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STAPHYLOCOCCI Staphylococcus aureus Clinical importance: Upper respiratory tract infections, gastrointestinal tract infections, genital tract infections, impetigo, cellulitis, bacteremia, osteomyelitis, toxic shock syndrome, wounds, burns, food poisoning, endocarditis. Specimens: Sputum, urine, nose, ear, eye, bedsores, vaginal swabs, blood cultures, body fluids, CSF, stool, food. Microscopic examination: Gram positive cocci in clusters. Culture: Facultative anaerobic. 370C (Body temp.) optimal temperature. Blood agar. Nutrient agar. Mannitol agar. Macconkey agar without crystal violet. Tpey agar. Coagulation test: positive. DNAse: positive . Catalase test: positive. Novobiocin: sensitive. Haemolysis: haemolytic. Susceptability testing: Penicillin. Erythromycin. Cloxicillin. Bactrim. Augmentin. Clindamycin. Flucidin. Amikacin. Vancomycin.
Staphylococcus epidermidis Clinical aspects: Urinary tract infections (hospital acquired), osteomyelitis (post operative), endocarditis (prosthetic valve), indwelling devices (catheters, prosthetic joints), bacteremia (neonates), endophthalmitis. Specimens: Urines, blood cultures, CSF, body fluids. Microscopic examination: Gram positive cocci in clusters. Culture: Facultative anaerobic. 370C (body temp.) optimum temperature. Blood agar. Nutrient agar. Mannitol salt agar. MacConkey agar without crystal violet. Coagulase: negative. DNAse: negative. Catalase: positive. Novobiocin: sensitive. Haemolysis: non-haemolytic. Susceptibility testing: see notes for Staph. aureus. Staphylococcus saprofiticus Clinical aspects: UTI in young females. Specimens: Urine. Microscopic examination: Gram positive cocci in clusters. Culture: Facultative anaerobic. 370C optimum temperature. Blood agar. Nutrient agar. MacConkey agar without crystal violet. Cled agar. Coagulase: negative. Dnase: negative. Catalase: positive. Novobiocin: resistant. Haemolysis: non-haemolytic. Susceptibility testing: See notes for Staph. aureus.
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Lancefield group A, B and D. Viridans spp. Pneumococci.
Streps can be differentiated by: beta-haemolysis and alpha-haemolysis. Beta-haemolytic: These strains produce a soluble haemolysin. Causes a clear zone of haemolysis around colonies on blood agar. (100 %) Alpha-haemolytic: Some species do not produce a soluble haemolysin. Causes partial haemolysis around the colonies creating a green color on blood agar. Non-haemolytic: Non-haemolytic strains do not produce soluble haemolysin and will not have a zone of haemolysis around the colonies. Streps microscopically: Gram positive cocci in chains. Streptococcus pyogenes (Group A) (β−hemolytic) Clinical aspects: Sore throat, scarlet fever, cellulitis, impetigo, vaginitis, otitis media, arthritis, wound infections; indirectly glomerulo-nephritis and rheumatic fever. Specimens: Throat swabs, wound swabs, vaginal swabs, blood cultures, sputum, urine. Culture: Blood agar with bacitracin disk: sensitive to bacitracin. Bile-aesculin agar: no growth. 6.5% NaCl: negative. Catalase test: negative. Susceptibility testing: Penicillin. Erythromycin. Streptococcus agalactiae (Group B) (β−hemolytic) Clinical aspects: Neonatal meningitis, neonatal septicaemia, endocarditis, cellulitis, arthritis, genetial tract, bovine mastitis. Specimens: Urine, blood culture, sputum, vaginal swabs, wound swabs. Culture: Blood agar with bacitracin disc – not sensitive. Bile aesculin agar: growth but no hydrolysis of aesculin. 6.5 % NaCl: negative. Catalase test: negative. Camp test: positive.
Susceptibility testing: Penicillin. Erythromycin. Streptococcus (Enterococcus) faecalis (Group D) (variable hemolysis) Clinical aspects: UTI, subacute bacterial endocarditis, ear infections, biliary tract infections, supperative abdominal lesions. Most enterococcal infections by E.faecalis. E.faecium resposible for most of the rest – around 5 – 10 %. Specimens: Urine, wound swabs, ear swabs, blood cultures. Microscopic examination: Gram positive cocci ovoid pairs or short chains, Culture: Blood agar with bacitracin – resistant. Bile aesculin agar: hydrolysis (black). 6.5 % NaCl: positive (yellow). Catalase: negative. Enterococcus agar: small pink colonies. Susceptibility testing: Penicillin 8. Vancomycin. Nitrofurantion (urine). High level of antibiotic resistance – plasmid mediated; can be transferred to other strains. Strepex kit test can be used for grouping of streps. (enzyme extraction.) Serology: ASOT (Antistreptolysin O titre.) NB: http://en.wikipedia.org/wiki/Enterococcus Streptococcus viridans (α -strep) Species: S. mutans, S. mitis, S. milleri, S. sanguis, S. bovis I, S. bovis II. Clinical aspects: Dental sepsis, dental caries, subacute bacterial endocarditis. Specimens: Throat swabs, sputum, blood culture. Microscopic examination: Gram positive cocci in chains or ovoid. Culture: Facultative anaerobic. 37oC (Body temperature) optimum temperature. Blood agar with penicillin, bacitracin.
Bile aesculin agar: different for specific species. 6.5 % NaCl: different for specific species. Biochemical reactions: different for specific species. Susceptibility testing: Penicillin. Erythromycin. Streptococcus pneumoniae (Pneumococcus) (α−strep) Clinical aspects: Lobar pneumonia, acute and chronic bronchitis, bronchopneumonia, otitis media, sinusitis, meningitis, peritonitis, arthritis, conjunctivitis. Specimens: Sputum, pus swabs, blood cultures, csf. Microscopic examination: Gram positive lanceolated diplococcus. Culture: Facultative anaerobic. 370C (body temperature) optimum. 5 – 10 % CO2. Blood agar with optochin: sensitive to optochin, draughtman colonies with coloration. Mueller Hinton agar with oxicillin disc. Bile solubility: bile soluble. Antigenic characteristics: Serotyping can be done, or a capsule swelling test Quelling reaction. Susceptibility testing: Penicillin. Erythromycin. Tetracyclines. Ceftriaxome. Chloramphenicol. Strep Q and A. NEISSERIA Neisseria gonorrhoea (Gonococci) Clinical aspects: Venereal disease, transmitted by direct, close sexual contact, acute gonorrhoea – males: urethra, and cervix uteri, blockage and scarring of fallopian tubes (salpingitis), anorectal and oropharyngeal – homosexuals, conjunctivitis – newborn infants, arthritis, meningitis. Specimens: Urethral swabs, eye swabs, blood culture, csf, sputum, urine, joint fluid, nasopharyngeal swabs.
Specimen transport: Stuart's transport media; survive 6 – 12 hours. Microscopic examination: Gram negative diplococci. Located intracellularly in polymorphonuclear leucocytes. Culture: Blood agar CO2. Chocolate agar CO2. Thayer Martin agar CO2. New York City agar CO2. 350 - 370 C (+/_ body temperature), 5 – 10 % CO2. Oxidase test: positive. Beta-lactamase test: positive or negative. Cystine-triptic digest agar (CTA sugars) – control, glucose, maltose, lactose, sucrose; will only take glucose. Susceptibility testing: Penicillin. Ciprofloxacin. Ceftriaxone. Tetracycline. Ofloxacin. Neisseria meningitidis (Meningococcus) Clinical aspects: Acute purulent meningitis, septicaemia with petechial rash. Specimens: Csf, blood culture, skin lesions, nasopharyngial swabs. Microscopic examination: Gram negative diplococci. Intra-cellular or extra-cellular. Culture: Blood agar CO2. Chocolate agar. Thayer Martin agar CO2. New York City agar CO2. 35 – 370C, 5 – 10 % CO2. Oxidase test: positive. Beta lactamase test: positive or negative. CTA sugars: glucose and maltose positive. Susceptibility testing: Penicillin. Ceftriaxone. Chloramphenicol. Neisseria lactamica Clinical aspects:
Primary habitat the nasopharynx of children (3m – 6 years), can cause meningitis, bacteremia. Rarely from genital tract. Specimens: Csf, blood cultures, vaginal swabs. Microscopic examination: Gram negative cocci. Culture: Same media as for N. meningitidis. Will grow on nutrient agar at 22oC (Room temperature). Glucose: positive, maltose: positive, lactose: positive. ONPG: positive. Serological cross agglutination between N. meningitidis and N. lactamica.
Moraxella catarrhalis Gram negative, oxidase positive diplococcus. Clinical aspects: Saprophytic organism of the upper respiratory tract, meningitis, bacteremia in immunosuppressed patients. Specimens: Sputum, throat swabs, body fluids, tracheal aspirates. Culture: Grow at RT (22oC). Ordinary media without serum. Oxidase test: positive. CTA sugars: no fermentative properties. Susceptibility testing: Cotrimoxazole. Co-amoxyclav. Amoxicillin. Ciprofloxacin. Ceftriaxone. Tetracycline.
CORYNEBACTERIUM Corynebacterium diphtheriae Clinical aspects: Diphtheria – a human pathogen, produces potent exotoxin with an affinity for heart muscle and peripheral nerves.
Inflammatory lesion with membranous exudate affecting the mucosa of the upper respiratory tract. Wound infections, skin infections. Specimens: Throat, nose and wound swabs. Microscopic examination: Pleomorphic gram positive rods, chinese lettering (coryneforms). Special stains to demonstrate the volutin granules – Neisser and Alberts stain. (Remember: Cocci are generally gram positive, except Neisseria and Moraxella. Bacilli are generally gram negative (GNB), except Corynebacteria, Listeria, Clostridia (anaerobes), acid fast and some others eg. Bacillus spp..) Culture: Facultative anaerobic. Optimum temperature 370C (Body temp). Grows best on medium containing blood or serum, eg. Loeffler serum, Tellurite agar (Hoyles medium), Chocolate agar. CTA sugars: ferment glucose and maltose. Nitrate: positive. Urease: positive. Catalase: positive. (NB) Elek plate: agar gel precipititation test for toxigenicity of C. diphtheriae. Susceptibility testing: Penicillin. Erythromycin. Do these sensitivities on Muller Hinton agar. Corynebacterium ulcerans Clinical aspects: Animal parasite, cause mastitis in cattle, spread to man by raw milk consumption, ulcerative exudative throat infections, cardiac and neurological complications. Microscopic examination: Gram positive pleomorphic bacilli. Culture: Blood agar – small zone of haemolysis. Loeffler serum. CTA sugars: ferments glucose and maltose. Nitrate: negative. Urease: positive. Catalase: positive. Susceptibility testing: Penicillin. Erythromycin.
Lysteria monocytogenes Clinical aspects: Meningo-encephalitis in people, listeriosis, food outbreaks eg. chicken. Pregnancy infections eg. septic abortion, neonatal sepsis (granulomatosis infantiseptica), cerebritis (brain abscesses, tumor, stroke), sepsis. Specimens: CSF, blood culture, liver tissue, faeces, food, exudates. Microscopic examination: Gram positive bacilli straight or slightly curved. Gram negative in older cultures. Non-spore forming. Culture: Aerobic and facultative anaerobic. 35 – 37 0C (body temperature) optimum temperature, Blood agar (sheep blood): small translucent with a narrow zone of complete haemolysis. Ordinary media, but better on media containing serum blood or liver extract. Catalase: positive. Motility at 250C (RT) – tumbling or head-over-heals. Esculin: hydrolysis. CTA sugars: ferment glucose, maltose and salicin promptly. Camp test: positive but a rectangular rather than an arrow head shaped zone of haemolysis. Susceptibility testing: Penicillin. Ampicillin.
MYCOBACTERIUM Mycobacterium tuberculosis Clinical aspects: Cause tuberculosis, chronic granulomatous lesions. Specimens: Sputum, laryngeal swabs, gastric lavage, bronchoscopic specimens, urine, pleural and peritoneal fluids, csf, pus, tissue. Microscopic examination: Gram positive bacilli, but because of the waxy material in the cell wall M. tuberculosis requires a mordant dye in the presence of heat; Ziehl-Neelsen stain, or use a fluorocrome stain – Auramine stain. TB is acid and alcohol fast, bacilli stain red against a blue background. We record our results according to the IUAT (International Units Against TB): Negative No acid and alcohol fast bacilli. Scanty positive 1 – 10 bacilli per smear. 1+ positive 10 – 100 bacilli per smear. 2+ positive 1 – 10 bacilli per field.
more than 10 bacilli per field.
Culture: Strict aerobe. Optimum temperature: 35 – 37 0C (Body temperature). Loewenstein-Jensen media: raised, dry, wrinkled. At first whitish, later buff colored or yellow. Blood agar. Serum agar. Media which contain whole egg or vegg yolk. Catalase: positive. Niacin test: positive. Nitrate reduction: positive. Susceptibility testing: INH. Rifampicin. Ethambutal. See also: http://en.wikipedia.org/wiki/MDR_TB http://en.wikipedia.org/wiki/XDR_TB Exert from TYGERLAND, Faculty of Health Sciences, University of Stellenbosch: Paediatric tuberculosis, When necessity is the mother of invention With the cooperation of Profs Rob Warren and Ben Marais, Prof Colleen Wright recently set out to implement and test a new technique that provides a rapid and accurate diagnosis of mycobacterial tuberculosis, using routinely collected fine needle aspiration samples (from swollen lymph nodes) which were directly inoculated into the liquid transport medium developed in the Faculty. The new technique used utilizes the melting profile or dissociation of double-stranded DNA, which is specific for each species of mycobacteria, using fluorescent dye as a marker. “This technique is rapid and simple and the equipment required for high resolution melting is relatively inexpensive and can be used in routine laboratories. Up to 72 samples may be processed in a cycle and the whole process takes approx. three hours”, she says. With Warren and Marais as her supervisors, Wright recently completed a PhD thesis on the above approach. She says this whole new approach to TB diagnosis was developed in response to a real problem faced by clinicians who work with TB patients in resource-limited environments every day... Mycobacterium leprae Clinical aspects: Cause leprosy – affects skin, mucous membranes. Specimens: Sputum, scrapings from nasal mucosa, ulcerated nodule on skin. Scraping of tissue after skin incision – particularly of ear lobe. Microscopic examination: Stain with modified ZN using 20 % H2SO4 as decolorant. Long slender bright red bacilli arranged like match bundles (globi or globus shape).
HAEMOPHILUS Haemophilus influenza Clinical aspects: Meningitis, laryngoepiglottitis (croup), otitis media, sinusitis, upper respiratory tract infections, pneumonia, bacteremia, endocarditis, urogenital, maternal and perinatal infections. Specimens: Csf, sputum, ear swabs, throat swabs, eye swabs, blood cultures, vaginal swabs, urethral swabs, amniotic fluid, placental tissue. Microscopic examination: Small gram negative pleomorphic bacilli. Culture: Aerobic. 370C (body temperature) optimum. Requires both X and V factors. X-factor is heat resistant, present in haemin. V-factor is a respiratory coenzyme destroyed by heat at 1200C, but released from blood cells by heating at 700 – 800C (chocolate agar). Blood agar with Staph. streak: blood agar provide X-factor; Staph. hemolyses the RBC to release the V-factor. Colonies form sattelism around the Staph. streak. Nutrient agar with Staph. streak: no growth. Catalase: positive. Oxidase: positive – slow reaction. Beta-lactamase: positive or negative. Susceptibility testing: Cotrimoxazole. Co-amoxyclave (Augmentin). Amoxicillin. Ciprofloxacin. Ceftriaxone. Tetracycline. Do these sensitivities on Haemophilus Test Medium (HTM) – has X and V without blood. Haemophilus parainfluenza Clinical aspects: Endocarditis, meningitis, brain abscesses, peritonitis, purulent urethritis. Otherwise a normal commensal in the throat and mouth. Specimens: As for Hflu. Microscopic examination: As Hflu. Culture: Use same media as for Hflu.
Requires only V-factor for growth – grow on blood agar with Staph. streak and also on nutrient agar with Staph streak (Does not require X factor.). Colonies are bigger and more opaque than Hflu. On MH does not grow around X, as it only requires V factor for growth) – only method to differentiate from Hflu (as at 2003). Susceptibility testing: As for Hflu.
ENTEROBACTERIACEAE Properties of enterobacteriaceae: gram negative bacilli; grow in presence of bile salts (MacConkey agar), ferment glucose aerobically or anaerobically; reduce nitrates to nitrites, oxidase negative; non-sporing, motile or non-motile; catalase positive. Includes: E. coli, Klebsiella, Enterobacter spp., Serratia, Proteus, Providencia, Citrobacter, Salmonella, Shigella, Yersinia. Natural habitats: widely distributed on plants and in soil, water and intestines of humans and animals. S. typhi species only found in man – typhoid fever. Klebs. pneumonia can be found in the environment but can also cause urinary tract infection (UTI) and upper respiratory tract infection (URTI).
1. Escherichia coli Clinical aspects: UTI-cystitis, pyelonephritis, gastro-enteritis, respiratory tract infections, neonatal meningitis, endotoxic shock. Specimens: Urine, stool, sputum, csf, wound and pus swabs, water. Microscopic examination: Gram negative bacilli, non-sporing. Culture: Aerobic and anaerobic. 370C. (body temperature) optimum. MacConkey agar: lactose fermenter – smooth, rose-pink in color. CLED agar: bright pink in color. Blood agar: can be haemolytic. Biochemical reactions: Lactose: fermenter. Motile. Indole: positive. Mannitol: fermenter. Forms gas from glucose. Citrate: negative. KCN: negative.
Methyl red: positive. Voges Proskauer: negative. Public health: Do IMVEC for drinking water supplies: Indole positive, MR positive, VP negative, citrate negative. Eijkman test: ability to form gas at 440C from lactose. Susceptibility testing: Cotrimoxazole. Co-amoxyclav. (Augmentin) Amikacin. Amoxicillin. Gentamycin. Ciproflox / Ofloxacin. Ceftriaxone. Nitrofurantoin. (urine) Naladixic acid. (urine) Cefoxitin. Antigenic structure: Serotyping done. E. coli in diarrhoea: Enteropathogenic strains (EPEC) Enterotoxogenic strains (ETEC) Enteroinvasive strains (EIEC) Enterohaemorrhagic strains (EHEC). 2. Klebsiella species Clinical aspects: UTI, URTI – pneumonia, bacteremia. Specimens: Urine, stool, sputum, blood cultures, wound, pus and burn swabs. Microscopic examination: Gram negative bacilli, non-sporing. Culture: Aerobic and anaerobic. 370C (body temperature) optimum. MacConkey agar: pink mucoid colonies. CLED agar: light pink mucoid colonies. Blood agar: large grey mucoid colonies. Biochemical reactions: Lactose fermenter. Non-motile. Indole negative. Klebs. oxytoca is indole positive. Mannitol: fermenter. Forms gas from glucose. Citrate: positive. (remember E.coli is neg.) KCN positive. (E.coli neg.)
Methyl red: negative. (E.coli and others pos.) Capsulated: can do capsule swelling test with specific antisera for klebsiella. Susceptibility testing: as for E.coli. 3. Enterobacter spp. Clinical aspects: Opportunistic pathogens, they are frequent colonizers of hospitalized patients – specifically those on antibiotics, burn wound, urinary and respiratory infections. Microscopic examination: Gram negative bacilli. Culture: Very similar to Klebsiella spp. Motile. MacConkey agar: non-lactose fermenting. (late lactose fermenting.) ONPG: positive. (late lactose fermenters.) Susceptibility testing: as for E.coli. Enterobacters are usually resistant to first generation cephalosporins.
Clinical aspects: UTI, septicemia, wound infections. Specimens: Urine, blood culture, csf, wound and pus swabs. Microscopic examination: Gram negative bacilli, coccal forms to long filaments. Culture: Aerobic and anaerobic. 370C (body temperature) optimum. Blood agar: fishy smell and swarming over plate. MacConkey agar: non-lactose fermenting colonies. CLED agar: non-lactose fermenting (transparent colonies). Stop swarming-PNPG: 0.215 g p-nitrophenylglycerol to 500 ml media, 4% agar, MacConkey agar or DCA. Biochemical reactions: Non lactose fermenter. (NLF) Urease positive. (Singers turn blue.) Motile. Indole negative.(Note: other Proteus spp. and Providencia and Morganella are indole positive.) Mannitol negative.
H2S positive. Gas from glucose. Citrate positive. KCN positive. PPA positive. (Phenyl-alanine agar.) Methyl red: positive. Voges proskauer: positive or negative. Antigenic structure: P.mirabilis can be serotyped by their O-antigens into 49 O-groups Susceptibility testing: same as for E.coli. Resistant to Nitrofurantion in urines.(differentiate E.coli.)
Clinical aspects: Diarrhoea, food poisoning, septicemia. Specimens: Stool, vomit, foodstuffs, blood cultures. Microscopic examination: Gram negative bacilli. Culture: Aerobic and anaerobic. 370C (body temperature) optimum. Blood agar: grey, smooth colonies. Nutrient agar: colorless smooth colonies. MacConkey agar: non lactose fermenting. SS agar: NLF with H2S (black). Differentiate S.typhi which is H2S negative. XLD agar: red colonies with black centers. DCA agar: colorless with black centers. Enrichment media. Biochemical testing: Non lactose fermenter (nlf). (NB) Motile. (NB) Indole negative. (NB) Mannitol: ferment. (NB) Forms gas from glucose (S.typhi non gas-forming), ferments glucose.(NB) H2S produced. S.typhi not. Citrate positive. S.typhi citrate negative. KCN: negative. Dulcitol: positive. S.typhi non dulcitol fermenting. Susceptibility testing: see S.typhi.
- Salmonella typhi Clinical aspects: S.typhi only a pathogen of man. Cause typhoid (do not confuse with rickettsial disease typhus), diarrhoea, seticemia. Specimens: Stool, urine, blood culture. Microscopic examination: Gram negative bacilli. Culture: Aerobic and anaerobic. 370C (body temperature) optimum. Blood agar: grey, smooth colonies. Nutrient agar: colorless, smooth colonies. MacConkey agar: non lactose fermenting. SS agar: colorless. (very tiny black – H2S – cannot see it well. H2S negative.) DCA agar: smaller, pale, smooth, shiny. Enrichment media: selenite F, Rappaport media, Tetrathionate broth. Biochemical reactions: Non lactose fermenter. Motile. (NB) Indole negative. (NB) Mannitol: ferment. No gas from glucose (anaerogenic), ferment glucose. (NB) H2S: little or none. Citrate negative. KCN: negative. Dulcitol: not fermented. Antigenic structure: Use the Kauffmann-White classification for serotyping of Salmonella. The ID of serotypes depends on the detection of the O (somatic) and H (flagella) antigens by means of agglutination with specific antisera. O-antigens represent the side chains of sugar polymers projecting from the outer lipopolysaccharide layer of the bacterial cell wall. 60 different types, heat stable, unaffected by heating at 1000C (water boiling point) for 2.5 hrs, alcohol-stable, withstand 96% alcohol at 370C for 4 hrs. (this destroys the flagellar antigens.) H-antigens represent determinant groups on the flagellar antigen. Heat and alcohol labile – if 30 min at 100 0C it will detach the flagella from the bacteria = no agglutination with H-antisera. Flagella antigens can be diphasic: phase 1 (specific), phase 2 (nonspecific). Vi-antigen: S.typhi forms Vi antigen – covering the layer outside the cell wall. Heat-labile, removed by heating 1 hr at 1000C. O-groups A - Z : A= 2, or D= 9. Groups A – E are human pathogens. Eg. Kauffmann-White: S.typhi group A-; Oantigen 9, 12, Vi; H-antigen phase 1-d; phase 2-.
O, H and Vi antigens are positive with S.typhi. Note: do not use colonies grown on MacConkey agar for typing. Typing procedure: Emulsify organism in saline. Use clean glass slide and place two drops on each end of slide. Add poly O antisera to one drop and poly H to drop 2. Look for agglutination. If positive in both = Salmonella. Start typing with specific antisera. Serology: Widal test. Susceptibility testing: Cotrimoxazole. Amoxacillin. Ofloxacin. Ceftriaxone. Chloramphenicol. (NB) SHIGELLA S.flexneri. S.boydii. S.sonnei. S.dysenteriae.
1. 2. 3. 4.
Clinical aspects: Bacillary dysentry (shigellosis), abdomonal pain, fever, watery stools, mucoid and blood, rarely spreads from mucosa into blood. Specimens: Faeces, rectal swabs, rarely vomit. Microscopic examination: Gram negative bacilli. NB: do a wet prep and look for numerous polymorphs (pus cells) and erythrocytes (blood). Culture: Aerobic or facultative anaerobic. 370C (body temperature) optimum. Blood agar: smooth, grey colonies. Nutrient agar: smooth, colorless colonies. MacConkey agar: nlf , but S.sonnei become pink with prolonged incubation – a late lactose fermenter. DCA agar – selective medium: pale small colonies. SS agar: colorless colonies. Selenite F broth – for growth and enrichment of S.flex. and S.sonnei. Biochemical reactions: S.flex & S.boydii Motility Indole non +/-S.sonnei non -S.dysenteriae non +
Mannitol Gas H2S Citrate KCN Dulcitol ONPG
Note: Klebsiella and Shigella are the only non-motile Enterobacteriaceae. Shig.sonnei is a LLF.(otherwise Shigella and Salmonella are NLF) Shig.dysenteriae very pathogenic. Dysentry is a notifiable disease. Antigenic structure: Shigellas are differentiated by their somatic (O) antigens into serotypes identified by agglutination with specific antisera. Typing method: Emulsify organism in a drop of saline. Check first for auto-agglutination before adding 1 drop of specific antisera. Mix or rotate 1 – 2 min. Look for agglutination. Susceptibility testing: Cotrimoxazole. Amoxacillin. Ofloxacin. Ceftriaxone. Chloramphenicol. Naladixic acid. SERRATIA
Looks exactly like Enterobacter on MacConkey. To differentiate: Dnase: positive with Serratia. Negative with Enterobacter. Gas producer: Serratia – no. Enterobacter – yes. Serratia marcescens has bright red pigment. YERSINIA ENTEROCOLYTICA
Clinical aspects: Enteritis, mesenteric lymphadenitis, infected animal bite, contamination of stored food. (seems to be rarely encountered in humans in S.A.)
Hereditary haemochromatosis sufferers more susceptible. (http://en.wikipedia.org/wiki/Yersinia_enterocolitica ) Specimens: Blood, food, soil, faeces. Microscopic examination: Gram negative cocco-bacilli; pleomorphic in older cultures. Culture: Aerobe. Blood agar: non-haemolytic, smooth translucent colonies. MacConkey agar: medium pin point colonies. SS agar: nlf. Biochemical reactions: Motile at 220C (RT), non-motile at 370C (body temperature). Indole: negative or positive. Urease: positive. Catalase: positive. ONPG: positive. Antigenic structure: Serotyping can be done with specific antisera for Yersinia enterocolytica. Susceptibility testing: Chloramphenicol. Tetracycline. Usually rt penicillin, ampicillin and 3rd generation cephalosporins.
NON-FERMENTERS PSEUDOMONAS AERUGINOSA
Clinical aspects: Natural habitat: environmental – water, soil, plants, hospital environment, pathogenic in swimming pools, contact lens fluid, cosmetic solution, bowel coloniser, coloniser of upper respiratory tract, UTI, ear, sinus, eye infections, endocarditis, septicaemia. Specimens: Urine, blood, pus swabs, sputum, burn swabs. Microscopic examination: Gram negative bacilli. Culture: Strict aerobe. Temperature range: 50C – 420C. (Ps. aeroginosa 420C) Blood agar: large colonies.
MacConkey agar: non lactose fermenting. Pigment production: pyocyanin (blue-green, soluble in water and chloroform), fluorescin (yellowgreen, soluble in water; not chloroform), pyorubin (dark brown). Distinctive musty smell. Sheen in reflective light. Biochemical reactions: Oxidase positive. Motile. N2 gas produced. Citrate positive. KCN positive. O/F test (Hugh Leiffson): oxidative. Sellars media (stab plus slope innoculation): slant:green, but: blue or no change. Susceptibility testing: Amikacin. Gentamycin. Ciprofloxacin. Ofloxacin. Ceftazidime. ACINETOBACTER ANITRATUS (= ACINETOBACTER BAUMANNII)
Clinical aspects: Free living saprophyte in soil and water, regular contaminants in hospital environment, bronchopneumonia, septicaemia, skin and wound infections. Specimens: Blood cultures, pus swabs, burn swabs, endotracheal tube, urine. Microscopic examination: Gram negative bacilli. On solid media – cocco-bacilli. Culture: Strict aerobe. 370C (body temperature) optimum. Blood agar: some can be haemolytic, white to cream smooth colonies. MacConkey agar: non lactose fermenting colonies, slightly pink. Biochemical reactions: Oxidase negative or slow oxidase positive. Non-motile. No N2 gas produced. Citrate: positive. KCN: positive. O/F test: oxidative. Sellars media: slant: blue, butt: no change, yellow band with 10% dextrose. Susceptibility testing: All strains are resistant to penicillin. Hospital strains are rt Pg, ceph, E, T, C. Use same sens as for gram negative bacilli, plus also report Ceftazidime.
ANAEROBES ANAEROBIC BACILLI
Clinical aspects: Human actinomycosis – chronic granulomatous infection, lumpy jaw. http://en.wikipedia.org/wiki/Actinomyces_israelii Specimens: Pus, pus swabs, blood culture. Microscopic examination: Gram positive branching bacilli. Looks like fungal hyphae. Non-acid fast with Ziehl Neelsen. Non endospore forming. Culture: Anaerobic or facultative anaerobic. Responds to penicillin, amoxicillin. BACTEROIDES FRAGILIS
Clinical aspects: Part of normal flora in mouth, GIT and female genital tract. Only 3% of normal gut flora, but highly pathogenic – cause 80% of anaerobic infections. Predominate in anaerobic abscesses. Intraabdominal infections, brain abscesses, oral infections, pleuro-pulmonary infections, pelvic infections (usually complicated by mixed infection – eg. due to gut leakage), bacteremia, endocarditis, skin and bone infections. Virulence factors include: polysaccharide capsule, lipopolysaccharide, agglutinins, histolytic enzymes, oxygen tolerance, beta-lactamases. Clinical features include foul odor, tissue necrosis and abscesses. Has often been missed in culture because organism is very fastidious. Specimens: Pus swabs, throat swabs, cervical swabs, body fluids, blood cultures. Microscopic examination: Gram negative pleomorphic bacilli. Non-motile, non-sporeforming (large vacuoles may resemble spores.) Culture: Anaerobic. 370C (body temperature) optimal. Blood agar: convex white to grey, semi-opaque and glistening. Can be hemolytic.
Brucella agar: grey glistening colonies, foul smell. Aesculin agar: hydrolyse (black). Growth stimulated by bile.(Bile salt 20%) Laboratory identification: Aerobic and anaerobic. GNB (gram negative bacillus), pleomorphic. Brucella agar with id 8-ring: Penicillin R, Kanamycin R, Vancomycin R, Colistin R, Erythromycin S, Rifampicin S. Aesculin agar: contains esculin and ferric citrate – hydrolysis (blackening around the colony). Catalase positive. Indole negative. Susceptibility: Use Wilson Chalgren media with metronidazole disc. In vitro susceptibility: Metronidazole (some resistant strains have been reported). Carbapenems eg. imipenem,meropenem,ertapenem. Beta-lactam/beta-lactamase combinations eg. piperacillin/tazobactam,ampicillin/sulbactam,amoxicillin/clavulanate. Tigecycline. Prophylactic antibiotics given if medical procedure will disrupt the mucosa or if mucosa has been disrupted by trauma. CLOSTRIDIUM PERFRINGENS
http://en.wikipedia.org/wiki/Clostridium Clinical aspects: Gas gangrene, myonecrosis (necrosis of individual muscle fibers), food poisoning (C.botulinum and C.perfringens). http://en.wikipedia.org/wiki/Clostridium_perfringens Specimens: Wound and pus swabs, tissue, food samples, blood culture. Microscopic examination: Gram positive bacilli. Large with stubby or square ends. Spores are formed only in absence of fermentable carbohydrates: subterminal and bulging. Looks like Bacillus spp. (aerobic gram positive sporeforming rods, Bacillus spp. catalase pos.) Biochemical reactions: Divided into saccharolytic and proteolytic clostridia on biochemical reactions. Saccharolytic clostridia: grow rapidly in carbohydrate media with production of acid and abundant gas; ferment glucose, cooked meat broth (Robertsons meat), but won't digest the meat; sour smell and the meat is reddened, with acid and gas – eg. C.perfringins. Proteolytic clostridia: digest meat, liquify gelatin, coagulate serum; cooked meat (Robertsons) – blackening of the meat and the volume will reduce with formation of foul-smelling products – eg. C.tetani. Culture: Anaerobic. 37 – 45 0C. Blood agar: double zone of haemolysis (1st zone of complete haemolysis due to the theta toxin;
2nd wider zone of incomplete haemolysis due to alpha toxin). Colonies are large, round, smooth, regular and slightly opaque. Nagler plate (Willis and Hobbs media): selective media for clostridia. If neomycin is added it makes media more selective for C.perfringens. The media is a lactose egg-yolk milk agar medium with neomycin sulphate; the indicator is neutral red; the neomycin inhibits staphs and bacillus species. Several clostridia produce phospholipase enzymes (lecithinase) that will give rise to opalescence in both human serum and egg yolk media. The Nagler reaction produced by C.perfringens is neutralized by C.perfringens antitoxin. Biochemical reactions: Non-motile. Catalase: negative. Ferment glucose, lactose, sucrose, maltose, mannitol. Dulcitol not fermented. Gelatin: liquified. H2S produced.(Sulphide positive) Milk media: stormy clot (acid clots the milk causing the protein to coagulate; the gas breaks up the clot). Indole negative. Urease negative. Susceptibility testing: Pg G preferred drug for gas gangrene. Metronidazole. Food poisoning: C.perfringens type A produce heat resistant spores. Between 4 – 8 hrs after having eaten contaminated food, develop acute abdominal pain and diarrhoea; nausea may occur but no vomiting. Symptoms last for 12 – 18 hrs. Enterotoxin is synthesized during sporulation. Types of contaminated foods: roasts, poultry, fish and stews. Also raw mwat at slaughter. Clostridium germinates at cooking; multiply at cooling. 106 to 107 viable cells / gram of food will cause symptoms. ANAEROBIC STREPTOCOCCI 1. PEPTOSTREPTOCOCCUS spp 2. PEPTOSTREPTOCOCCUS ANAEROBIUS 3. PEPTOCOCCUS NIGER Clinical aspects: These species are normal flora in 1) the mouth, 2) urogenital tract and 3) gastrointestinal tract. Human infections: 2nd most common encountered in human infections. Very important in pleuropulmonary disease, brain abscesses, gynecological infections. Specimens: Pus swabs, cervical swabs, body fluids, blood cultures. Microscopic examination: Gram positive cocci.
Culture: Anaerobic. Blood agar: small white colonies. To identify – culture AER and ANAER. Make a smear: gram pos. cocci. Brucella agar with SPS (sodium polyanethol sulfonate). Peptostreptococcus species: resistant to SPS (< 12 mm). Peptostreptococcus anaeobius: sensitive to SPS (< 12 mm). Peptococcus niger: black pigment. Susceptibility testing: No need to test. Treatment of choice is Penicillin. YEAST CANDIDA ALBICANS
Clinical aspects: Oral candidiasis (thrush), cutaneous candidiasis (hands, feet, trunk), candida vaginitis, paronychia (cuticle), respiratory tract, UTI, meningitis. Specimens: Sputum, skin, pus swabs, vaginal swabs, csf, biopsies, tissue, blood culture. Microscopic examination: Gram positive yeast. C.albicans forms multicellular branched filaments from unicellular forms. Culture: Aerobe. 370C (body temperature) and RT. Blood agar: raised, rounded white colonies; larger with prolonged incubation, dull appearrance. Sabouraud Dextrose agar (contains dextrose, peptone, pH 5.4 – 5.6): white dull colonies. Incubate day 1 at 370C, then RT. Corn meal agar with Tween 80: inoculate with yeast, put coverslip on and incubate at 250C; examine microscopically for chlamydospore formation. Germ tube test: Bovine or human serum – 9 drops. Emulsify yeast in serum. Incubate at 370C for 1 – 2 hrs. Look for germ tubes or pseudomycelium. If positive, id as C.albicans. If negative, do further tests to id the yeast. Treatment: Nyastatin, Amphotericin B. CRYPTOCOCCUS NEOFORMANS
Clinical aspects: Cryptococcosis, meningeal cryptococcosis, pulmonary cryptococcosus, cutaneous (skin) lesions, HIV patients.
Specimens: CSF, sputum, skin swabs, autopsy tissue, biopsies. Microscopic examination: Yeast-like fungi. Heat fixation causes yeast to distort badly. If numerous, can be seen on gram stain. They produce an extracellular polysaccharide capsule. Do indian ink preparation: 1 drop of CSF onto a glass slide with diluted indian ink. Look for dark yeast cells surrounded by a clear halo (refractile capsule). Never forms a pseudomycelium. Culture: Aerobe. 220 (RT) and 370C (BT)(best at 37). Blood agar: greyish slow growing colonies. Sab dex agar: 1 at RT and 1 at BT: non descriptive colonies, slightly opaque; slime-producer like klebsiella. Potato dextrose agar: to enhance the growth and capsular formation. Selective media: Tryptan blue: dark blue colonies at RT. Niger seed agar: dark brown colonies at 370C (BT). Urease: positive. Treatment: Amphotericin B.
PART 2: MICROBIOLOGY: TESTS AND MEDIA : http://www.scribd.com/document_collections/2374607 NOTES FROM CAPE PENINSULA UNIVERSITY OF TECHNOLOGY (FMR. CAPE TECH.), BIOMEDICAL TECHNOLOGY, 2003. MORE COURSE NOTES AT: http://www.scribd.com/document_collections/2374607
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