Lecture 11 Microbiology 20-05-2021 PDF

Lecture 11 Microbiology: Some other important bacteria 1  Content Overview: biological characters, infection and immunity, pathogenesis and diagnosis, treatment and prevention. 1. infection caused by Hemophilus influenza,...

Lecture 11 Microbiology: Some other important bacteria 1  Content Overview: biological characters, infection and immunity, pathogenesis and diagnosis, treatment and prevention. 1. infection caused by Hemophilus influenza, 2. Bordetella and Brucella, 3. Bacillus anthracis, Bacillus cereus 4. Legionellae pneumophila 2  Hemophilus influenza Formerly called Pfeiffer's bacillus or Bacillus influenzae Described by Richard Pfeiffer during an infuenza pandemic in 1892 Mistakenly considered to be the cause of influenza (flu) until 1933 when the viral cause of influenza became apparent. Richard Pfeiffer (1858-1945) 3  Hemophilus influenzae (Greek: haema-blood, philos-loving) Smallest bacteria (1.0-1.5µm) Gram-negative rods- cocobacilli Facultative anaerobes Non-motile May or may not have a capsule Cultivation requires media enriched with blood – chocolate agar Growth factors: X factor –hematin i.e. hemin; V factor - nicotine amid (NAD) present in erythrocytes Transmitted via respiratory droplets, or direct contact with contaminated secretions Normal flora of the human respiratory tract and oral cavity 4 Colonies on chocolate agar media  Cultivation Staphylococcus aureus Growth factors: X factor –hematin i.e. hemin; V factor - nicotine amid (NAD) present in erythrocytes Erythrocytes are the only source of hemin, NAD may be provided by other bacteria such as Haemophilus influenze Staphylococcus or yeasts Satellite phenomenon - satellitism 5  H. Infuenzae serotypes Six capsulated serotypes - typing based on capsule polysaccharide a → f Type b capsule comprises polyribose-ribitol phosphate (PRP - capsular polysaccharide) capsule , which is strongly associated with virulence, particularly H. infleunzae type b (Hib) The outer membrane include proteins, lipopolysaccharades (LPS) and lipoolygosaccharades(LOS) Noncapsulated and thus nontypable H. influenzae (NTHi) can be classified by various typing schemes based on outer membrane proteins and other factors. H. infuenzae produces no known exotoxins 95% of invasive disease is caused by type b Nonencapsulated (nontypeable) organisms are part of normal flora of the respiratory tract 6  Clinical importance H. influenzae -type b is an important human pathogen H. ducreyi -sexually transmitted pathogen (chancroid) Other Haemophilus are normal flora - H. parainfluenzae – pneumonia & endocarditis - H. aphrophilus – pneumonia & endocarditis - H. aegyptius – pink eye (purulent conjunctivitis) 7  Public health aspects H. influenzae type b incidence has fallen 99% post-vaccine Pre-immunization – Serotype b was the most common invasive species 8  Epidemiology H. influenzae – strictly human pathogen Found in nasopharyngeal flora of 20-80% of healthy people, depending on age, season and other factors Spread by respiratory droplets Before introduction of effective vaccines 1 out of 200 children developed invasive disease by the age 5 y. Meningitis develops in children under 2 y of age, highest incidence is recorded between 6-18 months. Infants in the age of 0-6 moths are protected by maternal immunity. >90% of these cases are due to serotype Hib Immunization where implemented has dramatically reduced disease Person-to-person spread requires prophylaxis 9 Immunization 10  Invasive Diseases Only capsulated strains are invasive More than 90% of invasive strains are type b Attachment to respiratory cells is mediated by pili and outer membrane proteins (adhesions) Invasion goes between cells Capsule prevents phagocytosis 11  DISEASES: Septic arthritis Osteomyelitis Cellulitis Pericarditis Pneumonia - most frequent is serotype f Otitis media Mixed infection with Streptococcus pneumoniae and then non-typeable H 12  Acute purulent meningitis may follow sinusitis or otitis Symptoms: vague malaise, lethargy, irritabilty and fever Mortality (3-6%) and neurologic sequelae are significant 13 Acute epiglottitis The onset is sudden with fever, hoarseness and muffed cough and rapid progression to severe prostration within 24 h. Cherry-red, swollen epiglottitis (can be visualized in lateral X rays) , and stridor are hallmarks 14 Cellulitis is usually facial 15 Cellulitis 16  Immunization The Hib vaccine is classified as a polysaccharide conjugate vaccine, which is a type of inactivated bacterial vaccine. It is made by joining a piece of the polysaccharide capsule that surrounds the Hib bacterium to a protein carrier. This joining process is called conjugation. Conjugating a protein carrier to a piece of the polysaccharide capsule from a Hib bacterium creates an effective vaccine. 17  Pathogenesis – host factors Hib anticapsular antibody is bacteriocidal and protective Hib protein conjugate vaccine (polyribosylribitol phosphate (PRP) capsule) is the most effective The Hib conjugate vaccine does not protect against nontypeable strains. Persons at risk for invasive H influenzae disease – Asplenia (absence of normal spleen function and is associated with some serious infection risks) – Immunocompromised 18  Diagnosis and treatment Blood culture are useful in systemic infections Bacteriologically, small cocobacilli grown on chocolate agar but not on blood agar strongly suggest H. influenzae Confirmation and specification depends on demonstration of requirements in X (hematin) and V (NAD) factors and biochemical tests Treatment: immunization, rifampin chemiprophylaxis is indicated for non-immunized close contacts (adults and children) 19  Haemophilus ducrey (Chancroid) Haemophilus ducrey is a fastidious, Gram-negative, facultative anaerobic coccobacilli which dies rapidly outside the human body. Haemophilus ducrey causes sores on the genitals and Disease rate is more common in men than in women. It is characterized by painful necrotizing genital ulcers that may be accompanied by inguinal lymphadenopathy. It is a highly contagious but curable disease. 20 Transmission, diagnose and treatment H. ducreyi is transmitted sexually by direct contact with purulent lesions and by autoinoculation to nonsexual sites, such as the eye and skin. The organism has an incubation period of 1 day to 2 weeks, with a median time of 5-7 days. There is no blood test for chancroid. Chancroid symptoms can be confused with other infections, such as herpes and syphilis. Definitive diagnosis of chancroid requires the identification of H. ducreyi on special culture media that is not widely available from commercial sources. Chancroid is treated with antibiotics including ceftriaxone, and azithromycin. Large lymph node swellings need to be drained, either with a needle or local surgery. Both partners should be treated at the same time. 21  Bordetella Strict aerobe Gram negative Small coccobacillus (0.5-1.0 μm) -singly or in pairs Transmission by aerosolized droplets Non-invasive Strictly human pathogen Jules Bordet and Octave Gengou contributed for 22 discovery of B.pertussis1900  B. pertussis Small, transparent hemolytic colonies on Bordet-Gengou medium Bordet-Gengou Media includes: nicotinamide charcoal antibiotic (to inhibit the growth of other respiratory flora) B. pertusis is a slow growing bacteria, requires 3-7 days in the best conditions 23 Bordetella pertusis The cell wall of B. pertusis has typical structure of Gram – bacteria Outer membrane contains different type of lipopolysaccharaide The surface exhibits a rod-like protein – filamentous hemagglutinin (FHA), because of its ability to bind to and agglutinate erythrocytes FHA binds amino-acid sequences found in host cells Pili and prertactin (membrane protein) are adhesins 24 25 26  Pathogenesis B. pertussis has a remarkable tropism to cilliated broncheal epithelium attaching to the cells themselves. The adherence is mediated by FHA, pili, pertactin, and the binding subunits of PT Cilliates cells are progressively destroyed Mucous becomes devoid of cilliary blanket 27  Virulence factors Primarily a toxin-mediated disease Pertussis toxin (PT), and adenylate cyclase toxin (AC) attack immune cells, paralyze them and even kill them Absorbed PT acts on multiple cell types: neutrophils, lymphocytes, macrophages. 28  Toxins Pertussis toxin (PT) is a major virulence factor for B. pertusis It is an A-B toxin produced from a single operon as an enzymatic subunit and five binding subunits hat are assembled in the complete toxin on the bacterial surface The enzymatic subunit is then internalized and ADP-ribosylates - a G protein that affects adenylate cyclase – is activated, which disrupts cell regulation 29  Other toxins Pore forming adenylate cyclase (AC) enters host cell, catalyses the conversion of ATP to cyclic AMP The activity interferes with cellular signaling, chemotaxis, superoxidate generation and function of immune effectors cells, including PMNs, lymphocytes, macrophages and dendritic cells. AC also induces programmed cell death (apopthosis) Tracheal cytotoxin (TCT) – a monomer of B.pertusis peptidoglican generated during cell wall synthesis. TCT is directly toxic to ciliates tracheal epithelial cells causing their extraction from the mucose and eventual death 30 31  Epidemiology - Whooping cough Predominately a pediatric disease Highest in the 1st year of life Highly contagious Spread by airborne droplets Maternal antibodies are not protective Females suffers more than males Worldwide distribution Epidemics occurs periodically Immunization reduces disease, but outbreaks continue Atypical adult disease facilitate spread Infants have high mortality Non immune rarely escape infection Waning immunity needs boosting 32 Epidemiology of whooping cough 33  Pertussis Among Adolescents and Adults Disease often milder than in infants and children Infection may be asymptomatic, or may present as classic pertussis Persons with mild disease may transmit the infection Older persons often are source of infection for children 34  Pertussis Pathogenesis Two-stage process of disease – Respiratory colonization 7-10 days NO symptoms Positive cultures toward the end of this stage – Toxin-mediated disease 35 Clinical progression of pertussis 36 Manifestation: Persistent cough Lymphocytosis Histamine sentization Insulin secretion Complications: Atelectasis (collapse or closure of a lung resulting in reduced or absent gas exchange) Superinfection with Streptococcus penumoniae Convulsions Subconjuctival or cerebral bleeding are related to venous pressure effects of paroxymal coughing 37  Diagnosis Based on symptoms Culture of respiratory secretions on Bordet-Gengou medium Direct fluorescent antibody (DFA) testing – rapid diagnostic PCR Slide agglutination 38  Treatment Erythromycin - effective at catarrhal stage Vaccine Killed whole bacterial cell suspension – given with DTP (combined with diphtheriae and tetanus) vaccine The vaccine had side effects – local inflamation, fever and fibrille seizure Vaccine- induced immunity wanes after five to ten years Today acellular vaccines (DTaP) are developed containing PT, FHA (some pertactin or pili) Multicomponent acellular vaccines Not long lasting effect Full primary vaccination at 2,4,6 months, boosters at 15-18 months and between 4-6 years 39  Brucella Brucellosis is one of the most widespread zoonotic disease's globally. The disease is caused by gram-negative bacteria, which are interacelluar coccobacilli. Brucella spp. are rod-shaped, aerobic, non-motile, urease +, that lack a diffuse sugary capsule around their membrane Brucella spp. are capable of causing disease in a variety of animal species, including humans 40  Medically important species There are currently 8 recognized species of Brucella, of which six are capable of infecting humans. Brucella melitensis – high pathogenicity for humans B. abortus – moderate pathogenicity for humans B.suis - high pathogenicity for humans B.canis – moderate pathogenicity for humans B. ceti B. pinnipedialis 41  Reservoirs, epidemiology Organism Reservoir B. abortus Cattle, buffalo, camels B.melitensis Sheep, goats, camels B. suis Swine B. canis Dogs Causes abortions in cattle, goats and pigs In most cases, human infections occur through ingestion of contaminated milk, and unpasteurized dairy products, breathing the organism, or having the bacteria enter the body through skin wounds Occupational disease for veterinarians 42 Human brucellosis and associated species 43  Lab infection and bioterrorism The efficient transmission of Brucella via inhalation of contaminated dust or aerosols makes brucellosis one of the most common laboratory-acquired infections. The original weaponization, by the US and the former Soviet Union in 1950. 44 Culture Grows slowly (2-3 days) Aerobic conditions Enriched blood agar Produces catalase, oxidase, urease, but do not ferment carbohydrates The lipid composition of the Brucella envelope is unusual – dominant phospoholipid component (phosphatidylchlorine) is most typical for eukaryotic cells than to prokaryotes 45  Pathogenesis-Virulence Factors Pathogenic potential is dependent on two major virulence mechanisms: Host cell invasion Intracellular survival and replication 46  Pathogenesis Granuloma developed by B. abortus After penetrate the skin or mucous membranes, B. abortus is able to evade toll-like receptors and multiply in macrophages in the liver sinusoids, spleen, bone marrow and other components of reticuloendothelial system and form granulomas. Inhibits apoptosis thus prolonging the life Intracellular stage of B. abortus of the host cell where it is replicating. 47  Clinical Diagnosis Symptoms and signs of acute brucellosis: Starts 7- 21 days after infection Fever 39- 40 C Night sweats Chills Myalgia (muscles) Arthralgia (joints) Headache Weight loss (up to 20 kg during prolonged illness) Malaise Weakness 48  Clinical Diagnosis Chronic manifestations of brucellosis: Musculoskeletal system Gastrointestinal system Genitourinary system Hepatobiliary system Cardiovascular system Central and peripheral nervous system Because of mimicking nature of brucellosis, treatment can be difficult depending on the stage of the disease and the organs involved. Fatigue 49  Diagnosis and treatment of human brucellosis Plate agglutination test (a.k.a., Brucella ring test) diagnosing Brucella Drop of serum mixed with drop of Brucella antigen Clumping indicates infection If the mixture remains clear, the result is negative. Treated with combination of tetracycline or rifampin and doxycycline for 6 weeks period Up to 10% of patients have relapses in the first 3 months after therapy For infants, tetracycline is toxic, so children are treated with trimethoprim-sulfamethoxazole. 50 Control and prevention of brucellosis In 1934, the U.S. Department of Agriculture (USDA) established the National Brucellosis Eradication Effort which is managed by Animal, Plant, and Health Inspection Service (APHIS) APHIS certifies states as brucellosis-free, classes A, B, or C of which all states are currently classified A Serology & confirmatory bacterial culture to identify infected animals Positive animals/herds are destroyed Vaccination is available but is not a 100% effective and is costly to cattle ranchers 51  Bacillus anthracis Bacillus ~ 60 species; Gram-positive or Gram-variable bacilli Large (0.5 x 1.2 to 2.5 x 10 um) Nonhemolytic Most are saprophytic contaminants or normal flora Bacillus anthracis is most important member Produce endospores Aerobic or facultatively anaerobic Catalase positive (most) Rapidly differentiates from Clostridium Bacillus spp. are ubiquitous Soil, water, and airborne dust Thermophilic (< 75°C) and psychrophilic (>5-8°C) Can flourish at extremes of acidity & alkalinity (pH 2 to 10) Meduse heads 52  Bacteriology Endospore survives in nature Polypeptide capsule is antiphagocytic Exotoxin complex has multiple components and actions Exotoxin complex: Edema factor Lethal factor Receptor binding protein – protective antigen 53 Diseases associated with Bacillus 54  Epidemiology of B.anthracis Rare in the US (1974-1990, 17 cases reported by CDC) Enzootic in certain foreign countries (e.g., Turkey, Iran, Pakistan,and Sudan) Anthrax spores infectious for decades Biologic warfare experiments (annual tests for 20 years) ✔ Gruinard, off western coast of Scotland ✔ 4 x 10e14 fully virulent spores exploded ✔ Eliminated in 1987 (formaldehyde & seawater) Three well-defined cycles Survival of spores in the soil Animal infection Infection in humans 55 Epidemiology of B. anthracis (cont’s) Primarily a disease of herbivorous animals Most commonly transmitted to humans by direct contact with animal products (e.g., wool and hair) Also acquired via inhalation & ingestion Increased mortality with these portals of entry Still poses a threat Importing materials contaminated with spores from these countries (e.g., bones, hides, and other materials) Usually encountered as an occupational disease Veterinarians, agricultural workers 56 Epidemiology of anthrax in animals and human hosts 57  Laboratory diagnostic On blood agar Large, spreading, gray-white colonies, with irregular margins Many are beta-hemolytic (helpful in differentiating various Bacillus species from B. anthracis) Smears with large Gram-positive rods are suggestive Hemolysis and motility extracts anthrax Spores seen after several days of incubation, but not typically in fresh clinical specimens 58 Clinical manifestation of B. anthracis Cuteneous anthrax 95% human cases are cutaneous infections Begins 1 to 5 days after contact Small, pruritic, non-painful papule at inoculation site Papule develops into hemorrhagic vesicle & ruptures Slow-healing painless ulcer covered with black eschar surrounded by edema Infection may spread to lymphatics w/ local adenopathy Septicemia may develop 20% mortality in untreated cutaneous anthrax 59 Clinical manifestation of B. anthracis Inhalation anthrax In pulmonary anthrax the inhaled spores are taken up by alveolar macrophages but apparently do not germinate inside them at least until they drain to the mediastinum via lymphatics The most lethal of anthrax from is manifested in the lung as a mediastinal process and systematically via virulent bacteriemia Virtually 100% fatal (pneumonic) Meningitis may complicate cutaneous and inhalation forms of disease Pharyngeal anthrax Fever Pharyngitis Neck swelling 60 Clinical manifestation of B. anthracis Gastrointestinal (Ingestion) Anthrax Virtually 100% fatal Abdominal pain Hemorrhagic ascites Paracentesis fluid may reveal gram-positive rods 61 Treatment and prophylaxis Treatment Penicillin is drug of choice Erythromycin, chloramphenicol acceptable alternatives Doxycycline now commonly recognized as prophylactic Vaccine (controversial) Laboratory workers Employees of mills handling goat hair Active duty military members Potentially entire populace of U.S. for herd immunity 62  Key Characteristics to Distinguish between B. anthracis & Other Species of Bacillus Characteristic Bacillus anthracis Other Bacillus spp. Hemolysis Neg Pos Motility Neg Pos (usually) Gelatin hydrolysis Neg Pos Salicin fermentation Neg Pos Growth on PEA blood agar Neg Pos 63 Gram-Variable Stain of B. cereus with Endospores 64 Foodborne Diseases of B. cereus Intoxication Foodborne infection 65  Legionellae pneumophila Legionella Gram stain Legionella on Buffered Charocal yeast Agar (BCYE) Agar Plate 66  LEGIONELLA The bacterium got its name after a 1976 outbreak, when many people who went to a Philadelphia convention of the American Legion suffered from this disease, a type of pneumonia (lung infection). Although this type of bacterium was around before 1976, more illness from Legionnaires' disease is being detected now 67  Disease Patterns Each year, between 8,000 and 18,000 people are hospitalized with Legionnaires' disease in the U.S. However, many infections are not diagnosed or reported, so this number may be higher. More illness is usually found in the summer and early fall, but it can happen any time of year. 68  Legionella pneumophila Legionella pneumophila is a thin, aerobic, pleomorphic, non-spore forming, non capsulated, Gram-negative bacterium, motile with polar, subpolar and lateral flagella staining with silver impregnation methods Legionella pneumophila has 16 serogroups L. pneumophila (Philadelphia strain, serogroup 1) is the primary human pathogenic bacterium in this group and is the causative agent of legionellosis or Legionnaires' disease 69  Culture Growth on Buffered charcoal yeast extract agar (BCYA) with L cysteine and antibiotics Slightly acidic conditions ≈pH 6,9 Slow growing for 2-5 days 70  Legionnaires’ Disease Legionnaires’ Disease is a pneumonia like illness caused by the Legionella bacteria and can be fatal. The infection is caused by breathing in small droplets of water contaminated by the bacteria. The disease cannot be passed from one person to another. 71  Epidemiology Legionella bacteria (Legionella pneumophila) are found in the natural environment and may contaminate and grow in water systems, including domestic hot and cold water systems. They survive low temperatures and thrive at temperatures between 20 – 45°C if the conditions are right. They are killed by high temperatures at 60°C or above. Amoebas in fresh water habitat act as reservoir Disease rate among exposed is low Human infection is typically by inhalation of aerosols provided by cooling towers and air conditioners Shower heads Outcome of infection depends on size of infective dose 72 73  Higher risk properties are those which have hot and cold water storage tanks especially where there are deposits that can support bacterial growth providing a source of nutrients for the organism e.g. rust, sludge, scale, organic matter. 74  Risk groups The risk increases with age (> 50) but some people are at higher risk including: smokers and heavy drinkers people suffering from chronic respiratory or kidney disease diabetes, lung and heart disease anyone with an impaired immune system like cancer or kidney failure People who take drugs that suppress (weaken) the immune system (like after a transplant operation or chemotherapy) 75  Pathogenesis Strong tropism for the lung L. pneumophila invades and replicates in alveolar macrophages. Lysosomal fusion is blocked that would otherwise degrade the bacteria Host endoplasmic reticulum (ER) is incorporated into the legionella-containing vacuole (LCV). In this protected compartment, the bacteria multiply. This process is similar to those that occur in environmental protozoa. 76  Manifestation Severe toxic pneumonia begins with myalgia and headache, followed with rapidly rising fever and dry cough Chills, pleuritic chest pain, vomiting, diarrhea, confusion Liver function test often indicate some hepatic disfunction Mortality is high in immunocompromised patients up to 50% 77  Pontiac fever Pontiac fever is a non- pneumonic form of L. pneumophila infection Symptoms are flu-like, including fever, tiredness, myalgia, headache, sore throat, nausea, and cough may or may not be present. Pontiac fever is self limited and requires no hospitalization or antibiotic therapies. There are no reported deaths associated with Pontiac fever. 78  Diagnostic Specimens: Sputum, Bronchial aspirate, Lung biopsy Culture Florescent methods Serology ELISA Urine Antigen Test 79  Treatment Respiratory disease - azythromycin or fluoroquinolones and the newer macrolides are used to treat L. pneumophila pneumonia. Treatment typically lasts 7-10 days or in the case of immunosuppressed patients, 21 days. Pontiac fever usually does not require antimicrobial therapy. 80  Prevention Preventing Legionella aerosols is a primary goal Heat, hyperchlorination and metal ions may be needed in institutions 81 Reading: Chapters 18, 22 82 83