Bacteriology

Bacteria
Bacteria are single-celled organisms Contain no membrane bound nucleus
Termed prokaryotes = pre nucleus

Pro = pre karyote = nucleus

Do not contain any other organelles (such

as ER or Golgi or mitochondria) Cytoplasm is surrounded by a lipid membrane with is surrounded by a rigid cell wall DNA bundled in a region called the nucleoid

The Bacterial Cell

Flagella

Ribosomes Capsule Cell wall Plasma membrane

Nucleoid region (DNA)

Pili

The Bacterial Cell

Nucleoid DNA

Bacterial Nomenclature
Binomial naming system
Two word naming system

First word is genus name

Always capitalized Escherichia

Second word is species name

Not capitalized coli

When writing full name genus usually

abbreviated
E. coli

Full name always italicized

Or underlined

Bacterial Morphology
Morphology classification by shape

Morphology is often expressed in the binomial

naming system
Ex. Bacillus subtillis, the word Bacillus means rod Ex. Streptococcus pyogenes, Streptococcus means

a chain of spheres Ex. Helicobacter pylori, helix shaped bacterium Many names of bacteria do not reference there shape such as E. coli, Pseudomonas aeruginosa, or Burkholdaria cepacia.

Bacterial Morphology

Examples of Morphology
Bacteria exhibit a variety of shapes
Coccus Spherical Bacillus Rod or cylinder shaped

Examples of Morphology
Other shapes
Coccobacillus Short round rod Vibrio Curved rod Spirillum Spiral shaped Spirochete Helical shape

Bacterial Anatomy and Structures

Cytoplasm- fluid that fills the cell where most

reactions and activities of the cell take place Cell Membrane- barrier between the internal cell fluid (cytoplasm) and the exterior environment. It is selectively permeable only allowing in selected comounds Cell Wall- rigid structure surrounding the cell membrane that gives bacteria there shape. Stronger than the cell membrane helps hold the cell together and maintain structural integrity. The cell wall can be of varying degrees of thickness depending on the type of bacteria Cell capsule- slime layer that protects bacteria and helps bacteria to adhere to surfaces

Bacterial Anatomy and Structures

Nucleoid region- location of the genetic material

(DNA) in the bacterial cell. Typically bacterial DNA is one large circular DNA molecule that is packed together by supercoiling.
*Bacterial DNA should not be referred to as a

Plasmid- a small genetic element of circular DNA

chromosome because it is not packed together using chromatin and does not resemble the organization of DNA in eukaryotes. The alternate name for bacterial DNA is genophore

that is separate and distinct from the nucleoid DNA. The plasmid is typically thousands of times smaller than the nucleoid DNA but often very important as it may carry genes for antibiotic drug resistance

Bacterial Anatomy and Structures

Flagella- long structure made of protein that

propels the bacteria when it spins. Pili- structure made of protein subunits that extend out of the cell. Function for attachment to surfaces and gliding motility. Inclusions- small structure used to store excess material typically as nutrient reserve. Stored materials include phosphates, polysaccharides, nitrogen, sulfur, proteins and biopolymers Ribosome- a structure consisting of protein and rRNA. It translates mRNA into a sequence of amino acids (proteins)

Size Distribution

Peptidoglycan
Polymer of disaccharide

N-acetylglucosamine (NAG) & N-acetylmuramic acid (NAM) Linked by polypeptides

Figure 4.13a

Figure 4.13b, c

Gram-positive cell walls

Thick

peptidoglycan Teichoic acids In acid-fast cells, contains mycolic acid

Gram-negative cell walls

Thin peptidoglycan No teichoic acids Outer membrane

Gram-Positive cell walls

Teichoic acids:
Lipoteichoic acid links to plasma membrane Wall teichoic acid links to peptidoglycan

May regulate movement of cations Polysaccharides provide antigenic variation

Figure 4.13b

Gram-Negative Outer Membrane

Lipopolysaccharides, lipoproteins, phospholipids. Forms the periplasm between the outer membrane and

the plasma membrane. Protection from phagocytes, complement, antibiotics. O polysaccharide antigen, e.g., E. coli O157:H7. Lipid A is an endotoxin. Porins (proteins) form channels through membrane

Gram-Negative Outer Membrane

Figure 4.13c

Gram Stain Mechanism

Crystal violet-iodine crystals form in cell Gram-positive
Alcohol dehydrates peptidoglycan
CV-I crystals do not leave

Gram-negative
Alcohol dissolves outer membrane and leaves holes in

peptidoglycan CV-I washes out

Atypical Cell Walls

Mycoplasmas
Lack cell walls Sterols in plasma membrane

Archaea
Wall-less, or

Walls of pseudomurein (lack NAM and D amino acids)

Damage to Cell Walls

Lysozyme digests disaccharide in peptidoglycan.
Penicillin inhibits peptide bridges in peptidoglycan. Protoplast is a wall-less cell. Spheroplast is a wall-less Gram-positive cell. L forms are wall-less cells that swell into irregular

shapes. Protoplasts and spheroplasts are susceptible to osmotic lysis.

Plasma Membrane

Figure 4.14a

Plasma Membrane
Phospholipid bilayer

Peripheral proteins
Integral proteins Transmembrane proteins

Figure 4.14b

Fluid Mosaic Model

Membrane is as viscous as

olive oil. Proteins move to function Phospholipids rotate and move laterally

Figure 4.14b

Typhoid fever

Organism
Salmonella typhi, a Gram-negative bacteria. Similar but often less severe disease is caused by

Salmonella serotype paratyphi A. Many genes are shared with E. coli and at least 90% with S. typhimurium, Polysaccharide capsule Vi: present in about 90% of all freshly isolated S. typhi and has a protective effect against the bactericidal action of the serum of infected patients. The ratio of disease caused by S. typhi to that caused by S. paratyphi is about 10 to

Pathogenesis
Entry in GIT localisation in Gut associated

lymphoid tissue Lymphatic channel thoracic duct circulation primary silent bacteremia localisation in macrophages of RES in spleen, liver, bone marrow (incubation period 8-14 days) secondary bacteremia

Acute non-complicated disease

Characterized by Prolonged fever, Disturbances of bowel function Headache, malaise and anorexia. Bronchitic cough Exanthem (rose spots), on the chest, abdomen and back.

Complicated disease
10% of typhoid patients

GIT: occult blood in 10-20% of patients, and

malena in up to 3%. Intestinal perforation has also been reported in up to 3% of hospitalized cases. CNS: Encephalopathy, Typhoid meningitis, encephalomyelitis, Guillain-Barr syndrome, cranial or peripheral neuritis and psychotic symptoms Others: Hepatitis, myocarditis, pneumonia, disseminated intravascular

Diagnosis
Culture: blood, bone marrow, bile Bone marrow aspirate culture is the gold standard

for the diagnosis of typhoid fever Failure to isolate the organism

(i) the limitations of laboratory media (ii) the presence of antibiotics (iii) the volume of the specimen cultured (iv) the time of collection, patients with a history of fever for 7 to 10 days being more likely than others to have a positive blood culture.

Widal Test
O antibodies appear on days 6-8 and H antibodies on

days 10-12 Negative in up to 30% of culture-proven cases of typhoid fever S. typhi shares O and H antigens with other Salmonella serotypes and has cross-reacting epitopes with other Enterobacteriacae, and this can lead to false-positive results. Such results may also occur in other clinical conditions, e.g. malaria, typhus, bacteraemia caused by other organisms, and cirrhosis This is acceptable so long as the results are interpreted with care in accordance with appropriate local cut-off values for the determination of positivity.

New serological test

Specific antibodies usually only appear a week

after the onset of symptoms and signs. This should kept in mind when a negative serological test result is being interpreted. New serological tests
IDL Tubex
Typhidot (better), high negative predictive value Dipstick test,

Typhoid epidemiology

Treatment of uncomplicated typhoid

Treatment of severe typhoid

Oral drugs
Ofloxacin: 15-20 mg / kg for 7-14 days

Azithromycin:8-10 mg/kg for 7 days

Cefixime: 20 mg /day for 7-14 days Chloramphenicol: 50-75 mg /kg/day for 14-21

days

Fluoroquinolones
Optimal for the treatment of typhoid fever Relatively inexpensive, well tolerated and more

rapidly and reliably effective than the former first-line drugs, viz. chloramphenicol, ampicillin, amoxicillin and trimethoprim-sulfamethoxazole. The majority of isolates are still sensitive. Attain excellent tissue penetration, kill S. typhi in its intracellular stationary stage in monocytes/macrophages and achieve higher active drug levels in the gall bladder than other drugs. Rapid therapeutic response, i.e. clearance of fever and symptoms in three to five days, and very low rates of post-treatment carriage.

Chloramphenicol
The disadvantages of using chloramphenicol

include a relatively high rate of relapse (57%), long treatment courses (14 days) and the frequent development of a carrierstate in adults. The recommended dosage is 50 - 75 mg per kg per day for 14 days divided into four doses per day, or for at least five to seven days after defervescence. Oral administration gives slightly greater bioavailability than intramuscular (i.m.) or intravenous (i.v.) administration of the succinate salt.

Cephalosporins
Ceftriaxone: 50-75 mg per kg per day one or two

doses Cefotaxime: 40-80 mg per kg per day in two or three doses Cefoperazone: 50-100 mg per kg per day

Dexamethasone for CNS complication

Should be immediately be treated with high-dose

intravenous dexamethasone in addition to antimicrobials Initial dose of 3 mg/kg by slow i.v. infusion over 30 minutes 1 mg/kg 6 hourly for 2 days Mortality can be reduced by some 80-90% in these high-risk patients

GI complication
Patients with intestinal haemorrhage need intensive

care, monitoring and blood transfusion. Intervention is not needed unless there is significant blood loss. Surgical consultation for suspected intestinal perforation is indicated. If perforation is confirmed, surgical repair should not be delayed longer than six hours. Metronidazole and gentamicin or ceftriazone should be administered before and after surgery if a fluoroquinolone is not being used to treat leakage of intestinal bacteria into the abdominal cavity. Early intervention is crucial, and mortality rates increase as the delay between perforation and surgery lengthens. Mortality rates vary between 10% and 32%.

Relapse
5-20% of typhoid fever cases that have

apparently been treated successfully. A relapse is heralded by the return of fever soon after the completion of antibiotic treatment. The clinical manifestation is frequently milder than the initial illness. Cultures should be obtained and standard treatment should be administered.

Thank you

Size in the Microbial World

Tremendous range in size Smallest virus approximately 1/1,000,000th size of largest eukaryotic cell

Prokaryotic Cells
Comparing Prokaryotic and Eukaryotic Cells
Prokaryote comes from the Greek words for

prenucleus. Eukaryote comes from the Greek words for true nucleus.

Prokaryote
One circular

Eukaryote
Paired

chromosome, not in a membrane No histones No organelles Peptidoglycan cell walls Binary fission

chromosomes, in nuclear membrane Histones Organelles Polysaccharide cell walls Mitotic spindle

 Average size: 0.2 -1.0 m 2 - 8 m Basic shapes:

 Unusual shapes
Star-shaped Stella Square Haloarcula

Most bacteria are monomorphic A few are pleomorphic

Figure 4.5

Arrangements
Pairs: diplococci,

diplobacilli
Clusters:

staphylococci
Chains:

streptococci, streptobacilli

Glycocalyx
Outside cell wall Usually sticky

A capsule is neatly

organized A slime layer is unorganized & loose Extracellular polysaccharide allows cell to attach Capsules prevent phagocytosis
Figure 4.6a, b

Flagella
Outside cell wall Made of chains of

flagellin Attached to a protein hook Anchored to the wall and membrane by the basal body

Figure 4.8

Flagella Arrangement

Figure 4.7

Figure 4.8

Motile Cells

Rotate flagella to run or tumble Move toward or away from stimuli (taxis)

Flagella proteins are H antigens

(e.g., E. coli O157:H7)

Motile Cells

Figure 4.9

Axial Filaments
Endoflagella In spirochetes Anchored at one end

of a cell Rotation causes cell to move

Figure 4.10a

 Fimbriae allow

attachment Pili are used to transfer DNA from one cell to another

Figure 4.11

Cell Wall
Prevents osmotic lysis Made of peptidoglycan (in bacteria)

Figure 4.6a, b

Vaccination
Vi polysaccharide, is given in a single dose

Protection begins seven days after injection,

maximum protection being reached 28 days after

injection when the highest antibody concentration is obtained. Protective efficacy was 72% one and half years after vaccination and was still 55% three years after a single dose. In Asian countries where Vi-negative strains have been reported at the low average level of 3%.

live oral vaccine Ty2la

three doses two days apart on an empty

stomach. Protection as from 10-14 days after the third dose. > 5 years. Protective efficacy of the enteric-coated capsule formulation seven years after the last dose is still 62% in areas where the disease is endemic; Antibiotics should be avoided for seven days before or after the immunization

Antibiotic resistance
MDR is mediated by plasmid

Quinolone resistance is frequently mediated by

single point mutations in the quinoloneresistancedetermining region of the gyrA gene Nalidixic acid resistant: MIC of fluoroquinolones for these strains was 10 times that for fully susceptible strains.