o. progress from the portal of entery, via lymphatic channels and regional L.N., to the thoracic duct and the bloodstream, which distributes them to the parenchymatous organs. Granulomatous nodules that may develop into abscesses form in lymphatic tissue, liver, spleen, bone marrow, and other parts of the RES. In such lesions the m.o. is intracellularly. Osteomyelitis, meningitis,orchitis, arthritis, vertebral collapse or cholecystitis ma occur
Histological reaction in brucellosis consists of proliferation of mononuclear cells, exudate of fibrin, coagulation necrosis, and fibrosis. The granulomas consist of epithelioid and giant cells, with central necrosis and peripheral fibrosis. B. abortus usually causes mild disease without suppurative complications; non-caseating granulomas of the RES are found. B. melitensis infection is more acute, severe and causes granulomatous lesions. These two species are common in Iraq. Placentas and foetal membranes of cattle, sheep and goats contain erythritol (a growth factor for Brucella). The proliferation of m.o. in pregnant animals leads to placentitis and abortion (economic loss). There is no erythritol in human placents, and abortion is NOT part of human brucellosis.
Clinical Finding:The I.P. Of brucellosis is 1- 6 weeks. The onset is insidious, with malaise, fever, weakness, aches, and sweat.The fever usually rises in the afternoon; its fall during the night is accompanied by drenching sweat. There may be GI and nervous symptoms. L.N. enlarge, and the spleen becomes palpable. Hepatitis may be accompanied by jaundice. Deep pain and disturbances of motion, particularly in vertebral bodies suggest osteomyelitis. The manifestations may suside in weeks or months , although localized lesions and symptoms may continue.Following the initial infection, a chronic stage may develop; characterized by weakness, aches and pains, low grade fever, nervousness or depression and other nonspecific manifestations including psychoneurotic symptoms. Brucellae usually can not be isolated from such patients except in only 2% of them. The diagnosis of “chronic brucellosis” is difficult to establish.
Diagnostic Laboratory Tests: A. Specimens: Blood and biopsy material B. Culture: Mentioned above. Subcultures are made from at intervals from blood culture or broths. C. Serology: IgM antibody levels rise during the FIRST week of acute illness, peak at 3 months, and may persist during chronic disease. Even with approperiate treatment, high IgM may persist up to 2 years. IgG antibody levels rise 3 weeks after onset of acute disease, peak at 6- 8 weeks and remain high during chronic disease. IgA levels parallel the IgG levels; could be blocking antibody (causes prozone pheno.).
The serological tests used for diagnosis of brucellosis are: 1. Agglutination test (tube or slide); usig: a. Heat-killed phenolized smooth standerized brucella antigens. b. Rose bengal antigens (stained red). The significant titre = 1/160 or higher. Cross reaction may occur with cholera vaccine, S.typhi /paratyphi , or Yersinia. 2. 2-Mercaptoethanol test; 2-ME can destroy IgM and leaves IgG. This may be used to differentiate between current /recent brucellosis from previous /old brucellosis. Also, is useful in chronic active brucellosis.
3. Blocking antibodies; These are IgA antibodies that interfere with agglutination by IgG & IgM and cause false negative result (prozone phenomena). These antibodies appear during the subacute stage of infection tend to persist for many years, and are detected by Coomb’s antiglobulin method. Also, high dilution agglutination test can overcome the problem of prozone phenomena.4. Skin test (Brucellergen or Brucellin); it is DHS reaction to I.D. Injection of a protein brucella extract. Erythema, oedema, and induration develop witin 24 hours. It is unreliable test & is rarely used.5. ELISA , RIA or Immunofluorescent ; for Ab detection.
Immunity: Resistance to subsequent attack may be produced. Both cellular and humoral immunities are induced. Treatment: Combined anti-microbial drugs are used, taking in consideration that the m.o. is an intracellular one: 1. Ampicillin or tetracycline with Streptomycin give good result. Cotrimoxazole may be added. 2. Refampin (900 mg once daily) with doxycycline (200 mg once daily) gives 97% cure rate. Also, an aminoglycoside drug could be added. Treatment should be continued for at least 6 weeks and for bone or joint infections the treatment continues for at least 8 weeks. For neuro-Brucellosis chloramphenicol or ampicillin could be used with other drugs.
Epidemiology, Prevention & Control: 1. Brucellae are animal pathogens transmitted to humans accidentally. 2. Eradication of brucellosis in animals by immunization (using avirulent live strain 19) of heifers is an important issue. Immunization of humans is experimental, but was attempted in some countries for individuals at high risk of getting brucellosis. 3. Pasteurization of milk and milk products and reduction of occupational hazards are also important steps.