Bacte 2 - GOODLUCK! PDF

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GRAM POSITIVE COCCI GRAM POSITIVE COCCI LIST OF GRAM-POSITIVE COCCI CATALASE POSITIVE ➢ Staphylococcus spp  S. aureus  S. epidermidis  S. lugdunensis  S. haemolyticus  S. saprophyticus ➢ Micrococcus spp CATALASE NEGATIVE ➢ Streptococcus spp  S. pneumoniae  S. mitis  S. bovis  S. pyogenes  S. agalactiae ➢ Enterococcus spp  E. faecium  E. faecalis CLASSIFICATION Mahon: Staphylococcus are members of the family Staphylococcaceae. Streptococcus and Enterococcus belongs to Streptococcaceae. Bailey: Staphylococcus are combined with Bacillaceae, Planococcaceae and Listeriaceae after the discovery of molecular analysis that Micrococcus and Staphylococcus are not closely related. GENERAL CHARACTERISTICS ➢ Micrococcus  Gram positive cocci in tetrads (4)  Under micrococcaceae

 Catalase positive, Oxidase negative  Common normal flora of skin, mucosa and oropharynx with no known virulence ➢ Staphylococcus  “staphle” - bunches of grapes  Seen as round, smooth, raised and glistening on solid media  Gram positive cocci in clusters  Young cocci stain strongly as gram positive but old specimen stains as gram negative once old. (Date of inoculation/ 48-72 hours routinely to get the young cocci)    

Catalase positive, oxidase negative Carbohydrates slow fermenters, produces lactic acid (DNAse) Facultative anaerobes, resistant to drying, heat (50C for 30 mins) Common normal flora which can be found on nasopharynx, skin and other mucosal surfaces

➢ Streptococcus  Gram positive cocci in chains    

Catalase negative Facultative anaerobes Most common encountered infections to man Catalase test (+ cocci) can separate negative and positive. Gives differentiation in staph and streph.

MICROCOCCUS Gram positive cocci in tetrads

MICROSCOPY

White, small colonies Positive Sensitive Resistant

CULTURE MICRODASE TEST BACITRACIN LYSOSTAPHIN

LIST OF COMMON Staphylococci COAGULASE POSITIVE ➢ ➢ COAGULASE NEGATIVE

STAPHYLOCOCCUS Gram positive cocci in clusters Creamy, medium colonies negative Resistant sensitive

➢ ➢ ➢ ➢ LIST OF COMMON Streptococci ALPHA HEMOLYTIC ➢ ➢ ➢ ➢ BETA HEMOLYTIC ➢ ➢ NON-HEMOLYTIC ➢ ➢ STAPHYLOCOCCUS SPP. / STAPHYLOCOCCACEAE 1. Staphylococcus aureus ➢ NF: Anterior nares, nasopharynx, skin, mucosal surfaces, perineal area ➢ MOT: Through surgical wound or abrasions, fomites, air or unwashed contaminated hands ➢ VIRULENCE FACTOR  Enterotoxin – causes food poisoning, non-invasive pathogen ▪ A-D: pathogenic tissue toxin ▪ E: less significant ( death ➢ LABORATORY DIAGNOSIS o G/S: intracellular and extracellular gram negative diplococcic o 1ml of CSF centrifuged at 1000g for 10 minutes o Blood agar: medium sized, gray and convex, encapsulated strains are mucoid, green tinge o

Neisseria lactamica ➢ The only Neisseria that uses lactose that can exhibit delayed lactose utilization Neisseria mucosa ➢ Large, very mucoid and adheres to the agar which can cause pneumonia in children Neisseria elongata ➢ Rod shaped that gives weakly positive catalase test Neisseria weaver ➢ Normal oral microbiota in dogs and can be found in man following dog bites Moraxella catarrhalis ➢ Opportunistic pathogen and can cause lower respiratory infections ➢ Third most common cause of acute otitis media and sinusitis in children ➢ CHOC agar: smooth, opaque, gray to white colonies with hockey puck appearance and remains intact if pushed ➢ Older colonies gives “wagon wheel” appearance ➢ Oxidase and catalase positive, assacharoloytic and differentiated by DNAse and Butyrate esterase reactions

GENERAL CHARACTERISTICS NEISSERIACEAE True pathogens 1. Salmonella enterica 2. Shigella spp 3. Yersinia spp

General characteristics

➢ ➢ ➢ ➢ ➢ ➢ ➢

Gran negative bacilli All are glucose fermenter All are normal Gl flora except Yersinia pestis All are catalase positive except Shigella dysenteriae Al are oxidase negative except Plesiomonas spp All are nitrate reducers except Photorhabdus and Xenorhabdus Antigenic structure o K antigen ▪ Capsular antigen ▪ Heat labile ▪ Vi: Salmonella spp o O antigen ▪ Somatic antigen ▪ Heat stable ▪ Antibodies to O antigens (IgM) o H antigen ▪ Located in flagella ▪ Denatured or removed by alcohol or heat ▪ H agglutinates with anti H antibodies (IgG)

OPPURTUNISTIC PATHOGEN Averyella dalhousiensis ➢ Isolated in blood and wound specimens ➢ Commonly mistakes as Salmonella enterica and Kluyvera ascorbata ➢ Diarrhea (magkakasundo siilang lahat) Citrobacter spp ➢ Resembles Salmonella spp but are ONPG positive and LDC negaive ➢ Grows on Simmon’s citrate agar ➢ Belongs to tribe salmonellae o Salmonella o Citrobacter o Arizonae ➢ Clinical significance o C. koseri ▪ Causes nursery outbreaks of neonatal meningitis and brain abscess o C. freundii ▪ Isolated in diarrhea stool cultures ▪ Associated with hospital acquired infection, UTI, pneumonia, intra-abdominal abscess ▪ Endocarditis among intravenous drugs abusers ▪ Deiffentiated to Salmonella by

• • •

Urease (+) LDC (-) Grows on KCN agar

Cronobacter spp ➢ LIST OF ORGANISMS o C. mustjensii o C. turicensis o C. malonaticus ▪ Adult infection o C. sakazakii ▪ Neonatal infections ▪ Metabolizes sialic acid found in breast milk, infant formula, mucis and gangliosides ▪ Yellow pigmentations ▪ Enterobacter sakazakii

Edwardsiella tarda ➢ Isolated from the environment and cold blooded and warm blooded animals ➢ E. tarda o Commonly isolated in man o Dofferentiated with E. coli by ▪ H2S production (+) ▪ Lactose fermentation (-) ➢ Laboratory result o Urea (+) o LDC (-) o Indole (+) o Does not grow on citrate agar

GRAM NEGATIVE BACILLI

LABORATORY DIAGNOSIS Enterobacteriaceae IDENTIFICATION SCHEMES

Gram stain > Mac Conkey Medium > Oxidase Test > Biochemical tests and Carbohydrate fermentation tests > Enzyme detection GENERAL CHARACTERISTICS ➢ All are glucose fermenters ➢ All are nitrate reducers EXCEPT o Photorhabdus o Xenorhabdus ➢ Motile by flagella at 37C EXCEPT o Shigella – room temp o Klebsiella room temp o Yersinia - Cold temp (4C) ➢ All are catalase positive EXCEPT: o Shigella dysenteriae ➢ Oxidase negative EXCEPT: o Plesiomonas spp ➢ PRIMARY PATHOGENS o Salmonella enterica o Shigella spp o Yersinia spp ➢ CPON: enterobacteriaceae ➢ CNON: Shigella dysenteriae ➢ CPOP: Plesiomonas

LACTOSE FERMENTATION Rapid Lactose Fermenters (EKE) (24 hrs) ➢ Escherichia spp ➢ Klebsiella spp ➢ Enterobater spp Late Lactose Fermenters (CHSYSS) (48 hrs) ➢ ➢ ➢ ➢ ➢ ➢

Citibacter spp Hafnia spp Serratia spp Yersinia spp Salmonella arizonae – 48 HRS incubation Shigella sonnei - 48 HRS incubation

Non-Lactose Fermenters (SSPPMM) (72 hrs)

➢ ➢ ➢ ➢ ➢ ➢ ➢

Salmonella spp Shigella spp Proteus spp Providecia Morganella spp Edwardsiella spp Erwinia spp

MICROSCOPY ➢ Non-definitive test ➢ Gram negative bacilli or coccobacilli ➢ Capsulated EXCEPT: o E. coli o Klebsiella spp o Salmonella spp

MACROSCOPIC/CULTURE MEDIA A. Non-Selective Media 1. Sheep Blood Agar 2. Chocolate Agar B. Selective/Differential Media 1. • • • • • •

Mac Conkey (MAC) Agar Selective: Gram (-) enteric bacilli Differential: Lactose fermentation CHO: lactose Inhibitor: crystal violet, bile salts pH IND: neutral red RESULTS o LF: pink colonies o NLF: colorless colonies

***has color change if they can ferment lactose 2. • • • •

Eosin Methylene Blue (EMB) Agar Selective: Gram (-) enteric bacilli Differential: Lactose fermentation CHO: Lactose, Sucrose INH: Eosin, methylene blue dyes

• •

IND: precipitated eosin and methylene blue RESULTS o LF: pink to purple colonies o NLF: colorless colonies

*** green metallic sheen – E. coli •

SPECIAL CHARACTERISTICS o E. coli – pink purple colonies with green metallic sheen (lactose fermenter) o Klebsiella – pink mucoid colonies o Enterobacter – pink colonies with dark centers, fish eye colonies. o _________ - School appearance

HektoenEnteric Agar (HEA) Selective: Gram (-) enteric bacilli Diffrential: lactose fermentation, H2S CHO: Lactose, Sucrose, salicin INH: bile salts pH IND: bromthymol blue (ralated in lactose fermentation) H2S IND: ferric ammonium citrate (Fe NH4 citrate) (black precipitate) RESULTS: • LF, H2S (-): yellow colonies, no black centers • LF, H2S (+): yellow colonies with black centers • NLF, H2S (hydrogen sulfide) (-): green colonies, no black centers • NLF, H2S (+): green colonies with black centers ➢ *** H2S positive – Salmonella (green NLF), proteus (green NLF), Citrobacter (yellow LLF), edwardsiella (green NLF), (black precipitate) 3. ➢ ➢ ➢ ➢ ➢ ➢ ➢

C. Salmonella and Shigella Isolation ➢ Salmonella-Shigella agar (SSA) • Selective: Salmonella, Shigella • Differential: presence of H2S • CHO: Lactose • INH: brilliant green, bile salts • pH IND: neutral red • H2S IND: Ferric ammonium citrate • RESULTS: o NLF, H2S (+) Salmonella

o NLF, H2S (-) Shigella ➢ Xylose Lysine Desoxycholate (XLD) Agaro • Selective: Salmonella, Shigella • Differential: Presence of H2S • CHO: xylose, lactose, sucrose • INH: sodium desoxycholate • pH IND: phenol red • RESULTS: o NLF, H2S (+) o NLF, H2S (-)

D. Other Culture Media ➢ Sorbitol Mac Conkey (SMAC) Agar o MAC with D-sorbitol o E. coli consumes sorbitol EXCEPT E. coli 0157:H7 strain ➢ Bismuth Sulfite Agar o Detection of Salmonella in endemics or epidemics ➢ Cefsulodin Irgasan Novobiocin Agar o Selective for Yersinia (bull’s eye) ➢ Gram negative broth o Enrichment medium for enteric pathogens ➢ Selenite F broth o Enrichment for Salmonella spp

BIOCHEMICAL TESTS A. Routine Biochemical Tests

1. Triple Sugar Iron o Initial step in identifying Enterobacteriaceae o Carbohydrate fermentation is indicated by the change of pH based on color changes (alkaline to acid) o CHO: • Lactose (10 parts/1%) • Sucrose (10 parts/1%) • Glucose (1 part/0.1%) o pH IND: phenol red o H2S IND: ferrous sulfate o Sulfur source: sodium thiosulfate o RESULTS:  Yellow – acidic (A)  Red – alkaline (K) RESULT A/(A) H2S (-) “ “ “ K /(A) H2S (+)

K/(A) H2S (-)

K/A H2S (-)

INTERPRETATION Slant: yellow Butt: yellow Gas: With space H2S: no blackening Slant: red Butt: yellow Gas: with space H2S: blackening Slant: red Butt: yellow Gas: with space H2S: no blackening Slant: red Butt: yellow Gas: no space H2S: no blackening

2. Lysine Iron Agar (purple color)

ORGANISM Escherichia Klebsiella Enterbacter Salmonella Proteus Citrobacter Edwardsiella Morgenella Serretia Providencia Plesiomonas shigella Yersinia

o Principle: determines the ability of organism to deaminate (slant) or to decarboxylate (butt) lysine (***LDA: LYSINE deamination/ LDC: lysine decarboxylation) (LDA – saw is slant) (LDC – butt) (cadaverine – neutralize glucose) o Differentiates non lactose fomenters (NLF) o pH IND: Bromcresol purple o H2S IND: ferric ammonium citrate o Mechanism of LDA and LDC  In deamination of lysine, a compound is formed that forms a burgundy color in the presence of H2S indicator, coenzyme and flavin mononucleotide  Cadaverine is formed if the organism has lysine decarboxylase. This cadaverine can neutralize glucose fermentation returning an acidic environment back to the original alkaline state (alkaline-acid-alkaline) o RESULTS:  Lysine deaminase (LDA) ▪ Red (+) - acidic ▪ Purple (-) if alkaline  Lysine decarboxylase (LDC) ▪ Purple (+) - alkaline (neutralize) ▪ Yellow (-) - acidic (no neutralization) RESULT

INTERPRETATION

K/K H2S (+) LDA: - LDC: +

Slant: purple Butt: purple H2S: blackening Slant: purple Butt: yellow H2S: no blackening Slant: red Butt: yellow H2S: No blackening

K/A H2S (-) LDA: - LDC: +

R/A H2S (-) LDA: + LDC: -

3. IMViC Reaction ➢ Indole o Detects the enzyme trytophase o MED: Tryptone broth/SIM o IND: kovac/ ehrlichs rgt o RESULT: red ring (+) • E. coli

ORGANSIM

salmonella

Shigella Proteus Providencia Morganella

• P. vulgaris ➢ Methyl red o Indicates mixed acid fermentatin o IND: methyl red o RESULT: red color + • E. coli ➢ Vogues Proskauer o Indicates butylene glycol fermentation or 2,3 butanediol fermentation o End product: acetin o IND: 40% KOH, alpha naphtol o RESULT: red color + (alk) • KES-H group *** immense relationship: Methyl red: + / Vogues P - (or vice versa) ➢ Citrate o Ability to utilize citrate as single carbon source o MED: Simmon citrate agar o Nitrogen source: NH2 o PH IND: Bromthymol blue o RESULT: Blue + • Enterobacter • Klebsiella

Escherichia + + - Klebsiella - - + + Enterobacter - - + + Salmonella - + - + Shigella - + - Proteus mirabilis - + - v Proteus vulgaris + + - + Providencia + + - + Morganella + + - Citrobacter - + - + Hafnia – v v -

Serratia – v + + Yersinia v + - Edwardsiella + + - Klebsiella oxitoca + - + + *** v – variable reaction/ indefinite

B. Other Biochemical Tests ➢ Sulfide Indole Motility o Sulfide: H2S production o Indole: Red ring appearance o Motility: Dispersion of organism ➢ Motility Indole Ornithine o Ornithine decarboxylation: formation of putrescine, purple + o Indole – red ring appearance o Motility – dispersion of organism ➢ Urea Hydrolysis/Urease Test o Detects enzyme urease o MED: Christensen’s urea agar o pH IND: phenol red o RESULT: ▪ Red: (+) alkaline, K ▪ Yellow: (-) acidic, S o After 2-4 hours: rapid urease o >4 hours: slow urease

UREASE HYDROLYSIS ➢ Rapid Urease Producers o Proteus o Providencia o Morganella ➢ Late Urease Producers o Citrobacter o Klebsiella o Enterobacter o Yersinia o Serratia

➢ ONPG Test o Rapid test to detect beta galactosidase or the LLF o Breakdowns ONPG to galactose and nitrphenol o RESULT ▪ LLF: yellow (+) ▪ Other salmonella (-) ➢ Nitrate Reduction o Determines the ability of organism to reduce nitrate to nitrite by adding sulfanilic acid and a-napthylamide. o RESULT ▪ Red: water soluble (+) ▪ Confirmation with zinc dust for negative results ➢ Decarboxylase Test/Moeller’s Method o Ability of organism to decarboxylate amino acid forming amine o Requires acid pH and anaerobic environment o Three carboxylase broths o pH IND: bromcresol purple o RESULT: (+) returning to alkaline

AA

enzyme

End prod.

Bacteria

Arginine Lysine

ADH LDC

Citruline Cadaverine

Ornithine

ODC

Putrescine

E. cloacae Klebsiella E. aerogenes E. aerogenes E. cloacae

➢ Phenylalanime deaminase (PAD) Test o Deaminase test: either lysine or tryptophan is used o Ability of organism to deaminate forming phenylpyruvic acid o RESULT: (+) green slant ▪ Proteus spp ▪ Providencia spp ▪ Morganella spp

➢ Gelatin Liquefaction Test o Determine the ability of the bacteria to produce proteases that hydrolyzes gelatin and liquefies solid gelatin medium releasing amino acids o RESULT: liquefaction

▪ ▪

Serratia spp (Serratia macesens) Proteus spp (Proteus vulgaris)

➢ MUG Testo o Detects beta-d-glucoronidase o Fluoresces blue under UV lights o RESULT: ▪ All E. coli EXCEPTE. coli 0157:H7

Additional: Semiautomated tests API test kits...