School of Dentistry

FACULTY OF MEDICINE AND HEALTH

DSUR3210 Illness and Well-Being

Classification and identification of

bacteria of medical importance

Dr Thuy Do
Division of Oral Biology
Why learn medical microbiology?

• To study microbes that cause

infections and diseases.
• To help with diagnosis,
prevention and treatment.
Aims

 To discuss the major groups of bacteria that cause disease in humans

 To describe the methods by which bacterial human pathogens are sub-

classified

Learning outcomes

 Be able to describe the principles of the methods used to sub-classify

bacterial human pathogens

 Be able to provide examples of the basic methods used to identify human

pathogens
 The germ theory of disease

Louis Pasteur (1822–1895) was a French Heinrich Hermann Robert Koch

chemist and microbiologist, discovered the (1843–1910) was a German
principles of vaccination, microbial physician who developed the
fermentations and pasteurisation. Koch's postulates.
He is remembered for his remarkable
breakthroughs in the causes and preventions
of diseases.
https://en.wikipedia.org/wiki
Examples of bacterial pathogens and diseases recognized or
re-emerged since 1977:

Bacterium Disease
Legionella pneumophila Legionnaires' pneumonia
Listeria monocytogenes listeriosis
Campylobacter jejuni gastroenteritis distributed world-wide
Staphylococcus aureus toxic shock syndrome
E. coli O157:H7 hemorrhagic colitis; hemolytic uremic syndrome
Borrelia burgdorferi Lyme Disease and complications
Helicobacter pylori gastric and duodenal ulcers
Ehrlichia chaffeensis human ehrlichiosis
Clostridium difficile antibiotic induced diarrhea; pseudomembranous colitis
Vibrio cholerae O139 epidemic cholera
Salmonella enterica Serotype salmonellosis
Typhimurium DT 104
Bartonella henselae cat scratch fever
Streptococcus pyogenes (Group A necrotizing fasciitis (GAS); streptococcal toxic shock
Strep) syndrome
Multiple drug resistant S. aureus (e.g. nosocomial and community associated infections
MRSA)
Chlamydia pneumoniae atherosclerosis
Clostridium botulinum sudden infant death syndrome (SIDS)
Vibrio vulnificus wound infection, septicemia, gastrointestinal disease
Parachlamydia pneumonia
Corynebacterium amycolatum hospital-acquired endocarditis
Klebsiella pneumoniae blood stream infections
Linezolid-resistant enterococci (E. nosocomial infections
faecalis and E. faecium)
Multiple drug resistant Acinetobacter nosocomial infections
baumannii
 Note: *DALY = Disability-Adjusted Life Years, the years of healthy life lost
due to disability, sickness or premature mortality. N/A = not available.
Source: Global Health Council 2009.

http://www.cdc.gov/
 http://en.wikipedia.org/wiki/File:Phylogenetic_tree.svg

Eukaryotes: Fungi, Protozoa

10-100m
Prokaryotes: Eubacteria
(Archaebacteria)
1-10 m
Viruses: non-cellular
50-100nm
Cell size and scale: https://youtu.be/l7kZjdeo0Cs
Generalised structure of a bacterium

Capsule
Cell wall
Rods/bacilli
Cytoplasmic
membrane
Cocci

Cytoplasm
Coccobacilli
Chromosome
(nucleoid) Vibrio/curved rods

Fimbriae/pili Spirilla

Flagellum
Laboratory identification of bacteria
Isolation in pure culture

Identification using differential characteristics

Bacterial colonies on a Columbia Blood Agar

(CBA) plate, incubated in CO2
(non-selective medium)
Differential characteristics:

Colony morphology
Shape,
Haemolysis -haemolysis β-haemolysis  -haemolysis
Pigment

Cell morphology
Shape, spores,
Gram stain
Gram staining

Fixation

Crystal violet

Iodine

Decolourisation
(alcohol/acetone)

Counter stain
(safranin)
GRAM + GRAM -
Gram’s stains
Differential characteristics:
Metabolic activities:
Fermentation, acid or gas production, enzymes, utilisation of
carbohydrates or proteins (e.g. catalase, coagulase, oxidase tests)

Antigens:
binding to specific antibodies

Cellular composition:
lipids, amino acids, peptidoglycan. (e.g. Gram stain,
acid-fast stain)
DNA (molecular techniques):
PCR, nucleic acid sequences, homology, probes.
e.g. 16S rDNA sequence data, and genomics using
next generation sequencing technologies (Illumina,
Oxford Nanopores Technologies, Roche, PacBio
etc)
HiSeq3000

http://www.illumina.com/

http://www.nature.com/articles/srep01843
http://www.delta.tudelft.nl/article/dawn-of-the-age-of-genomics/24767
http://www.nasa.gov/mission_pages/station
/research/news/dna_sequencing
 The Ziehl–Neelsen stain (or acid-fast stain)

Mycobacterium tuberculosis visualization

using the Ziehl–Neelsen stain.

1) Carbol fuchsin
2) Decolourisation (methanol)
3) Methylene blue
 Catalase test

https://www.youtube.com/watch?v=2oKq0ZTmD30
Coagulase test

Staphylococcus aureus = coagulase positive.

Oxidase test

Oxidase reagent:
Tetra-methyl-paraphenyl-di-amine
(TRMPPD)
Gram-positive bacteria

Gram-positive bacilli

Acid-fast Mycobacteria, which include human pathogens such as Mycobacterium

tuberculosis, Mycobacterium leprae. Mycobacterium avium-intracellulare and
Nocardia spp. cause disease in immunocompromised patients.

Gross appearance of tuberculous Leprosy caused by Mycobacterium leprae

dissemination in the lungs. http://emedicine.medscape.com/article/22
(Patient G, male, aged 35 years) 0455-overview
http://www.iomcworld.com/ijcrimph/i
jcrimph-v01-n02-01-f.htm
Gram-positive bacteria

Spore producing Gram-positive bacilli

Bacillus anthracis causing anthrax, Bacillus cereus causing food poisoning

(associated with the consumption of rice).

Clostridium spp. e.g. Clostridium perfringens (causing gas gangrene), Clostridium

tetani causing tetanus, Clostridium botulinum causing botulism.

A skin lesion caused by anthrax Sir Charles Bell's portrait of a soldier dying of tetanus.
https://en.wikipedia.org/wiki http://textbookofbacteriology.net/clostridia_3.html
Gram-positive bacteria

Motile and non-sporing bacilli

Listeria spp.
The major human pathogen in the Listeria genus is Listeria monocytogenes, often
found in foods such as cheeses and pâtés, it causes listeriosis.

Scanning electron micrograph

of Listeria monocytogenes.
https://en.wikipedia.org/wiki
 Gram-positive bacteria

Non-motile and non-sporing bacilli

Corynebacteria and Propionibacteria are catalase positive

e.g. Corynebacterium diphtheriae causing diphtheria. Propionibacterium acnes is
associated with acne.

Lactobacilli are catalase negative, associated with milk. They are important
members of the human microbiota, and are being explored as potential probiotics.

Case of diphtheria
http://www.healthline.com/health/diph
theria#Overview1 Propionibacterium acnes in sebaceous glands.
http://www.medical-labs.net/propionibacterium-2448/
Gram-positive bacteria

Gram-positive cocci

Facultative anaerobic cocci: staphylococci (catalase positive), and streptococci

(catalase negative).

Important human pathogens: Staphylococcus aureus (catalase and coagulase

positive); Streptococcus pneumoniae (catalase negative,  -haemolytic, optochin-
sensitive); Streptococcus pyogenes (β-haemolytic).

Streptococcus pyogenes. Left: Gram stain of Streptococcus

pyogenes in a clinical specimen. Right: Colonies on blood agar
Staphylococcus aureus viewed under exhibiting β-haemolysis (clear).
a microscope http://textbookofbacteriology.net/streptococcus.html
Gram-negative bacteria

Gram-negative cocci

Veillonella spp. (obligately anaerobic), important members of the human oral

microbiota, often isolated in dental plaque, role in infection remains speculative.

Parvobacteria and acinobacters (aerobic cocco-bacilli), opportunist pathogens

causing infection in compromised hospital patients, frequently resistant to multiple
antibiotics.

Neisseria spp. (required 5-10% CO2 for growth in culture)

Neisseria meningitidis causes meningococcal meningitis. Neisseria gonorrhoeae
causes gonorrhoea.
 Gram-negative bacteria

Gram-negative bacilli

Haemophilus spp.
Haemophilus influenza, associated with respiratory infections, may cause
meningitis in unvaccinated children. May also cause epiglottitis (rare infection
causing swelling of epiglottis which could lead to death by suffocation).

Legionella spp.
Legionella pneumophila, causing Legionaire’s disease, first described in 1976.
Bordetella spp.
Bordetella pertussis causes whooping cough.

Bordetella pertussis cultured on charcoal agar

Transmission electron microscopy

image of Legionella pneumophila
https://en.wikipedia.org/wiki
Gram-negative bacteria

Gram-negative bacilli

The family enterobacteriaceae (oxidase negative)

Escherichia coli, a member of the commensal gut microbiota.

Yersinia pestis causes the plague.
Shigella dysenteriae is responsible for bacterial dysentery.

Salmonella spp.
Salmonella enterica causes gastroenteritis, Salmonella typhi causes typhoid fever
(not to confuse with typhus, caused by Rickettsia).

Providencia spp.
Members of this genus cause urinary tract infections in people with indwelling
catheters.
 Gram-negative bacteria

Other Gram-negative bacilli

Oxidase positive:

Campylobacter spp. are the most common cause of bacterial diarrhoea.

Vibrio spp. (e.g. Vibrio cholera causing cholera).
Brucella spp. cause brucellosis (“undulant fever”).
Flavobacterium spp. (refer to yellow colonies) are a group of environmental
bacteria causing infection in compromised individuals.

Pseudomonas spp. (e.g. Pseudomonas aeruginosa,

causing opportunistic infections in immunocompromised
people).
Aeromonas spp. (e.g. Aeromonas hydrophila causes
diarrhoea).

Vibrio cholera