BACTERIAL IDENTIFICATION METHODS

Cell Wall

● Protects internal structures of cell

● Maintains cell shape

● Referred as Peptidoglycan or Murein layer

● Basis of Gram Staining

● Vary in composition with species of bacteria

Mycoplasma & Ureaplasma spp.

● No cell wall

● Have sterols in cell membrane

Gram Stain

● Developed by Hans Christian Gram

● Differential Stain

● Identified using Light Microscope

1. Fix culture on slide with methanol or heat

2. Flood slide with Crystal violet for 1 minute. Rinse with water.

3. Flood with Iodine for 1 minute. Rinse with water.

4. Flood with decolorizer for approx.

5 seconds and rinse with water immediately

5. Apply Safranin for 30 seconds. Rinse with water and gently blot the slide dry with paper towels.

6. Allow to dry for 5-10 minutes. Examine

Steps for Gram Stain

Blue to Purple

Color at the end pf the gra, staining procedure in Gram-Positive Bacteria

Pink to red

Color at the end pf the gra, staining procedure in Gram-Negative Bacteria

thick layer

Peptidoglycan in cell walls of Gram-positive

thin layer

Peptidoglycan in cell walls of Gram-negative

Gram-positive bacteria

Teichnoic acids and lipoteichnoic acids are present in cell walls

Gram-negative bacteria

Teichnoic acids and lipoteichnoic acids are absent in cell walls

Lipopoccharide

In Gram-positive Bacteria it is absent, while in gram negative it is present in cell walls

Acute Necrotizing Ulcerative Gingivitis (ANUG)

● Aka Vincent’s angina / Trench Mouth

● Synergistic infection

Fusobacterium nucleatum

Treponema vincentii

Common bacteria involved in Acute Necrotizing Ulcerative Gingivitis (ANUG)

Streptococcus pyogenes:

Streptococcus mutans:

Corynebacterium diphtheriae:

Chlamydia pneumoniae:

Infection in the Oral Region

Chlamydia pneumoniae:

Pharyngitis

Corynebacterium diphtheriae:

Respiratory diphtheria

Streptococcus mutans:

Dental Caries

Streptococcus pyogenes:

Strep Throat

Staphylococcus spp. (Catalase Positive)

● Grape-like clusters

● Facultative anaerobe

● Nonmotile, non-spore forming

● 3 Major Pathogens

- Staphylococcus aureus

- Staphylococcus epidermidis

- Staphylococcus saprophyticus

3 Major Pathogens in Staphylococcus spp. (Catalase Positive)

Staphylococcus aureus (Coagulase positive)

● Normal microbiota of the skin

● Most virulent species of staphylococcus

● Most commonly encountered

● Mode of Transmission: Person-to-person, Respiratory Droplets, Fomites

● MRSA (Methicillin Staphylococcus aureus)

- Resistant to methicillin, oxacillin

- Treatment: Vancomycin

S. epidermidis

- Slime-producing staphylococci

- Most frequently encountered among CONS

- Primarily associated with HAIs (Hospital Acquired Infections)

- Infections: Bacterial Endocarditis, Infections from medical devices (Prosthetic heart valves, Prosthetic Joints, IV lines), UTI

S. saprophyticus

- Usually associated with community-acquired UTI in young, sexually active

females but not in HAIs

- Infections: UTI in young, sexually active women

Capsule:

Inhibits phagocytosis; appear as slime layer/biofilm

Protein A

Inhibits complement cascade, Has high affinity to Fc receptor on IgG, antiphagocytic, used in coagglutination assay

Coagulase

fibrin clot formation; protect bacteria from phagocytosis

Staphylokinase

promotes fibrinolysis

Hyaluronidase

spreading factor; hydrolyze hyaluronic acid in connective tissue

B-lactamase

resistance to beta-lactam antibiotics

a-Hemolysin

targets eukaryotic cell membranes

b-hemolysin

degrades sphingomyelin

y-Hemolysin

lyses white blood cells

Panton-Valentine Leukocidin (PVL)

Toxic to white blood cells; important in CA-MRSA infections

Exfoliative Toxins

generalized desquamation of skin; responsible for the Scalded Skin Syndrome (Ritter Disease)

Toxic Shock Syndrome Toxin-1 (TSST-1)/Enterotoxin F:

causes toxic shock syndrome; Superantigen (causing massive activation of T cells resulting to sever inflammation, tissue damage and systemic symptoms)

Enterotoxins

Superantigens; heat-stable and resistant to gut enzymes. Causes food poisoning

• Skin Scalded Syndrome (Ritter’s Disease)

• Toxic Shock Syndrome

• Impetigo

• Folliculitis

• Carbuncle

• Furuncle

  ● Pneumonia

  ● Meningitis

  ● Osteomyelitis

  ● Focal Suppuration or abscesses

  ● Bacteremia

  ● Food Poisoning and Diarrhea

  ● Empyema

  ● Endocarditis

  ● Sepsis

Staphylococcus aureus Clinical Manifestations

Penicillin and Vancomycin

Treament for Staphylococcus aureus

Streptococcus species (Catalase Negative)

● arranged in chains except S. pneumoniae (diplococci)

● Facultative anaerobe

● Categorized mainly based on

- Hemolytic Pattern

- Lancefield Classification

Streptococcus pyogenes (Group A)

● Prototypical human pathogen

● Most virulent streptococcus spp.

● Beta-hemolytic

Streptolysin O:

Can cause lysis in the absence of oxygen

Streptolysin S:

Can cause lysis in the presence of oxygen

• Erysipelas

• Necrotizing Fasciitis

• Cellulitis

• Stre

• StrepThroat

• Impetigo

• Scarlet Fever

• ● Streptococcal Toxic Shock Syndrome

  ● Acute Glomerulonephritis

  ● Rheumatic Fever

Streptococcus pyogenes (Group A) Clinical Manifestations

Penicillin

Treatment for Streptococcus pyogenes (Group A)

Streptococcus agalactiae (Group B)

● Beta-hemolytic

● Normal Flora: Vaginal Tract,, Lower GIT

● Usually associated with neonates

● Meningitis

● Sepsis

● Respiratory Distress Syndrome

Streptococcus agalactiae (Group B) Clinical Manifestations

Penicillin

Treatment for Streptococcus agalactiae (Group B)

Enterococcus spp.

● Normal Flora: Oropharynx; Female GIT, Skin

● E. faecalis, E. faecium

Non-Enterococcus spp.

● S. bovis: most important spp.

- associated with colon cancer

● S. equinus, S. gallolyticus

Viridans Streptococci

● Mostly are alpha hemolytic

● Normal Flora: Oral Cavity, Upper Respiratory Tract

● S. mutans, S. sarguis, S anginosus

● Subacute bacterial endocarditis (S. sanguis)

● Dental Caries (S. mutans)

Clinical Manifestations of Viridans Streptococci

Streptococcus pneumoniae

● lancet-shaped diplococci

● only encapsulated streptococci

● Normal Flora: Nasopharynx Oropharynx

● Lobar Pneumonia

● Meningitis

● Otitis Media

● Sepsis

Streptococcus pneumoniae Clinical Manifestations

Against capsular Ag

Vaccine for Streptococcus pneumoniae

Neisseria species

● Kidney/ Coffee-Bean shaped Diplococci

● Capnophiles; Cold-sensitive

● Non-motile

Neisseria gonorrhoeae

● Not considered a normal microbiota

● MOT: Sexual Contact, Vertical Transmission, Fomites

Neisseria meningitidis

● Encapsulated

● Normal Flora: Nasopharynx

● MOT: Respiratory Droplets

● Urethritis

● Urethral Stricture

Clinical Manifestations (N. gonorrhoeae) in MEN

● Urethritis

● Pelvic Inflammatory Disease

Clinical Manifestations (N. gonorrhoeae) in WOMEN

Ophthalmia Neonatorum

Clinical Manifestations (N. gonorrhoeae) in INFANTS

Ceftriaxone

Treatment (N. gonorrheae)

Spore-forming Bacilli (Bacillus species)

● Spore: Central spore

● B. anthracis, B. cereus

Spore-forming Bacilli (Clostridium species)

Causes botulism, tetanus, gas gangrene, pseudomembranous colitis

C. tetani:

terminal spore

C. botulinum

subterminal spore

C. perfringens

central spore

C. difficile

subterminal spore

Non Spore-forming Bacilli (Corynebacterium diphtheriae)

● Has metachromatic granules

● MOT: Respiratory Droplets; Direct Contact

Non Spore-forming Bacilli (Listeria monocytogenes)

● Psychrophile

● Umbrella Motility in semisolid agar

● Tumbling Motility in direct wet mount

● MOT: Contaminated Food, Vertical Transmission, Direct Contact

Bacillus anthracis

● Largest Pathogenic Bacteria

● Non-motile, encapsulated

● MOT: Inoculation, Ingestion, Inhalation

● Causes anthrax

Bacillus cereus

● Motile, non-encapsulated

● Mainly causes Food Poisoning

● Opportunistic pathogens

● Two Forms: Emetic and Diarrheal

● Cutaneous Anthrax:

● Gastrointestinal Anthrax:

● Inhalation Anthrax:

Clinical Manifestations (B. anthracis)

Ciprofloxacin

Treatment (B. anthracis)

● Food poisoning

● Eye infections

● Meningitis, Endocarditis, Pneumonia,

Osteomyelitis

Clinical Manifestations (B. cereus)

Ciprofloxacin

Treatment (B. cereus)

Clostridium botulinum

● Usually found in soil and animal feces

● Causes botulism

● MOT: Ingestion

Clostridium tetani

● Usually found in soil and horse manure

● Causes tetanus

● MOT: Contaminated materials

Clostridium perfringens

● Causes gas gangrene

● MOT: Ingestion, Direct contact

Clostridium difficile

● Normal Flora of colon

● Causes pseudomembranous colitis

● MOT: Ingestion, Direct contact

● Flaccid Paralysis

● Adult Botulinum

● Infant Botulism

Clostridium botulinum (Clinical Manifestations)

● Spastic Paralysis

● Trismus (Lockjaw)

● Risus sardonicus (devil’s grin)

● Opisthotonus (arching of back)

Clostridium tetani (Clinical Manifestations)

● Gas Gangrene (clostridial myonecrosis)

● Diarrhea

Clostridium perfringens (Clinical Manifestations)

● Pseudomembranous colitis

● Diarrhea

Clostridium difficile (Clinical Manifestations)

Antitoxin therapy

Clostridium botulinum (Treatment)

Toxoid / DPT Vaccine

Clostridium tetani (Treatment)

● Extensive surgical debridement

● Drug of Choice: Penicillin

● Antitoxin therapy

Clostridium perfringens (Treatment)

Vancomycin

Clostridium difficile (Treatment)

Corynebacterium diphtheriae

● Has metachromatic granules

● MOT: Respiratory Droplets; Direct Contact

● Respiratory diphtheria

● Cutaneous diphtheria

Corynebacterium diphtheriae Clinical Manifestations

Toxoid / DPT vaccine

Drug of Choice: Penicillin, Erythromycin

Treatment Corynebacterium diphtheriae

Enterobacteriaceae Family

● gram-negative bacilli inhabiting the GIT

● E. coli is part of the normal flora but salmonella and shigella are regularly pathogenic

● Facultative anaerobes

● All are motile except Shigella, Klebsiella, Yersinia

● All ferment glucose

O antigen

most external part of the cell wall lipopolysaccharide; Heat and

alcohol resistant

K antigen

external to O antigen; some are polysaccharide

H antigens

located on flagella: Heat and alcohol labile

Klebsiella pneumoniae

● present in respiratory tract and feces

● Causes pneumonia (Currant-jelly sputum)

● Causes hemorrhagic necrotizing consolidation of the lung

● UTI

Klebsiella pneumoniae subspecies ozaenae

Causes atopic rhinitis

Klebsiella pneumoniae subspecies rhinoscleromatis

Causes rhinoscleroma

Klebsiella pneumoniae granulomatis

Causes granuloma inguinale

Ampicillin or Tetracycline

Treatment for Klebsiella species

Shigella species

● Non-motile

● Causes Bacillary Dysentery

● MOT: Direct Contact; Fecal-Oral Route

● Classified based on their O antigen