Gram Negative Bacilli Flashcards
(93 cards)
1
Q
Oxidase Test
A
- Positive - psuedomonas, Neisseria, Vibrio (purple colour)
- negative - enterobacteriaceae
2
Q
Fermentative vs Oxidative
A
- Fermentative - acid in aerobic and anaerobic conditions (enterobacteriaceae)
- oxidative - acid onl in aerobic conditions (psuedomonas)
- test: O-F glucose media
3
Q
MacConkey Agar Growth (Gram negative only)
A
- lactose fermenters: pink (e.coli, klebsiella)
- non-fermenters: colourless (salmonella, shigella)
4
Q
Fastidious bacteria
A
- require choc agar (x and v factors)
- example: heamophilus, neisseria
5
Q
MacConkey Agar
A
- primary medium for gnb differentiation
- base: peptone/tryptone
selective agents:- bile salts and crystal violet - inhibit gpb
differential agents: - lactose - fermentation indicator
- neutral red - pH indicator
Lactose fermenters - pink colonies (e.coli, Klebsiella)
Non-lactose fermenters - colourless colonies (salmonella, shigella)
- bile salts and crystal violet - inhibit gpb
6
Q
Xylose Lysine Desoxycholate (XLD) Agar
A
- Used for: Isolation & identification of Salmonella and Shigella in GIT samples.
- Selective agent: Desoxycholate → Inhibits non-enteric GNB & GPC.
Differential agents: - Lactose, Sucrose, Xylose → Fermentation indicators.
- Phenol red → pH indicator.
- Lysine → Helps differentiate Salmonella.
- Sodium thiosulfate & Ferric ammonium citrate → Detect H₂S production.
- Shigella → Pink colonies (No fermentation).
- Salmonella → Pink colonies with black centers (Ferments sugars, then decarboxylates lysine, producing H₂S).
7
Q
Primary Test: Oxidative/Fermentation (O-F) Test
A
- Semi-solid tryptone-based medium with single carbohydrate (e.g., glucose).
- pH indicator detects acid production
Test Setup:
1. Two tubes inoculated with the same bacterium.
2. One tube covered with oil → Anaerobic conditions.
3. One tube left open → Aerobic conditions.
Interpretation - Fermentative → Acid production in both tubes (e.g., Enterobacteriaceae) - - this bacteria is able to use glucose anerobically making the pH to go down and causing it to be yellow.
- Oxidative (Non-Fermentative) → Acid only in open tube (e.g., Pseudomonas) - will stay green
- Non-Saccharolytic → No color change in either tube (e.g., Alcaligenes).
8
Q
Secondary Test: Urease Test
A
- Purpose: Detects urease enzyme, which hydrolyzes urea into ammonia, water, and CO₂
- Test Setup:
- Medium: Urea-containing media with a pH indicator (phenol red).
- Reaction: Urease-positive bacteria increase pH, turning media pink
- Interpretation:
- Urease Positive → Pink color (Proteus, Helicobacter, Klebsiella).
- Urease Negative → No color change / yellow (Salmonella, E. coli).
9
Q
Secondary Test: Indole Test (IMViC)
A
- Purpose: Detects tryptophanase enzyme, which breaks down tryptophan into indole
Test Setup: - Medium: Tryptophan-containing broth.
- Reagent: Kovacs’ reagent (p-dimethylaminobenzaldehyde).
- Reaction: Indole reacts with Kovacs’ reagent, producing a red color at the surface.
Interpretation: - Indole Positive → Red layer (E. coli, Proteus).
- Indole Negative → No color change / yellow (Klebsiella, Enterobacter)
10
Q
Secondary Test: Methyl Red (MR) Test
A
- Purpose: Detects mixed acid fermentation, which lowers pH below 4.4
Test Setup: - Medium: Peptone, glucose, and phosphate buffer broth.
- Incubation: 48+ hours.
- Reagent: Methyl red (MR) indicator (2 drops added)
Interpretation: - MR Positive → Red color (pH < 4.4) → E. coli.
- MR Negative → Yellow color (pH > 6.0) → Enterobacter.
- Orange → Inconclusive, requires reincubation.
11
Q
Secondary Test: Voges-Proskauer (VP) Test
A
- Purpose: Detects acetoin & 2,3-butanediol fermentation, differentiating MR-negative bacteria
Test Setup: - Same tube as MR test (but performed separately).
Reagents: - Alpha-naphthol (600 µL, VP Reagent 1).
40% KOH (200 µL, VP Reagent 2)
Reaction: - Acetoin is oxidized in alkali + oxygen to form diacetyl, producing a red color
Interpretation: - VP Positive → Red color (Enterobacter, V. alginolyticus).
- VP Negative → No color change / yellow (E. coli, V. parahaemolyticus).
12
Q
Secondary Test: Citrate Test
A
- Purpose: Tests the ability of bacteria to use citrate as the sole carbon source and ammonium as the sole nitrogen source.
Test Setup: - Medium: Citrate agar with bromothymol blue as a pH indicator.
- Reaction: Alkaline products from citrate utilization turn the medium blue. Interpretation:
- Citrate Positive → Blue color (Klebsiella pneumoniae).
- Citrate Negative → Green color (E. coli).
13
Q
Secondary Test: Triple Sugar Iron (TSI) Agar
A
Purpose: Differentiates GNB based on:
1. fermentation of glucose, lactose or sucrose.
2. gas (co2) production
3. hydrogen sulfide (H2S production)
Test Composition:
- 1- parts lactose, 10 parts, sucrose, 1 part glucose
- peptone: protein source for non-fermenters
Indicators:
- phenol red - detects acid production (pH change)
- ferrous sulfate - detects H2S production (black precipitate
Intepretation:
- glucose fermentation only: red slant/yellow but
- acidic butt from glucose fermentation
- slant reverts to pink due to aerobic oxidation
- lactose or sucrose fermentation - yellow slant/yellow but (E.coli and Vibrio spp.)
- acid overwhelms any alkaline oxidation
- no fermentation - red slant/red but
- no acid production
- H2S production - black precipitate
- requires acidic environment; H2S reacts with ferrous sulfate.
- gas production - cracks/bubbls in the medium.
14
Q
APIe & API 20E System
A
Used for the biochemical identification of Enterobacteriaceae and other GNB.
**🧪 API 20E System:
20 mini biochemical tests in microtubes.
Tests include: Fermentation, enzyme activity, and amino acid metabolism.
Incubation: 24 hours at 35-37°C.
Result Interpretation:
Color changes based on metabolic reactions.
A 7-digit profile number is generated from positive/negative results.
Compared to a database for species identification.
🧪 APIe (Automated API System):
Uses API 20E but with automated reading & analysis.
Reduces human error and speeds up interpretation.
Often used in clinical microbiology laboratories.
✅ Advantages:
Quick & standardized identification of Enterobacteriaceae & other GNB.
Reliable & reproducible results with a large reference database.
⚠️ Limitations:
Requires pure culture.
Some rare species may not be in the database, requiring confirmation tests.
15
Q
Enterobacteriales – Description
A
Definition: The “enteric” Gram-negative bacilli (GNB).
🔬 Key Characteristics:
Non-spore forming GNB.
Catalase positive (except Shigella dysenteriae).
Oxidase negative (except Plesiomonas shigelloides).
Ferments glucose, with or without gas production.
Facultative anaerobes.
Most grow well on MacConkey agar.
🦠 Clinically Relevant Genera:
Intestinal Pathogens:
Escherichia, Shigella, Salmonella.
Nosocomial Pathogens (Non-GIT diseases):
Klebsiella, Enterobacter, Citrobacter, Serratia, Proteus/Morganella.
16
Q
Enterobacteriales – Clinical Significance
A
🔹 Very Common Pathogens in various infections:
🦠 Major Clinical Manifestations:
Gastrointestinal Infections – Salmonella, Shigella, E. coli.
Urinary Tract Infections (UTIs) – Klebsiella, Proteus, E. coli.
Wound and Respiratory Infections – Klebsiella, Enterobacter, Serratia.
🔍 Modes of Transmission:
Poor sanitation → Increases risk of primary GIT infections.
Nosocomial (hospital-acquired) infections:
Crowded environments promote close contact & transmission.
Iatrogenic transmission via medical procedures.
17
Q
Enterobacteriales – Identification
A
🧪 Identification Tests:
Gram stain → Gram-negative bacilli.
Catalase test → Positive (except S. dysenteriae).
Oxidase test → Negative (except P. shigelloides).
Glucose fermentation → Positive (facultative anaerobes).
MacConkey Agar → Most grow; lactose fermentation differentiates species.
18
Q
General Description GIT Pathogens - Escherichia Coli
A
Includes Escherichia, Salmonella, and Shigella
Escherichia: Five species, E. coli most common
Found in human & animal GIT
E. coli is an indicator of fecal contamination in food & water
Type species for Enterobacteriaceae—other members classified based on relatedness to E. coli (e.g., 16S rRNA)
19
Q
Clinical Significance - E.coli
A
Normal flora of GIT but can cause:
UTI & PID (if transmitted to UT/G-UT; requires fimbriae)
Diarrheal diseases (diarrheagenic E. coli)
Pathogenic Strains & Diseases:
STEC (EHEC): Shiga toxin-producing → dysentery, HUS, renal failure
ETEC: Enterotoxigenic → traveler’s diarrhea, affects children
EPEC: Enteropathogenic → pediatric diarrhea
EIEC: Enteroinvasive → invades colon, causes dysentery
20
Q
E. coli Identification
A
Difficult to differentiate pathogenic from commensal strains
Biochemical Characteristics:
Lactose fermenting
Indole (+), Methyl Red (+)
Voges-Proskauer (-), Citrate (-)
TSI: K/A or A/A, sometimes gas production
Motile, Urease (-)
STEC O157 Identification:
Sorbitol MacConkey (SMAC) agar → Non-fermenter of sorbitol
Confirmed by specific antisera
21
Q
Shigella – General Description & Habitat
A
Four serogroups/species:
S. dysenteriae (A) – Type strain
S. flexneri (B)
S. boydii (C)
S. sonnei (D)
Non-motile, non-active
Human pathogen, transmitted via faeces-contaminated water
22
Q
Shigella - Clinical Importance
A
Causes Shigellosis
Symptoms: Bloody diarrhea, cramps, abdominal pain
Low infectious dose – highly contagious
23
Q
Shigella - Identification
A
Similar to non-active E. coli
Lactose non-fermenter
Indole mostly negative, MR positive
VP negative, citrate negative
Non-motile
TSI: K/A (Alkaline/Acid, no gas, no H₂S)
XLD: Pink colonies, no H₂S production
Urease negative
Species differentiation: Serotyping, amino acid & carbohydrate utilization
24
Q
Salmonella - Overview
A
Two species:
S. enterica (Subspecies I–VI) – Includes human pathogens
S. bongori
S. enterica (subspecies I): Major human pathogen
2435 serotypes, 1453 in serogroup 1
Serogroup names are not italicized
25
Salmonella - Clinical Importance
Typhoid fever (systemic disease)
Diarrhea, foodborne illness
Transmitted via feces-contaminated water, food, and animals
26
Salmonella - Identification Features
IMViC: -,+,-,+
Lactose non-fermenter
Xylose and/or sucrose positive
Lysine decarboxylation restores neutral pH
H2S positive → XLD agar: pink colonies with black centers
Motile
TSI: K/A, H2S positive, with or without gas
Urease negative
Confirmed via serotyping (antisera) and further biochemical tests
27
Non-GIT Pathogens Overview
Includes Klebsiella, Enterobacter, Citrobacter, and Serratia
Found in:
GIT (high carriage after antibiotics/hospital stays)
Skin, mucous membranes, nasopharynx
Widespread in the environment
28
Clinical Importance of Klebsiella, Enterobacter, Citrobacter, and Serratia
Nosocomial infections (hospital-acquired)
Transmission: Mostly via hands, person-to-person, equipment
Common Infections:
Surgical wound infections (Klebsiella & Enterobacter—common intra-op causes)
Sepsis (less common than GP but high mortality)
UTIs (with E. coli, especially in catheterized patients)
Pneumonia
29
Biochemical Identification of Genera
Klebsiella:
Indole (-) (K. oxytoca is +)
Methyl Red (-)
Voges-Proskauer (+)
Citrate (+)
Non-motile
Citrobacter:
Indole (-)
Methyl Red (+)
Voges-Proskauer (-)
Citrate (+)
Variable motility
Enterobacter:
Indole (-)
Methyl Red (-)
Voges-Proskauer (+)
Citrate (+)
Motile
Some species (e.g., E. agglomerans) produce yellow pigment
Serratia:
Indole (-)
Methyl Red (-/+)
Voges-Proskauer (+)
Citrate (+)
Motile
S. marcescens produces red pigment
Escherichia coli:
Indole (+)
Methyl Red (+)
Voges-Proskauer (-)
Citrate (-)
Motile
30
Edwardsiella tarda – Clinical Significance
GIT pathogen
Causes disease similar to Salmonella
31
Edwardsiella tarda – Identification
Biochemically similar to Salmonella
Indole (+) (key differentiating factor)
32
Hafnia alvei – Clinical Importance
Nosocomial pathogen
Possible GIT pathogen
Causes Salmonella-like disease
33
Hafnia alvei – Identification
Biochemically almost identical to Salmonella
RT Methyl Red (-)
RT Voges-Proskauer (+) (when incubated at 18–22°C)
34
Yersinia spp. – Overview
Separated from Pasteurella in 1944
Small, non-spore-forming Gram-negative bacilli (NSF GNB)
Motility:
Most species motile at 22–30°C
Not motile at 37°C
Yersinia pestis is non-motile
10 species, 3 pathogenic to humans:
Y. pestis, Y. enterocolitica, Y. pseudotuberculosis
35
Yersinia pestis – Clinical Significance
Natural Habitat: Rodents and their arthropod ectoparasites
Transmission:
Fleas or direct contact with rodents
Human-to-human transmission (more intense disease)
Plague Types:
Bubonic: Swollen lymph nodes (buboes)
Pneumonic: Severe lung infection, highly contagious
Septicemic: Blood infection, high mortality
Virulence factors "switched on" in humans
Intracellular replication in lymph nodes
36
Yersinia enterocolitica & Yersinia pseudotuberculosis
Natural Habitat: Found in animals, humans, and the environment
Y. pseudotuberculosis mainly in rodents
Pigs are an important reservoir for human infections
Y. enterocolitica serotypes O:3 and O:9
37
Yersinia enterocolitica & Yersinia pseudotuberculosis – Clinical Disease
Infects gut lymphatic tissue
Symptoms:
Pain mimicking appendicitis
Watery diarrhea
Invasive disease
Complication:
Reactive arthritis (in HLA-B27 patients)
38
Yersinia Identification
Y. pestis: Identified through clinical association with plague
Microscopy:
Bipolar staining (resembles safety pins)
Culture:
Selective agar: CIN (Cefsulodin-Irgasan-Novobiocin)
Pinpoint colonies at 24 hours
Best growth at 25°C
Y. enterocolitica has bull’s-eye colonies
39
Yersinia Biochemical Identification
Motility:
All motile except Y. pestis
Metabolically active at 25°C
IMViC Pattern:
Yersinia enterocolitica: -, +, +, -
Fermentative metabolism (Sugar utilization varies)
40
Acinetobacter – Overview
Order: Pseudomonadales (not Enterobacteriales)
Morphology: Oxidase negative, non-fermentative, non-pigmented Gram-negative coccobacilli
Species: Over 50 species in the genus
Habitat:
Found in wet environments like soil, ponds, water treatment plants, and fish farms
Common in wastewater
Pathogenic Species:
A. baumannii (most common pathogen)
A. calcoaceticus
A. lwoffii
Transmission:
Spread through environmental surfaces and transient colonization of healthcare workers’ hands
41
Acinetobacter – Clinical Aspects
Infecting Species:
A. baumannii is the primary pathogen (around 10 species infect humans)
Low virulence but common in hospital infections
Cultured from:
Sputum or respiratory secretions
Wounds
Urine (high fluid content)
Prefers:
Aquatic environments
Hospital irrigation and intravenous solutions
Key Challenge:
Distinguish between colonization and infection
Resistance:
A. baumannii is inherently resistant to multiple antibiotics
Environmental strains often harbor antibiotic resistance mechanisms, including carbapenemases and extended-spectrum β-lactamases
42
Acinetobacter – Identification
Gram Stain: May retain crystal violet stain and appear Gram-positive
Growth:
Grow on Blood Agar (BA) and MacConkey Agar (MAC)
May be β-haemolytic on BA
Motility: Non-motile
Fermentation: Non-fermentative
Oxidase: Negative (not Enterobacteriaceae)
Catalase: Positive
Indole: Negative
43
Oxidase Positive, Gram-Negative Rods Overview
Grow on MacConkey agar
Key Organisms:
Pseudomonas
Burkholderia
Vibrio
Aeromonas
Habitat:
Prefer wet environments
Not part of normal flora in healthy humans
Can be transient flora in hospital patients
Potential nosocomial pathogens
44
Oxidase Positive, Gram-Negative Rods – Metabolism
Carbohydrate Use:
Either do not ferment carbohydrates or degrade them through pathways other than fermentation
Key Pathways:
These organisms generally don’t rely on fermentation for energy production
45
Pseudomonads – Overview
Habitat:
Found in soil, vegetation, and water
Found in moist hospital environments (e.g., food, cut flowers, sinks, toilets, floor mops)
Can be found in respiratory therapy & dialysis equipment, disinfectant solutions
Species: ~140 species, mostly saprophytic
Human Interaction:
Uncommon to persist in healthy humans
Opportunistic pathogens in hospitalized or immunocompromised patients
Won't infect uncompromised, intact tissues
46
Pseudomonas aeruginosa – Clinical Significance
Opportunistic Pathogen:
Causes urinary tract infections
Respiratory infections (especially in cystic fibrosis patients)
Dermatitis, soft tissue infections, and bacteremia
Systemic infections in severely immunocompromised patients (e.g., burns, cancer, AIDS)
Nosocomial Pathogen:
Among the top 4 most commonly isolated nosocomial pathogens
~25% of all hospital-acquired Gram-negative infections
47
Pseudomonas aeruginosa – Culture and Morphology
Colonial Morphology:
Often β-haemolytic
Spreading, flat, mucoid colonies
May have a metallic sheen
Green pigment (Pyoverdin-yellow + Pyocyanin-blue)
Sweet, fruity odor
MacConkey Agar (MAC): Non-lactose fermenter (NLF)
Identification:
Oxidase positive
NLF with green pigment
Strong growth on Cetrimide agar
48
Pseudomonas aeruginosa – Pigments
Mucoid Strains:
Capsule presence
Common in cystic fibrosis patients
Pigments Produced:
Pyocyanin (blue)
Catalyzes toxic oxygen radical production, leading to tissue damage and inflammation
Pyoverdin/Fluorescein (yellow)
Pyorubin (red/brown)
Cetrimide agar: Acts as a cationic detergent, enhancing Pseudomonas resistance
49
Pseudomonas – Identification
Gram Stain: Straight or slightly curved Gram-negative bacilli
Motility: Highly motile via polar flagella
O-F Test: Oxidative
Oxidase Test: Positive (used to differentiate from Enterobacteriaceae)
Blood Agar: β-haemolytic
Cetrimide Agar: Growth on Cetr`imide
50
Burkholderia – Overview
History: Originally grouped with Pseudomonas (P. mallei and P. pseudomallei)
Genus Change: Now classified as Burkholderia
Cepacia Complex: 9 sub-species, associated with specific individuals (e.g., cystic fibrosis patients)
Human Diseases: Cause specific human diseases (e.g., glanders, melioidosis)
51
Burkholderia mallei – Clinical Significance
Disease: Causes glanders in horses
Survival: Cannot survive outside its host (due to genome reduction)
Primary Infection Site: Lungs
Transmission to Humans: Through contact with infected horses
52
Burkholderia pseudomallei – Clinical Significance
Agent of Melioidosis: Tropical disease affecting humans and mammals
Opportunistic Pathogen: Typically affects immunocompromised individuals
Transmission:
Skin wounds
Aerosols
Contact
Ingestion (rare)
53
Burkholderia pseudomallei – Culture
MacConkey Agar: Grows as a lactose fermenter (LF)
Ashdown Agar: Selective medium developed in 1979 by Les Ashdown
Contains crystal violet and gentamicin as selective agents
Forms large purple colonies after 48 hours
Dry, wrinkled colonies with neutral red and crystal violet uptake
Clinical Suspicion: Based on clinical presentation
Culture: Blood, pus, sputum cultures
Containment: PC3 required for isolation
Gram Stain: Gram-negative, oxidase positive
Ashdown Agar: Growth after 48 hours
Serology: Limited value (often negative early in disease)
IHA Titre > 1:40 indicates infection
PCR: No validated PCR assay yet
54
Family Vibrionaceae – Overview
Type Genus: Vibrio
Other Genera: Aeromonas (own family), Plesiomonas (now Enterobacteriaceae), Campylobacter, Helicobacter
Characteristics:
Motile
Aerobic/facultatively anaerobic
Catalase and oxidase positive
Fermentative, no gas produced
NaCl may be required for growth
Not normal human flora
Associated with GIT disease
55
Genus Vibrio – Description
Species: Over 30 species
Natural Habitat: Estuarine and marine environments globally
Common Hosts: Surfaces and intestinal tracts of marine animals
Diseases: Caused by contamination through food or water
Clinically Significant Species:
V. cholerae, V. parahaemolyticus, V. vulnificus
Other species can be opportunistic
56
Vibrio – Clinical Significance
Intestinal Infections:
Cholera (most well-known disease), caused by V. cholerae & V. parahaemolyticus
Transmitted via contaminated food or water
V. vulnificus isolated from feces of patients with diarrhoea, especially those consuming raw oysters
Extra-intestinal Infections:
Wound infections
Septicaemia
V. cholerae can cause both
57
Vibrio – Morphology
Cellular Morphology:
Small, comma-shaped rods
Shooting star motility
Colonial Morphology:
Grows on blood agar (BA)
β-haemolytic
Non-lactose fermenter (NLF) on MacConkey agar (MAC)
Isolate on TCBS selective agar
58
Vibrio cholerae – Clinical Presentation
Survival in Stomach:
Cannot survive acidic environment; high infectious dose (~103-106 cfu) required
Water or food dilutes stomach acid
Pathogenesis:
Adheres to epithelial cells in the small intestine
Produces enterotoxin
Leads to massive diarrhoea (rice water stools)
Non-O1/0139 Strains: Do not produce enterotoxin
59
TCBS Agar
Selective and Differential Agar for Vibrio species
Key Ingredients:
Thiosulphate: Provides sulfur source
Citrate: Detects H2S production
Bile salts: Inhibit Gram-positives
Sucrose: Fermentable carbohydrate source
Colony Appearance:
V. cholerae forms yellow colonies if sucrose is fermented
Useful for isolating V. parahaemolyticus & V. cholerae
60
Vibrio cholerae - Description
gram negative, curved rod-shaped bacterium
aquatic environments like freshwater and brackish water
cannot survive in highly acidic stomach environments; requires high infectious dose (103-106 CFU).
Adheres to epithelial cells of the small intestine and produces enterotoxin.
Causes "rice water" diarrhea due to massive fluid loss.
61
Vibrio cholerae - Clnical Significance
O1 serogroup responsible for widespread cholera outbreaks.
Non-O1 Strains: May cause cholera-like illness but do not trigger epidemics.
Subgroups: O1 isolates include serotypes Ogawa, Inaba, and rare Hikojima (hybrid subtype).
Biotypes: Classical and El Tor; Australian El Tor strongly β-haemolytic.
62
Vibrio cholerae
Growth Media: Produces yellow colonies on thiosulfate-citrate-bile salts-sucrose (TCBS) agar due to sucrose fermentation.
Biochemical Properties: El Tor biotype is β-haemolytic (Australian isolates) and has distinct epidemiological patterns.
Serotyping: Based on somatic O antigens (130+ serogroups).
Epidemiological Use: Differentiation of serotypes and biotypes helps in understanding cholera outbreaks.
63
Vibrio parahaemolyticus - Description
Organism: Vibrio parahaemolyticus (Gram-negative, curved rod-shaped bacterium).
Habitat: Natural inhabitant of temperate marine environments.
Growth Media: Green colonies (sucrose non-fermenting) on TCBS agar; non-haemolytic on blood agar (BA).
Halophilic: Requires 1% NaCl for growth.
64
Vibrio parahaemolyticus - Clinical Signficance
Disease: Associated with eating contaminated shellfish (not indicative of fecal contamination).
Symptoms: Self-limiting gastroenteritis; ranges from mild diarrhea to cholera-like illness.
Virulence Factors: Cytotoxin damages intestinal cells, leading to blood and WBC presence in diarrhea; haemolysin and enterotoxin also play roles.
Incidence: Responsible for 50-70% of foodborne enteritis cases.
Extra-Intestinal Infections: Can occur in wounds or ears exposed to contaminated seawater.
Bile Sensing: Uses bile as an environmental cue to upregulate virulence genes during infection.
65
Vibrio vulnificus - Description
Organism: Vibrio vulnificus (Gram-negative, curved rod-shaped bacterium, lactose-positive).
Habitat: Marine environments; halophilic (requires salt for growth).
Virulence: Highly virulent, associated with severe and potentially fatal infections.
66
Vibrio vulnificus - Clinical Significance
Wound Infections: Rapid, aggressive infections following exposure to contaminated seawater (e.g., oyster cuts).
Can cause tissue necrosis, systemic signs, and death within 48 hours.
Septicaemia: Acquired by consuming contaminated raw shellfish, particularly oysters.
Mortality rate up to 50% if untreated.
Death often due to septic shock.
67
Vibrio vulnificus - Identification
Sample Recovery: Commonly isolated from blood or skin lesions in infected individuals.
Lactose Fermentation: Unique among Vibrio species as a lactose-positive organism.
68
Aeromonas - Description
Organism: Aeromonas (genus of Gram-negative rods).
Habitat: Found in freshwater, estuarine, and marine environments; normal flora in leeches, oysters, fish, frogs, etc.
Species: 16 species identified, with 11 linked to human disease.
Transmission: Faecal-oral route or through open wounds.
Diseases: Causes diarrhea and aquatic wound infections.
Toxins: Produces haemolysin, adhesins, and proteases, though their roles in disease are not fully understood.
69
Aeromonas - Identification
Differentiation: Often mistaken for Vibrio and Plesiomonas spp.
Key Features: Motile, oxidase-positive, shows A/A (acid/acid) reaction on TSI.
TCBS Agar: Limited or no growth.
Blood Agar: Many pathogenic strains are β-haemolytic.
O/129 Resistance: Resistant to the Vibrio-static agent O/129 (2,4-diamino-6,7-diisopropylpteridine).
Aeromonas hydrophila: Grows on blood agar (BA) and may show β-haemolysis; shows limited growth on TCBS agar.
70
Pasteurella - Description
Organism: Pasteurella (Gram-negative, non-motile coccobacillus).
Habitat: Found in the oropharynx of healthy animals (especially cats and dogs).
Key Features: Facultative anaerobe, catalase-positive, oxidase-positive, indole-positive.
Species: ~17 species; human infections mainly caused by serogroups A and D.
Infection Course: Rapid, typically developing within 24 hours of exposure.
71
Pasteurella - Clinical Significance
Epidemiology:
Part of normal flora in animals’ mouths and upper respiratory tracts (URT).
Highest carriage rate in cats (70–90%) followed by dogs (20–50%).
Isolated in 75% of cat bite injuries and 50% of dog bite injuries.
Forms of Disease:
Localized soft tissue infection after animal bite (90%).
Chronic respiratory infections (rare, often in COPD patients).
Systemic infections (e.g., bacteremia, meningitis) in immunocompromised individuals, elderly, or neonates.
Transmission: Zoonotic infection via animal bites, scratches, or shared food.
72
Pasteurella - Morphology and Identification
Cellular Morphology:
Small Gram-negative rods with bipolar staining (safety pin appearance).
Colonial Morphology:
Grows well on blood agar (BA) and chocolate agar (Choc); variable growth on MacConkey agar (MAC).
Some species require Factor V.
Colonies: Flat, wet, gray on BA; buttery, 1–2 mm with a musty odor (due to indole production).
Key Differential: Distinguished from other bacteria by resistance to O/129 (a Vibrio-static agent).
73
Francisella tularensis - General Characteristics
Tiny, faintly staining Gram-negative coccobacilli (short round rods).
Non-motile, non-spore forming, and strictly aerobic (needs oxygen).
Surrounded by a thin, lipid-rich capsule (helps evade immune system).
Slow metabolism – produces acid from carbohydrates but no gas.
Requires cysteine or cystine for growth (cannot grow on normal media).
Slow growth – takes at least 3 days to appear on culture.
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Francisella tularensis - Clinical Significance
Causes tularemia ("rabbit fever"), a serious zoonotic disease.
Transmission routes:
Tick/insect bites (as few as 10 bacteria can cause infection!).
Direct contact with infected animals (especially rabbits).
Eating/drinking contaminated food or water.
Intracellular pathogen – survives inside macrophages and spreads to organs.
Forms granulomas (small areas of inflammation).
Different forms of tularemia:
Cutaneous – skin infection with ulcers.
Pneumonic – inhaled bacteria cause severe lung infection.
Glandular – ingestion leads to swollen lymph nodes.
Highly infectious! Tier 1 bioterrorism agent in the US.
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Francisella tularensis – Identification & Safety
Difficult to detect – biochemically inert (does not react in most standard lab tests).
Faint staining due to lipid-rich capsule.
Special detection methods needed:
Fluorescent antibody staining (glows under UV light).
Serology (blood tests) to detect antibodies.
Culture characteristics:
Slow growth (3+ days) on cysteine-enriched media.
Forms tiny "pinpoint" colonies that change shape over time.
Highly infectious – can penetrate unbroken skin or infect via inhalation.
Handled only in PC3 labs (high biosafety level) to prevent lab-acquired infections.
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HACEK Group – General Characteristics
HACEK Group Bacteria (Haemophilus parainfluenzae, Aggregatibacter, Cardiobacterium, Eikenella, Kingella)
Fastidious (hard to grow) Gram-negative coccobacilli.
Part of normal oral flora but can cause infections under certain conditions.
Slow-growing and require CO₂ for optimal growth.
Do not grow on MacConkey (MAC) agar.
Mostly oxidase-positive and catalase-negative.
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HACEK Group – Clinical Significance
Infections & Impact
Cause ~3% of infective endocarditis cases (especially subacute).
Infections often occur due to:
Poor oral hygiene.
Dental procedures (bacteria enter bloodstream).
Damaged or diseased heart valves.
May contribute to formation of fatty plaques in arteries (potential role in cardiovascular disease).
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HACEK Group – Identification & Diagnosis
Laboratory Features
Slow growth – can delay diagnosis.
Require CO₂ for growth (won’t grow well in normal conditions).
Do not grow on MacConkey agar (differentiates them from other Gram-negatives).
Mostly oxidase-positive and catalase-negative (helps with identification).
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Aggregatibacter – General Characteristics
Species: Aggregatibacter aphrophilus (formerly Haemophilus paraphrophilus)
Natural habitat: Found in the mouth and oropharynx of healthy individuals.
Part of normal dental plaque microflora.
Opportunistic pathogen – can cause infections when it enters the bloodstream.
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Aggregatibacter – Clinical Significance
Infections & Risk Factors
Causes subacute endocarditis, brain abscesses, sinusitis, and osteomyelitis.
Often linked to dental procedures that break the oral mucosal barrier.
Temporary bacteremia (bacteria in the blood) can lead to infection in susceptible individuals.
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Aggregatibacter – Identification & Culture
Laboratory Features
Requires CO₂ for growth.
Non-haemolytic (does not break down red blood cells).
Catalase-negative, indole-negative, urease-negative.
Ferments glucose (produces acid & gas) and ferments lactose (produces acid only).
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Aggregatibacter actinomycetemcomitans – General Characteristics
Most common HACEK member isolated in endocarditis cases.
Also linked to abscesses, osteomyelitis, and destructive periodontal disease.
Comitans means "accompanying" – often found with Actinomyces israelii.
Small, non-motile Gram-negative coccobacillus.
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Aggregatibacter actinomycetemcomitans – Clinical Significance
Infections & Risk Factors
Major cause of infective endocarditis (especially in dental-related cases).
Associated with periodontal disease, leading to severe gum infections.
Can also cause bone infections (osteomyelitis) and abscesses.
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Aggregatibacter actinomycetemcomitans – Identification & Culture
Laboratory Features
Slow-growing (takes 48–96 hours to grow).
Capnophilic (requires increased CO₂ for growth).
On Nutrient Agar (NA) – forms rough colonies with a star-shaped center (lost on subculture).
Ferments glucose and lactose, but not maltose.
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Cardiobacterium hominis – General Characteristics
Part of normal flora in the upper respiratory tract (URT) and bowel.
Opportunistic pathogen – can enter the bloodstream and cause infections.
Associated with infective endocarditis, often following dental procedures.
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Cardiobacterium hominis – Clinical Significance
Infections & Risk Factors
Causes bacteremia and subacute bacterial endocarditis (SBE).
Infections usually occur after dental work or in people with pre-existing heart conditions.
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Cardiobacterium hominis – Identification & Culture
Laboratory Features
Gram-variable (may not stain consistently).
Unique "flower" or "rosette" arrangement on Gram stain.
Grows on Blood Agar (BA) and Chocolate Agar (Choc) with CO₂.
Alpha-haemolytic (partial breakdown of red blood cells).
Slow growth – can take several weeks.
Oxidase-positive, indole-positive, catalase-negative.
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Eikenella corrodens – General Characteristics
Normal flora of the gingiva (gums) and bowel (found in 40-70% of people).
Opportunistic pathogen – can cause infections when introduced into wounds.
Commonly found with Streptococci in mixed infections.
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Eikenella corrodens – Clinical Significance
Associated with human bite wounds ("clenched fist injuries") and fist fights ("punch injuries").
IV drug users (IVDU) who lubricate needles with saliva ("skin popping") are at risk.
Can also cause dental infections, abscesses, and soft tissue infections.
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Eikenella corrodens – Identification & Culture
Laboratory Features
Grows on Blood Agar (BA) and Chocolate Agar (Choc) with CO₂ and high humidity.
Small grey colonies with a distinct bleach-like odour.
May "pit" the agar (dig into the surface).
Catalase-negative, urease-negative, indole-negative, oxidase-positive.
Does not ferment carbohydrates.
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Kingella kingae – General Characteristics
Part of normal oral microbiota but can cause infections when introduced into the bloodstream.
Enters the bloodstream via dental trauma (surgery, poor hygiene).
Gram-negative with short, plump cells that appear in pairs or chains.
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Kingella kingae – Clinical Significance
Infections & Risk Factors
Causes subacute bacterial endocarditis (SBE).
Affects bones and joints, causing osteomyelitis, septic arthritis, and discitis (spinal infections).
Children are at higher risk of bone and joint infections.
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Kingella kingae – Identification & Culture
Laboratory Features
Grows on Blood Agar (BA) after several days in CO₂.
β-haemolytic (destroys red blood cells).
Colonies have a "fried egg" appearance with pitting of the agar.
Oxidase-positive, catalase-negative.
