Clinical Bacteriology Lecture Notes PDF

Clinical Bacteriology Dr. San Diego | Lab I. ENTEROBACTERALES GENERAL CHARACTERISTICS ➔ Family - Genus - Species ➔ General Characteristics ✔ Fa...

Clinical Bacteriology Dr. San Diego | Lab I. ENTEROBACTERALES GENERAL CHARACTERISTICS ➔ Family - Genus - Species ➔ General Characteristics ✔ Facultative anaerobe or aerobe; Motile with II. STEPS in IDENTIFICATION peritrichous flagella except Shigella, Y. ➔ Culture Media - Fermentable CHO - Indicator - Fermenter - Non-Fermenter pestis, Klebsiella (except K. aerogenes- ➔ Addtl. Info motile) III. BIOCHEMICAL TESTS ✔ Motile at RT but Non-Motile at 37 deg: Genus ➔ Carbohydrate Fermentation Test Yersinia, except: Yersinia pestis ➔ ONPG Test ✔ Some are with pili or fimbriae; Grows well on ➔ Gelatin Hydrolysis Test ➔ IMViC Test Mc Conkey agar ➔ Urease Test ✔ All Ferments glucose and often with gas ➔ Deaminase Test production aerogenic except Shigella ➔ Motility Test ✔ Cytochrome oxidase negative except ➔ Malonate Utilization Test Plesiomonas (New member: Plesiomonas ➔ Test to Detect H2S Production shigelloides) ➔ Decarboxylase Test IV. PROTEUS-PROVIDENCIA-MORGANELLA ✔ Reduce nitrate to nitrites, except: Erwinia and ➔ Genus Proteus and Pantoea agglomerans ➔ Genus Providencia a. Intestinal Pathogens ➔ Genus Morganella - this is not normal flora ➔ Genus Yersinia - doesn’t normally reside in GIT ➔ Salmonella - Shigella V. RAPID LACTOSE FERMENTERS - Shigella (all), Salmonella (all), Y. ➔ Genus Enterobacter enterocolitica ➔ Genus Klebsiella b. Opportunistic Pathogens ➔ Genus Escherichia - these are GIT flora that can produce infections VI. OTHER GENERA/SPECIES outside GIT; once it leaves the GIT it becomes pathogenic ENTEROBACTERALES Virulence Factors: 1. Equipped with pili (adherence), common pili FAMILY-GENUS-SPECIES 2. Possession of plasmid- it can confer resistance to antibiotics 3. Possession of antigens, use to identify other groups STEPS IN IDENTIFICATION BOARD QUESTION: How Many Quadrants For Stool Streak? ○ Answer: 4 1 Clinical Bacteriology Dr. San Diego | Lab BIOCHEMICAL TESTS CARBOHYDRATE FERMENTATION TEST 1. To detect acid production 2. Can also detect ability of organism to produce H2S Media: TSI/Triple sugar Iron (Tubed Media) dispensed as Butt & Slant ○ Kliger’s Iron Agar- substitute for TSI - If wala TSI, you can use this - Same component but doesn’t contain sucrose 2 Clinical Bacteriology Dr. San Diego | Lab ○ it is an enzyme produce by both Lactose and Late Lactose Fermenter May be carried out using broth/disk (+) result: Yellow ○ (+) RLF ○ (+) LLF ○ (-) Non-LF ONPG Positive ONPG Negative Every Kool Entertainer Can Paborito ni Sam Si Papa Sing Songs, Yells Happily Yoyong Magsalita ng Yucky! Escherichia coli Proteus spp. Klebsiella spp. Salmonella spp. Enterobacter spp. Shigella spp Citrobacter spp. Providencia S. arizonae Y. pestis S. sonnei Morganella Y. enterocolitica Y.pseudotuberculosis H. alvei GELATIN HYDROLYSIS TEST BOARD QUESTION: VITEK SYSTEM- automation program for bacterial ID & susceptibility ○ Its principle is same to manual 1. Useful in the detection of the members of ○ Advantages: Serratia 1. Higher Sensitivity Organism is inoculated on Nutrient Gelatin 2. Faster Turnaround Time Media & incubated for 14 days at 35-37 degs ○ Within the incubation period, tubes are removed from the incubator daily & it is ONPG TEST placed inside the ref for 30 minutes or until the media had gelled ○ Gelatin Media - dispense as Butt, a semi-solid media ○ (+) result: Gelatin liquefaction ○ (-) result: complete solidification of media at 4c 1. Primarily to detect late lactose fermenters in Serratia we also use DNase test 2. To differentiate Citrobacter which are ONPG (+) ○ Triple Enzyme Positive - Genus Serratia from Salmonella which are ONPG (-) ➔ produces lipase, gelatinase, - Salmonella are ONPG (-) except S. DNase arizonae ○ Serratia = (+) DNase Test ONPG Test - specifically detect the enzyme Beta ○ DNase Test - usually use in detecting Galactosidase S.aureus 3 Clinical Bacteriology Dr. San Diego | Lab ○ (+) DNase - “SMS” (S.aureus, Moraxella ○ I.e MR (+) & VP (-) or vice versa catarrhalis, Serratia) MR tubes must be incubated for 48 hours before reading the result IMViC Test ○ reading the result too early can cause 1. Its primary purpose is to differentiate the Rapid false positive results Lactose Fermenters Consists of 4 test: UREASE TEST ○ Indole ○ Methyl Red ○ Vogues Proskauer ○ Citrate - Must use all together in a 4 different test tubes - However you could use the tests separately to detect other bacteria outside Enterobacterales uninoculated - the MR VP Broth is Light Yellow 1. Useful in the identification of PPM: - Urease BOARD QUESTION: - Deaminase Test (Priority Test for PPM) 2. Urease Test - to detect the ability of the bacteria IMViC Test - its primary purpose is to to produce the enzyme Urease differentiate the Rapid Lactose Fermenters - Based on the ability of the organism to ○ I.e, E.coli (+ + - -) → K.pneumoniae (- - + convert urea to ammonia thru the action +) of the enzyme urease The specimen you received is positive in PPM, no Vitek available, you should use Urease Test and Deaminase Test for faster detection. Rapid Urease Producers: does not require 24 hrs of incubation, in just 4 hrs they are already positive ○ Proteus ○ Providencia ○ Morganella Slow Urease (+): (+) after 4 hours are the following (KEY-SC) ○ Klebsiella ○ Enterobacter ○ Yersinia ○ Serratia ○ Citrobacter Process: ○ Media in Urease Test - Christensen Urea Agar /Urea broth ○ Indicator: Phenol Red ○ Results: - (+) result: pink, red, fuschia - (+): result after 4 hrs: orange ADDITIONAL INFORMATION - (-): yellow KOVAC’S - paradimethyl amonibenzaldehyde MRVP reactions are always opposite 4 Clinical Bacteriology Dr. San Diego | Lab Use of TTC (triphenyl tetrazolium chloride) colorless dye that turns red with organisms DEAMINASE TEST growth (to make the reading easier) All Salmonella are motile, All Shigella are non-motile MALONATE UTILIZATION TEST 1. Useful in the identification of PPM, these are the only deaminase (+) enterics Media use: ○ LIA - commonly use in the laboratory ○ Phenylalanine agar Based on the ability of the organism to use ○ Tryptophan agar sodium malonate as the only carbon source PAD + overnight culture + 10% Ferric chloride Indicator: Bromthymol Blue (color developer) Results: Results: ○ (+) result: blue ○ (+) result with Phenylalanine agar: - Salmonella Green ○ (-) result: green ○ (+) result with tryptophan agar: Brown - Shigella TEST TO DIFFERENTIATE SALMONELLA AND TEST TO DETECT H2S PRODUCTION SHIGELLA May be carried using any media provided that the Salmonella and Shigella are not GIT flora, via media contains an indicator for H2S detection fecal-oral route. Both cause diarrhea Examples of media with H2S indicator: ○ TSI MOTILITY TEST ○ LIA ○ SIM- most sensitive media for H2S detection ○ HEA H2S indicator in HEA, SSA, and LIA: ferric ammonium citrate H2S (+): Black H2S POSITIVE H2S NEGATIVE Very useful in detecting non-motile enterics Klebsiella and Shigella Si Edna at Cita, Shigella May be carried out using Sim Media Paborito ang Salmon! On a tube media: Salmonella ○ Motile: if growth is outside the line of Edwardsiella streak tarda ○ Non-motile: if growth is at the line of Citrobacter streak freundii Proteus vulgaris 5 Clinical Bacteriology Dr. San Diego | Lab Citrulline → Cleaved → Proteus mirabilis Ornithine → if the bacteria produces O-decarboxylase, putrescine will be removed Is there a need to perform separate tests just MOELLER’S BROTH to detect the ability of the bacteria to produce H2S? Need to prepare 3 tubes ○ No, because you can do it along with ○ 1st Tube: Lysine the other tests as long as there is the - Moeller’s Broth + AA + 1% indicator for H2S production. lysine = Lysine decarboxylase ○ 2nd Tube: Ornithine - Moeller’s Broth + AA + 1% DECARBOXYLASE TEST Ornithine = Ornithine Based on the ability of the organism to remove decarboxylase carboxyl group from specific amino acid through ○ 3rd Tube: Arginine - Moeller’s Broth + AA + 1% the release of the enzyme decarboxylase Arginine = Arginine There are 3 amino acids that can be used: decarboxylase ○ Lysine * Before incubating, you will overlay tubes w/ mineral oil ○ Ornithine - PURPOSE: Oxygen Barrier; To make environment ○ Arginine anaerobic (decarboxylation) Media: Moeller’s Broth, LIA Indicator: BOARD QUESTION: (+): Salmonella (-): Shigella What is the purpose of overlaying of tubes w/ AMINO ACID ENZYME CARBOXYL mineral oil ○ Oxygen Barrier Lysine lysine Cadaverine ○ To make environment anaerobic decarboxylase (decarboxylation) —------> Ornithine ornithine Putrescine Components: decarboxylase ○ Base Media: Base media + AA + overlay —-------> tubes with mineral oil CHO: glucose Arginine arginine Citrulline, Indicators: bromcresol purple, cresol red dihydrolase / Putrescine arginine Moeller’s Broth: decarboxylase decarboxylase (+) result: purple (Alkaline; decarboxylation) —------> (-) result: yellow (Acid; fermentation) LYSINE IRON AGAR REMOVAL OF ARGININE INVOLVES 2 STEPS: It is a media dispensed as slant & butt 1. ARGININE →Agmatine & CO2 = will only ○ Slant is observed for: deaminase test removed if the bacteria produce A-decarboxylase (upper) Product of degradation: - (+): Burgundy/Red Putrescine & Urea → if UREA - (-): Purple can produce UREASE, it can be ○ Butt is observed for: decarboxylase test converted to AMMONIA (lower) - (+): Purple (Alk) 2. ARGININE → Citrulline, Inorganic Phosphate & - (-): Yellow (Acid) Ammonia = will only removed if the bacteria Purpose: deaminase and decarboxylase test produces A-dihydrolase pH indicator: bromcresol purple H2S indicator: ferric ammonium citrate 6 Clinical Bacteriology Dr. San Diego | Lab PROTEUS-PROVIDENCIA-MORGANELLA “PPM GROUP” All are non-lactose fermenters Rapid Urease (+) within 4 hrs All are Deaminase (+) All causes RTI and UTI GENUS PROTEUS Use as source of antigens used in: ○ Weil Felix Test - a serologic test to detect rickettsial infections Species L O A - i.e Typhus fever (Lysine) (Ornithine) (Arginine - Specimen: Serum Klebsiella pneumoniae + - - - detects antibody by the use of Klebsiella oxytoca antigen from Proteus Salmonella typhi - Proteus vulgaris source of OX2, Escherichia coli + + - OX19 Enterobacter aerogenes Edwardsiella tarda - Proteus mirabilis source of Hafnia alvei OXK Serratia marcescens Proteus vulgaris - - - BOARD QUESTION: Providencia Which of the following Genera is involved in the Shigella sonnei - + - Proteus mirabilis development of Struvite Stone Morganella moganii ○ Genus Proteus Yersinia enterocolitica Citrobacter freundii - - + Proteus group is involved in the formation of Citrobacter koseri - + + renal stones/ kidney stones/ “Struvite Stone” Enterobacter cloacae Colonies on Mac Conkey agar develop colorless colonies with burnt chocolate/burnt gunpowder Species L O A odor (Lysine) (Ornithine) (Arginine Shows swarming on BAP Enterobacter cloacae - + + Enterobacter aerogenes + + - PROTEUS VULGARIS PROTEUS MIRABILIS Escherichia coli + + - Most frequently isolated and Causes nosocomial UTI may cause pneumonia and Citrobacter - + - septicemia Klebsiella pneumoniae + - - Source of OX2, OX19 Source of OXK Klebsiella oxytoca + - - P. agglomerans - - - 7 Clinical Bacteriology Dr. San Diego | Lab GENUS PROVIDENCIA SALMONELLA-SHIGELLA NO SWARMING on BAP, Normal Intestinal Flora Intestinal pathogens Causes RTI and UTI MOT: Spread via fecal oral route Species: NOT normal flora ○ P.rettgeri (urease +) ○ P.stuartii - causes RTI and UTI GENUS SHIGELLA TSI GENUS MORGANELLA ○ K/A = NLF (Non-Lactose Fermenter) NO SWARMING ON BAP ○ Late Lactose: S. sonnei Species: Morganella Can cause shigellosis/bacillary dysentery form Causes RTI and UTI of bloody diarrhea Virulence Factors: ○ Species are invasive PROTEUS PROVIDEN MORGANE CIA LLA ○ Produce endotoxin S. dysenteriae TSI Non-Lactose Fermenter ○ Most severe ○ Virulence factor: Can produce Urease + endotoxin & neurotoxin Deaminase + GENUS SALMONELLA Species can cause a number of infections GENUS YERSINIA ○ Diarrhea, Bacteremia and Gastroenteritis Motile at room temperature but non-motile at 37 and Enteric fever degC except Yersinia pestis Site of long-term carriage of S. typhi is the gallbladder (in recovered patients) Selective media: LIA Black colonies with metallic sheen Species: ○ S. enterica - human pathogen has several serotypes (i.e. S. typhi, S. paratyphi, S. choleraesuis) ○ S. bongori - animal pathogen CAUSES S. choleraesuis Bacteremia S. typhimurium Enterocolitis/Gastroenteritis Salmonella Typhi: Enteric fever/ Typhoid fever S. paratyphi A - Stool w/ PEA soup S. paratyphi B appearance S. choleraesuis - Rose spots skin 8 Clinical Bacteriology Dr. San Diego | Lab SALMONELLA SHIGELLA KLEBSIELLA PNEUMONIAE Resembles Citrobacter E. coli Also known as Friedlander’s Bacillus Can cause community acquired pneumonia Motility + - (currant jelly like sputum) ○ S. pneumoniae = rusty colored H2S + - (+) encapsulated: (+) Neufeld Quellung Malonate + - On plated media: it can develop Mucoid Colonies (+) String test – positive (+) result is due to the Invasive - + mucoid colonies because of capsule LIA K/K K/A ○ Direct On Plate: lift colonies using loop; - para mahiwalay (+ decarboxylate (- decarboxylase observe for tenacious string (parang si Shigella kay test) test) Salmonella, you sinulid) inoculate it sa ○ Slide Method: in a slide,,get a loopful of LIA colonies and add KOH then mix, then lift using a loop to observe tenacious string K. oxytoca biochemically resembles K. RAPID LACTOSE FERMENTERS (KEE) pneumoniae but can be differentiated by doing indole test (+) GENUS ENTEROBACTER Members of this genus are normal intestinal flora INDOLE TEST but may cause opportunistic infections E. cloacae most predominant/frequent isolate K. oxytoca + E. aerogenes is now Klebsiella aerogenes K. pneumoniae - E. sakazakii is now Cronobacter sakazakii ○ This is known to produce Yellow pigment that intensifies at room GENUS ESCHERICHIA temperature Most significant species is E. coli ○ Significant because it can cause necrotizing colitis often due to powdered infant milk formula (infected are babies) ESCHERICHIA COLI Other Enterobacter spp (usually encounter in a.k.a Colon Bacillus lab): #1 cause of community acquired UTI ○ E. taylorae ○ Staphylococcus saprophyticus - ○ E. gergoviae regarded as the common cause of UTI in ○ E. cloacae young women/young females Normal GIT flora, found in normal stools. Use as the primary marker of fecal contamination GENUS KLEBSIELLA ○ Stool culture: all are (+) in E. coli Significant species is Klebsiella pneumoniae ○ Presence in water is not okay as it is a Other species: sign of fecal contamination ○ K. oxytoca- biochemically resembles K. ○ Performs water analysis in soluble water pneumoniae but can be differentiated by to check the presence of E. coli doing indole test (+) Presence of E.coli in water - it means that the ○ K. ozaenae- “purulent sinusitis” water is fecally contaminated ○ K. rhinoscleromatis- granuloma of the Cause of: nose and nasopharynx ○ Bacteremia mere presence of bacteria in the blood 9 Clinical Bacteriology Dr. San Diego | Lab ○ Sepsis number of bacteria in blood is overwhelming, already causing harm in patients can be a blood pathogen, intensive report if acquired ○ E.coli in blood - considered significant finding Virulence Factors: ○ Pili ○ Cytolysins (inhibit immune effector cells) and Aerobactin (chelate iron) TSI report is A/A ➔ because it is a Rapid Lactose Fermenters IMViC reaction is: ++-- It has 2 serotypes: ○ Nephropathogenic E. coli / Uropathogenic E. coli (NPEC/UPEC) – OTHER GENERA/SPECIES this is the serotype that causes UTI ○ Diarrheagenic/Enterovirulent E. coli – Genus citrobacter Genus that resembles these are the serotypes known to cause Salmonella ○ ONPG (+) diarrhea ○ LDC (-) 1. Enterotoxigenic Escherichia coli (ETEC) Citrobacter koseri Citrobacter species documented as a cause of 2. Enteroinvasive Escherichia coli outbreak of neonatal (EIEC) meningitis 3. Enteropathogenic Escherichia coli (EPEC) Prodigiosin Red Pigment produced by Serratia marcescens 4. Diffusely adherent Escherichia coli (DAEC) Genus Serratia Genus known to produce a 5. Enteroaggregative Escherichia number of enzymes such as DNase, Gelatinase; coli (EAEC) Lipase 6. Enterohemorrhagic Escherichia coli (EHEC) - most Hafnia Alvei Considered as a late significant lactose fermenter, with delayed citrate activity ➔ AKA: Verotoxic Escherichia coli (VTEC) Plesiomonas Can cause secretory & Shiga shigelloides diarrhea in AIDS patients with Inflammatory bowel toxin-producing disease Escherichia coli (STEC) Oxidase (+) Edwardsiella tarda Can cause wound infections & bacteremia, most common human species Pantoeae Triple Decarboxylase agglomerans Negative 10 Clinical Bacteriology Dr. San Diego | Lab Plesiomonas Triple Decarboxylase shigelloides Positive Serratia Triple Enzyme Positive ROUTINE IDENTIFICATION TESTS FOR FAMILY ENTEROBACTERIACEAE IMViC, Motility, Growth in TSI/KIA (detection of Lactose fermentation, H2S and gas production) Urease, ONPG, Decarboxylase Test, Mannitol Fermentation OTHER GENERA/SPECIES 11