Lecture 4 Gram (–ve) coccobacilli, pleomorphic, non-motile. Many are animal pathogens. 10 species are human pathogens, most common pathogens are: Haemophilus Haemophilus aegyptius Aggregatobacter aphrophilus- Morphology: parainfluenzae Gram (–ve) coccobacilli, pleomorphic, none motile, may be capsulated(PRP).(PRP= polyribitol ribose phosphate) Habitat: Morphology: Morphology: Morphology: Other species of haemophilus are recently collectively None capsulated strains are present (25% - 75%) in throat at the nasopharynx as normal Gram –ve coccobacilli, Gram –ve coccobacilli, non - Gram –ve coccobacilli, non - motile, grouped under the flora non - motile, may be motile, may be capsulated (same may be capsulated ( the same as H. aggregatobacter The capsulated strains are present (1% - 5) in normal throat at the nasopharynx. capsulated ( the same as H. influenzae). influenzae). Culture: • The organism grows aerobically & anaerobicaly at 37oc for 24 hours and in CO2 as H. influenzae). These mostly colonizing the enriched atmosphere. upper respiratory tract, oral • It needs special factors to grow: X-factor and V-factor Habitat: Habitat: cavity X-factor: It is haemin, it is heat stable . present in: • It is a commensal of • It is a sexually transmitted Habitat: 1. X-disc (commercially available), 2. Blood agar. 3. Chocolate agar the upper respiratory disease(venereal ) •Not present in healthy individuals Causative agents of V-factor: It is Co-enzyme I (NAD) or Co-enzyme II (NADP). present in: tract. and a common cause of genital & 1- V-disc (commercially available). 2- Chocolate agar ulceration. not a normal flora. 3- Produced by Staphylococcus aureus •Pathogenicity: None capsulated, none typable Endocarditis, •Pathogenicity: Culture media: pneumonia, arthritis, ( ) Pathogenicity: - Blood agar: Growth only occurs around a streak of Staph. aureus. (provide the V-factor). Soft chancre.Ulcer to genital H.influenzae will exibit satellite phenomenon. organ, with swelling & tenderness It produces highly infectious ( the growth of haemophilus around the staph.aureus is called satellite phenomenon ) & easily bleeding. conjunctivitis often referred as (pink - Chocolate agar: provides both X & V factors needs factor Enlarged lyphnodes, painful. eye) mostly among children.. If organism is not capsulated: the colonies are 0.5-1 mm v If organism is capsulated: the colonies are 1-3 mm. only Culture: Culture: H.influenzae colonies: are transparent, and they have specific smell (bleach like odour). •Same as H. influenzae, but • The same as H. influenzae, needs X •The capsulated colonies show iridescence: seen as different shades of red, green, blue more fragile, should be cultured and V and orange. They could be only seen when the plate is obliquely examined with 1 within 10 minutes. factors, but slower in growth. illumination from below. • It needs X-factor for its growth It grows at 33o c on: amgrowon but not V-factor. • Chocolate agar: addition of 1% Biochemical reactions: chocolate It grows at 37o c on: isovitalex & Vancomycin. According to 3 biochemical reactions, (Indole, Urease and Ornithine decarboxylase) agar • Chocolate agar: consists of H.Influenzae is divided into 6 biotypes or biovars named from 1 to 6. blood+1% isovitalex + 3 µg/ml Antigenic structure: - Capsular Ag. vancomycin. •Capsulated H.influenzae have 6 different capsular antigens dividing the organism into 6 The colonies are very small, grow serotypes called serovars named from a - f. slowly. g • Serotype b is the most common and the most virulent type. • The capsule of serotype b is made up of Sugar phosphate called PRP(polyribitol ribose Biochemical reactions: phosphate) • Oxidase +ve - SomaticO antigen. • Catalase, urease and indole –ve • Ornithine decarboxylase -ve Pathogenicity: Capsulated strains are pathgenic,transmitted by droplet inhalation . H.influenzae type b, most serious. -Susceptible age group between 3months-5 years. - Adults at risk are those with allergy, smokers, viral infections and immunosupressed patients. Epiglottitis.-fatal Meningitis- fatal Pneumonia sinusitis, especially chronic one Septic arthritis Antibiotic sensitivity: The organism is sensitive to third generation chloramphenicol, and to but some strains are resistant. Vaccine against meningitis: It is a conjugate vaccine, given to infants at 2m, 4m, 6m &booster doses between age 1-1.5years. • PRP - D: Vaccine against H. influenzae and diphtheria. • PRP – OMPC: Vaccine against H. influenzae and . Note: Neonates & infants less than 3 months, have maternal antibodies. Howisitdiagnosed Lecture 5 Brucellae Bordetella Francisella Gram( –ve) cocco-bacilli, non-motile ,smooth colonies are capsuated. They include: Bordetella Species: • Brucella melitensis ….Goats&sheep • ….Cattle • ------huma • -----human • (type A) •Brucella canis ….Dogs •Brucella suis ….swine •Bordetella bronchiseptica------canine • Francisella holarctica (type B) Brucella suis danish Brucella suis America move • Francisella mediasiatica (type C) Growth characteristics: Bordetella pertussis: • Obligate intracellular, • fastedious • Aerobic, grow at 37oc for 24 hours. Morphology: Francisella tularensis • CO2 is essentiel for growth of Brucella abortus only. •Gram –ve coccobacilli, faintly stained, non-motile , capsulated. Morphology: •Catalase & oxidase (+ve). •Reduce nitrate to nitrite. •Bipolar metachromatic granules stained with toluidine blue. (safety pin Gram –ve coccobacilli. • H2S –ve in B.melitensis & B.canis • H2S +ve in B.abortus, B.suis appearance). Habitat: Habitat: • Found in animal reservoirs as; • Present in the respiratory tract of patients & nasopharynx of carriers. cats, dogs, donkeys, camels, Typicase Culture: rabbits, deer soy Bortedella pertussis is aerobic , needs enriched media to grow as: • Bordet-Gengou media: (Potatoe-blood-glycerol agar + penicillinG) Culture: Hes Has • Charcoal cephalexin blood agar(CCBA). • Aerobe , grows at 37o after 2-5 • Charcoal yeast extract agar It grows at 37°C in a moist environment. days on: Colonies appear within 3-7 days as small, white, pearl like or like mercury drops • Chocolate agar + 1% isovitalex Biochemical reactions: (make rich medium): • Oxidase +ve • Catalase +ve • Urease -ve • Nitrate -ve • Citrate -ve The colonies are non-haemolytic. Mode of transmission: Inhalation of droplet nuclei from patients at an early stage of The organism is highly infectious , Antigenic structure: the disease , or from carriers. very dangerous, should be • Polysaccharide surface Ag (O Ag), Two types of O Ag: M & N antigens, present in different - Incubation period: approx. 2 weeks processed in biological safety amounts in all the species. Pathogenicity: B.pertussis causes a disease called . cabinet type III. Pathogenicity: In animals: A respiratory tract infection that occurs in two stages: Biochemical reactions: •bacteremia •abortion. The organism could be discharged and infects the person Catarrhal; early stage, mild symptoms, highly contageous. • Oxidase –ve • Urease –ve labouring the animal. • The organism is excreted in milk of goats, cows, pigs Paroxysmal stage: • Tissue necrosis. • Mucus secreted obstructs the broncules • Catalase +ve leading to atelectasis & diminished oxygenation of the blood. Antibiotic sensitivity: The organism In human: It causes Malta fever, , undulant fever or Mediteranian fever. • associated with respiratory muscle spasms upon coughing.(cause of whoops) is sensitive to tetracycline. Infection occurs by drinking contaminated unpasteurized milk & dairy products. • Severe complications in children as bronchopneumonia, convulsions and brain •Lab acquired by contaminating aerosols. •Skin contact with infected animal tissues. damage, due to the lack of oxygen. • Secondary bacterial infections. Pathogenicity: •Incubation period: 1-4 weeks Laboratory diagnosis: Highly infectious, with infectious • The organism travels via the lymphatic chanels & regional lymphnodes to the blood stream, 1- Specimen : nasopharyngeal secretion. coughed secretions in a plate. dose=50 org. then paranchymal tissues (liver,spleen,kidneis) causing Granulomatous nodules that develop to 2- Films. 3- Culture. 4- Biochemical reactions. Infection is transmitted by: . Antibiotic sensitivity: • Arthropode bite (as ticks, deer • .fever, headache, night sweating, generalized pain, fatigue. • It is sensitive to tetracycline , erythromycin, chloramphenicol. flies) • Untreated cases get chronic symptoms as arthritis, endocarditis (Ag-Ab complex deposition in • Protection against whooping cough is by prophylactic vaccination.(DPTvaccine) • Direct contact with infected joints) , & meningitis. animal tissue Bruun Severity depends on sp., where B. melitensis is most severe & acute. While B. abortus cause Bordetella parapertussis: • Inhalation of aerosols. mildest infection. The same as Bortedella pertussis, but with milder symptoms of the disease. culture media to grow brucella spp Biochemical reactions. It produces which is •Brucella agar • Chocolate agar • Oxidase –ve • Urease +ve • Nitrate –ve • Citrate +ve characterized by severe fever, • Trypticase –soy agar (TSA): Selective media for all Brucella species. Cultural characteristics: enlarged tender lymph nodes, skin • Castaneda bottle. (Diphasic media) Colonial morphology is the same as Bortedelle pertussis, but the organism grows ulcers. Colonies are non - haemolytic, semi- transparent within 48 hours. -Bronchial inflammation & Laboratory diagnosis for brucellosis: Bordetella bronchiseptica: localized pneumonia. • Direct diagnosis: Specimen : blood Exibits the following differences than that of Bortedella pertussis: 1.Blood is cultured on special blood culture media, incubate at 37OC for 8 weeks (not less than •It is motile •Canines as host, occasionally infects humans. 8 weeks to be sure of the results). 2-biochemical reactions: Oxidase +ve Urease +ve Nitrate +ve Citrate +ve -Occuloglandular tularemia (Yellow 2. Film, biochemical reactions, sero-agglutination test using brucella antisera against A and M 3- - Cultural characteristics: granulomatous lesions of the eye antigens. Colonial morphology is the same as Bortedelle pertussis, but the organism grows lids) • Indirect diagnosis: Specimen : serum within 48 hours. The test is serum agglutination test. Detect the presence of IgG agglutinin titer. Antibiotic sensitivity: The organism is sensitive to many antibiotics. • Tetracycline combined with streptomycin is the usual treatment. • Some resistant strains have been reported.

zoonosis Tularemia

Maltafever

Lecture 6 Curved bacilli Divided into three groups: – : Vibrio spp. – Spirillaceae: Campylobacter spp. - Spirochaetaceae: Treponema spp., Leptospira spp. & Borrelia spp. Campylobacter Helicobacter Vibrios Most of the Campylobacter species are non- • 22 species exist, most common are: divided into: A- Non-pathogenic: thousands saprophytes, present in marine water, lakes, ponds & brakish water. pathogenic. The only pathogenic species are: • H. pylori • H. cinaedi • H. fennelliae B- Pathogenic: serogroup O1+O139 Vibrio cholerae serogroup non- O1 ,(2 biotypes 1 & 2 ) Morphology: V. fluvialis V.mimicus V. vulnificus V.alginolyticus Campylobacter coli. Gram-ve curved bacilli (formerly called Campylobacter pylori) Morphology: Morphology: Motile with tuft flagella. Gram –ve curved bacilli (comma-shaped), motile, having polar flagella. The high motility is like falling star or like fish Small Gram –ve bacilli, delicate,curved {single, or Pathogenicity in a stream. double (S-shape), or seagull wing appearance the Infection is widespread & number of cases is increasing. Habitat: – Fresh & brackish water, shellfish & other seafood. – Reservoir: Man…..V.cholerae O1&O139 No most common}, actively motile, darting motility. Transmission is mostly unknown. carriers Habitat: – by contaminated water and food.( Fecal – oral). Cultural characteristics: -Aerobic. - Halo-tolerant.(grow at 6% NaCl). -It needs alkaline medium.(6.8%) Grows Present in animals as; cattle, poultry, house pets – by mother to child. at 37°C on: and pigs.(main host for C.coli ). Cause acute & chronic gastritis which predispose the patient to peptic Alkaline peptone water: Enrichment broth: Vibrios grows rapidly in this broth, within 6 hours. The alkalinity Cultural characteristics: ulcer & gastric cancer. suppress the growth of intestinal commensals. Vibrios grow with thick pellicle layer at the surface of the broth. The organism is: H.pylori invades deep in the gastric mucosa, secretes: - urease TCBS (Thiosulphate - citrate bile salt sucrose agar): Selective and differential medium for the isolation of and – microaerophilic (need traces of O2, 5-10%), enzyme that buffers the mucosa by releasing ammonia. - differentiation between Vibrio species .It has bile salts which inhibits the growth of intestinal organisms other than – thermophilic ( grows at 43 oc) ,can survive Lipopolysaccharides exotoxin(vacA) secretory enzymes as vibrios. It differentiates between species on the bases of sucrose fermentation: refrigeration at 4ºC. mucinase , lipase & protease that damage the gastric mucosa. V.cholrae &V.fluvialis —> ferment sucrose giving yellow colonies – Capnophilic (need CO2) to grow after 5 days on: Symptoms: V.parahemolyticus & V.mimicus —> are sucrose nonfermenters that give green-blue colonies. – Campy agar media: A selective media for the Upper gastrointestinal inflammation with pain , nausea , vomiting & Blood agar: Growth of none hemolytic colonies. isolation of Campylobacter spp. ( Blood agar + fever V.cholerae antibiotics +antifungal drugs) to inhibit fecal Antigenic structure commensal organisms. E -H antigen is not specific. Certain species H antigens overlap Ex V.mimicus &V.cholerae share the same H antigen The colonies are small, mucoid, droplet like,& tend O antigen is specific .. There are 206 different O Ag dividing the organism into A subgroups (serovars). to spread along the streak line. Subgroup O1 &O139 V. cholerae are the causative strain of the classic . -biotype El Tor -biotype Biochemical reactions: classical – Oxidase +ve – Catalase +ve Subgroups O2 - O84 contains the remaining V.cholerae strains referred to as non-O1 V.cholerae, or non agglutinating – Hippurate hydrolysis: vibrios or non-choera vibrios Incubation period: 12hours – 5 days It is (+ve) in Campylobacter jejuni Pathogenicity: • Following the ingestion of food or drinks contaminated with organism;(fecal-oral) It is (–ve) in Campylobacter coli. • Infectious dose: 10^8-10 organisms. • Incubation period: 4-5days • Vibrios bypass the acidity of the stomach, and reaches the intestine. • Pathogenicity: Infection occurs following the • Strongly attaches to the epithelial cells lining the intestine. ingestion of food contaminated with the organism. • Secretes a powerful enterotoxin (cholera toxin) that activates the enzyme adenylate cyclase, increased cAMP, this - This organism produces severe gastroenteritis leads to secretion of large amounts of fluids & electrolytes into the lumen of the intestine , causing profused loss of (abdominal cramps , vomiting and bloody fluids(20l/d) and electrolytes (sodium, potassium bicarbonate, ). The rapid loss of fluid and electrolytes in vomitus diarrhea) as it invades the intestinal mucosa. and stools leads to severe dehydration which if not corrected can be rapidly fatal due to: – These manifestations are self limited, but • : (as 2 / 3 of the plasma volume is lost, thus the blood become concentrated and the heart could not complications if occurred are very dangerous, pump it), also, it is due to loss of potassium. • Renal failure which are ; • Hypovolaemic shock (due to abnormally low volume of circulating fluid (plasma) in the body). bacteremia , meningoencephalitis and • Acidosis due to loss of fluid and electrolytes (pH shifts toward acid side due to loss of bicarbonate buffering endocarditis. capacity). – It mostly infects children less than 2 years old. The patient is manifested by: Laboratory diagnosis: ➢ Abrupt onset of vomiting and life-threatening watery diarrhea (15-20 liters/day). ➢ Abdominal cramps. Specimen: stool ➢ As more fluid is lost, stool changes to rice-water stools: Colorless ,clear fluid, odorless, speckled with mucus, 1. Films: -Culture: (of gastric biopsy) The organism is: – microaerophilic (5-10% O2) epithelial cells & vibrios. El Tor biotype gives milder manifestations than the clasic biotype. a- Direct wet mount: RBCs, WBCs, motile Biochemical reactions: organisms. – capnophilic (need CO2 grow within 5 days on: Campy agar media: grey, translucent colonies. Oxidase +ve Urease +ve Nitrate +ve Indole +ve Sucrose +ve Mannose +ve L-arabinose –ve b- Gram stain film: Gram –ve bacilli. Curved single Immobilization test +ve or Prevention: • Sanitary disposal of excreta • Provide sanitary water supply • Combat of flies • Vaccine: killed O1 -Biochemical reactions: Oxidase +ve, Catalase +ve, Urease +ve S-shaped or seagull wing appearance. strain + exotoxin or killed O1 strain + killed O139 , given in 2 doses 14 days apart. Two weeks before traveling. • 2. Culture: Isolation of the cases -Antibiotic sensitivity: Resistant to many antibiotics, thus its – On B.A. at 37Oc for 24 hours: No growth Treatment: • Fluid & electrolyte replacement. • Antibiotics to kill the organism (Tetracycline or deoxycycline). treatment is difficult, requiring a triple antibiotic therapy, a – On Campy agar media at 42Oc for 5 days + Resistance is common. combination of metronidazole+ amoxicillin or tetracycline + bismuth CO2: Small, mucoid colonies. El-tor biotype of Vibrio cholera Morphology, culture, biochemical reactions, antigen structure: The same as Vibrio subcitrate. 3. Biochemical reactions. cholerae It differs from Vibrio cholerae in: 1. VP test (Voges-Proskauer test): El-tor biotype is +ve and classical biotype is -ve Antibiotic sensitivity: 2. Agglutination of sheep RBCs: El-tor biotype is +ve and classical biotype is –ve – Most Campylobacter strains are sensitive to : ramseswease not 3. Polymyxin B (50IU) sensitivity. EL-tor- resistan Classical – sensitive Erythromycin, vancomycin engine una Non-agglutinable Vibrio Morphology, culture, biochemical reactions: same as Vibrio cholerae It differs from Vibrio – Resistance has been reported to tetracycline and cholerae in: ampicillin. 1. Antigenic structure: It has no subgroup O1or O139, thus it doesn’t agglutinate with O subgroups 1&139 antisera. 2. Pathogenicity: mild diarrhea. : • Morphology: The same as Vibrio cholerae. • Culture: The same as vibrio cholerae, but on TCBC, the colonies are green,ie. no sucrose fermentation. • Biochemical reactions: The same as Vibrio cholerae except; sucrose –ve. • Antigenic structure: not subgroup O1 or O 139 antigen, • Pathogenicity: Gastroenteritis-food poisoning, slight diarrhea and vomiting. • Transmitted by the ingestion of shellfish and seafood Laboratory diagnosis of cholera: Specimen: stool - Direct wet film to detect motile organisms -Immobilization test.(+ve)

Assignment 1

HACEK

The HACEK organisms are a group of fastidious, gram-negative bacteria that are an unusual cause of , which is an inflammation of the heart due to bacterial infection. Or septicemia.

HACEK is an abbreviation of the initials of the genera of this group of bacteria:

H—> A—> (previously ) C—> E —> Eikenella K —>

The HACEK organisms are a normal part of the human microbiota, living in the oral-pharyngeal region. ​ Assignment 2

Immobilization test

It’s a procedure for identifying antibodies to motile microorganisms by ​ ​ ​ ​ measuring the ability of the antibodies to restrict the motility of the ​ microorganisms. Used for Vibrio cholerae diagnosis Along with the hanging drop ​ ​ ​ preparation that will show active motility.

• If +ve —> motility stops. • If -ve —> motile.

Agglutination reaction Endotoxin Exotoxin

Integral part of cell wall Excreted by organisms, living cell

Mostly in gram negative bacteria found in gram positive and gram negative

Lipopolysaccharide Polypeptide

Heat stable Heat labile

Moderately toxic Highly toxic

DIC, fever, shock, death Diarrhea, pneumonia,

What is the HACEK group and write a small note about it? Refers to a group of fastidious gram-negative coccobacillary organisms. HACEK stands for Haemophilus species, Aggregatibacter species, , , and Kingella species. - The HACEK group accounts for approximately 5%-10% of community-acquired native-valve endocarditis cases in patients who do not use intravenous drugs. - All of these organisms are part of the normal oropharyngeal flora, which grow slowly (up to 14 days), prefer a carbon dioxide–enriched atmosphere, and share an enhanced capacity to produce endocardial infections

cardybathomimimis

H A CEK skingena b Eikonella Aggregatobacter I condones

Hemophylis

Bacteriology assignments

Types and Examples of Flagella There are 4 types of flagellar distribution on bacteria

I. Monotrichous - Single polar flagellum - Example: Vibrio cholera II. Amphitrichous - Single flagellum on both sides - Example: Alkaligens faecalis III. Lophotrichous - tufts of flagella at one or both sides - Example: Spirillum IV. Peritrichous → All the members of family , if motile has peritrichous flagella. - Numerous flagella all over the bacterial body - Example: Salmonella Typhi

N.B: I should know their names, their numbers

What are they selective or differential? Why are they selective if they are? What organisms are retrieved from the media? Primary isolation media for fecal material

Selenite broth is used as a selective medium for the isolation of Salmonella species, inhibit growth of fecal coliforms. Selenite Broth is used as a selective enrichment for the cultivation of Salmonella spp. that may be present in small numbers and competing with intestinal flora.

Tetrathionate Broth selective enrichment liquid medium used for the cultivation of Salmonella typhi and Salmonella species from feces, urine, foods and other materials suspected of harboring these organisms.

What is it? Principle of it what are we reaction with what? why are we running this test? For which organism

Widal Test is an agglutination test which detects the presence of serum agglutinins (H and O) in patients serum with typhoid and . Principle Widal test is an agglutination test in which specific antibodies are detected by mixing the patient’s serum with killed bacterial suspension of Salmonella carrying specific O, H, AH and BH antigens and observed for clumping. Uses Use for both Salmonella Typhi and Salmonella Paratyphi Why are we running this test Rapid test for screening typhoid fever in endemic areas, When culture facilities is not available.