Bactriology Lecture 3 Wk7 Flashcards
Evolutionary timetable
Rise of oxygen stimulates rise of diverse range of living organisms
rRNA sequence - phylogenetic analysis
Bacteria -
Archaea
Eukarya
Bacterial evolutionary marker - ribosomal RNA
E. Coli 16s rRNA
16s rRNA gene amplification, sequencing
Cell culture -> DNA extract and PCR -> electrophoresis (check 16s DNA) -> sequencing -> align sequences, create phylogenetic tree.
Internal nodes - ancestor species
External nodes - extant known species
Branch - lengths represent evolutionary distance between species
Systematics, phylogeny and taxonomy
Systematics or phylogeny= study of evolutionary history of organisms
Taxonomy = science of classification to show similarities among organisms
Traditional taxonomy = practical aspects, visible properties
Modern taxonomy = DNA sequences + practical aspects
Modern bacterial taxonomy by polyphasic approach
1. Phenotypic 2. Genotypes 3. Phylogenetic
Petri dish. DNA. Tree
Biochemical test to separate bacterial species
Gram staining
Lactose fermentation
Oxidase
Catalase
Slide agglutination
Fatty acid analysis
Bacterial culture
Extract fatty acids
Derivatize to form methyl esters
Gas chromatography
Peaks from various fatty acid methyl esters
Compare pattern with database
Identify organism
Classes in bacteria
I. Saturated
II. Unsaturated
III. Cyclopropane
IV. Branched
V. Hydroxy
Genotype
GC content (%) = G+C/A+T+G+C X 100
GC-ratio= A+T/ G+C
Bacterial taxonomy y- genotype
Taxonomic Classification + nomenclature
- bacterial taxonomic hierarchy
Escherichia coli Staphylococcus aureus Streptomyces coelicolor
Domain Bacteria Bacteria Bacteria
Phylum Proteobacteria Firmicutes Actinobacteria
Class Gammaproteobacteria Bacilli Actinobacteria
Order Enterobacteriales Bacillales Actinomycetales
Family Enterobacteriaceae Staphylococcaceae Streptomycetaceae
Genus Escherichia Staphylococcus Streptomyces
Species coli aureus coelicolor
Genus epithet (=species)
Escherichia coli
Italicise the name
Escherichia coli
Genus name starts with upper case
A space between genus and species name
Species name starts with lower case
Gram-negatives - proteobacteria
Alphaproteobacteria
Proteobacteria
Alphaproteobacteria : caulobacter
Alphaproteobacteria : Hyphomicrobium
Hyphomicrobium cell cycle
Bacterial life cycle - binary fission vs budding
Alphaproteobacteria : diverse positioning of stalks in budding bacteria
Polar - small tail
Sub-polar - long tail
Bi-lateral - two tails either side
Bacterial diversity proteobacteria
- Alphaproteobacteria : Rizobiales (Agrobacterium, Rizobium)
Proteobacteria - gammaproteobacteria
Gammaproteobacteria : Pseudomonadales
Pseudomonas aeruginosa
Pyoverdine production by Pseudomonas aeruginosa
Biofilm formation of Pseudomonas aeruginosa
Enterobacteriales
Eschericia coli
Proteobacteria - Epsilonproteobacteria
Stomach
H.pylori
Actinobacteria/Firmicutes
Gram-positive bacteria
Firmicutes : lactic acid bacteria
Lactobacillus delbruekii
Lactate fermentation
Glucose —> 2pyruvate —> 2lactate
Glycolysis Lactate fermentation
Gram-positive bacteria
Firmicutes : endoscope forming bacteria (clostridium, Bacillus)
Gram-positive bacteria
Firmicutes : endoscope forming bacteria (Clostridium, Bacillus)
Exosporium nap (BcIA)
Exosporium basal layer
Firmicutes : staphylococcus
|| aureus = purple circles in clumps
Staphyloxanthin = beige rounds blobs
Hemolysis (=haemolysis)
- Alpha hemolysis
- Bete hemolysis
- Gamma hemolysis
Alpha hemolysis (α-hemolysis)
Incomplete / partial hemolysis, green hemolysis
Caused by hydrogen peroxide produced by bacteria
Oxidising hemoglobin to green methemoglobin
Beta hemolysis (β-hemolysis)
Complete hemolysis
Caused by enzyme (streptomycin or exotoxin)
Produced by bacteria
Gamma hemolysis γ-hemolysis
Non-haemolytic
Firmicutes : Enterococcus
6 VRE phenotype
Actinobacteria
Actinobacteria
Comparison of microbial genome sizes
Mycoplasma : ~0.6 Mb
Bifidobacteria : ~2 Mb
staphylococci : 2.8-3 Mb
Escherichia : 4.5 - 5 Mb
Actinomycetes : 8-9 Mb
Yesterday : ~14 Mb
Actinobacteria : Corynebacterium
Actinobacteria : Mycobacterium
Mycobacterium tuberculosis
Complex cell envelope of Mycobacterium
Actinomyces
-bovis
-israelii
Streptomyces
Free spore - germination + growth of substrate mycelium - development of aerial mycelium - growth of an apical aerial hypha - coiling of apical hyphal cell - sporulation-specific septum formation - completion of septation- maturation of spores - dispersal of spores
Streptomyces
Spore germination
Vegetative growth
Substrate mycelium
Antibiotic production
Formation of reproductive aerial hyphae
Aerial hypha
Chromosome segregation and separation
Spore mutation
Spore dispersal
- Actinobacteria : Streptomyces (Streptomyces coelicolor)
8,667,507bp
Respiratory infections
Streptococcus pneumoniae, Mycobacterium tuberculosis
Gastrointestinal infection
Camphylobacter jejuni, salmonella species, Shigella species, Escherichia coli 0157:H7, Helicobacter pylori
Skin infection
Enterobacter species, Pseudomonas aeruginosa, Enterococcus species, Staphylococcus species.
Upper respiratory trace
Nasal cavity
Pharynx
Larynx
Lower respiratory tract
Trachea
Primary bronchi
Lungs
Pneumonia
Tuberculosis
Pneumonia
Fluid filling air space in alveolus
Pleurisy
Infection irritating pleura (thin outer layer of lung)
Lung inflammation
Streptococcus pneumoniae
Symp- cough, chest pain, fever, diff breathing
Vaccination, various antibiotics
Streptococcus pneumoniae
Normal inhabitant of human respiratory tract, causes pneumonia
Common cause of bacterial meningitis, ear infections
Gram-positive 0.5-1.25 µm in diameter
doesn’t form spores, non-motile, alpha hemolytic when cultured on blood agar
Encapsulated coccoid bacteria
Polysaccharide capsule (virulence factor)
1 cirlcular chromosome = 2 base pairs, GC content of ~40%
Streptococcus pneumoniae : antibiotic resistance
- Commonly used antibiotics
: β-lactams (penicillin, amoxicillin, ampicillin)
: Cephalosporins (cefotaxime, ceftriaxone)
: Macrolides (erythromycin, azithromycin)
: Fluroquinolones
: Glycopeptides (vancomycin) - Antibiotic resistance
: Penicillin and Cephalosporins – resistance is common, mediated by altered PBP,
transmissible to other pneumococcal strains
: Macrolides – efflux pump or ribosomal mutations
: Quinolones – efflux pump or DNA gyrase mutations
: Vancomycin – no resistance yet
Pneumonia vaccine
- Pneumococcal polysaccharide vaccine (PPV or PPSV) : contains long chains of polysaccharide (sugar) molecules that make up the surface capsule of the bacteria
: a pure polysaccharide vaccine induces only short-term immunity and doesn’t work as well
in children younger than 2 years
: PPSV (Pnuemovax 23 = PPV-23 is known as the latest version) is the first vaccine
derived from a capsular polysaccharide
: widely used in high-risk adults - Pneumococcal conjugate vaccine (PCV)purified capsular polysaccharides from the bacteria that are “conjugated” (or joined) to a
harmless variety of diphtheria toxin
: produces an immune response in infants and antibody booster response to multiple
doses of vaccine
: the British immunisation schedule for infants, with the first dose given at two months old, and is now licensed to protect against invasive disease caused by Streptococcus
pneumoniae in adults aged 50 years and older
: PCV7, PCV13
Tuberculosis (TB)
Attacks lungs
Lethal infectious disease caused by mycobacteria
Spread through air
1/3 infected
1.3 M deaths 2022
Treatment long course of multiple antibiotics
Chronic cough, blood-tinged sputum, fever, night sweats, weight loss
Mycobacterium tuberculosis (MTB)
Causative agent
-rod-shaped, non-motile, related to actinomycetes
-Acid-fast gram-positive or gram-resistant bacteria - waxy coating on cell surface (mycolic acid) = impervious to gram staining
-slow growth: divides 15-20 hours
-strain variation: significant phenotypic differences between clinical isolates
-circular chromosome, GC content 65%: H37Rv strain (4 mega base with 3959 genes)
-multi drug-resistant TB (MDR-TB) extensively drug-resistant TB (XDR-TB) major problem
Chemotherapy protocol for treatment
Isoniazid
Rifampin
Pyrazinamide
Ethambutol
Isoniazid
Rifampin
Salmonella
Food borne illness: gastroenteritis, typhoid fever, paratyphoid fever
-two species, salmonella enterica + salmonella bongori
-zoonotic
-gram-negative, rod-shaped, non-spore-forming, anaerobic, motile with flagella
-0.7-1.5 µm in diameter, 2 - 5µm in length- chemoorganotrophes : obtaining energy from oxidation and reduction reactions using organic
sources
- produce hydrogen sulfide
Salmonella typhi
Salmonella enterica, serotype Typhi
-infects humans only, causes enteric bacterial infection, typhoid fever
-symptoms
High fever, sweating, diarrhoea, inflammation of stomach /+ intestines.
Enlargement of liver + spleen “rose spotted” skin rash on chest + abdomen
- 2 million cases, 500,000 deaths
-treatment
Drug resistance (MDR)
-prevention
Hygienic life style
Vaccines- the live, oral vaccine (Ty21a) + injectable tyhpoid polysaccharide vaccine
- 1st genome sequence S. typhi CT18 (MDR) strain
4.8 Mb circular chromosome, 2 plasmids pHCM1 (MRC) + pHCM2
Escherichia coli 0157:H7
- Gram-negative, rod shaped bacteria
- “O” refers to the cell envelope (LPS) antigen number, “H” refers to the flagella antigen
- Enterohemorrhagic E. coli (EHEC)
- Shiga-like toxin producing E. coli (STEC)
- symptoms of E. coli 0157:H7 infection : a low fever, nausea, vomiting, stomach cramps, and
bloody diarrhoea - fatal condition : hemolytic uremic syndrome (HUS)
- treatment
: non-specific supportive therapy, including hydration, is important
: no evidence that treatment with antibiotics is helpful, and taking antibiotics may increase
the risk of HUS
: antidiarrheal agents such as Imodium should be avoided
as it may prolong the duration of the infection
Helicobacter pylori
Causes gastritis, gastric ulcers + stomach cancer
Helix-shaped, motile, gram-negative, 3 3 µm long with 0.5 µm diameter
- genome : 1.6 Mb with 1590 coding genes, 29% of genome is involved in the pathogenesis
- virulence of Helicobacter pylori
: measured by the ability to produce cotoxin-associated protein (CagA) and active
vacuolating cytotoxin (VacA)
: Cag pathogenicity island : about 40 Kb long, responsible for pathogenesis, usually
absent from H. pylori strains isolated from humans
: other important virulent factors
- surface-exposed lipopolysaccharide
- iron-scavenging system
- urease
- motility for colonization
- type IV secretion system
Helicobacter pylori- treatment, prevention + vaccination
- therapy one week ‘triple therapy’
1 proton pump inhibitor
2 antibiotics - clarithromycin + amoxicillin, || + metronidazole - additional rounds of antibiotic therapy after failure ^
“Quadruple therapy” + bismuth colloid drug
Treatment of clarithromycin-resistant H.pylori vaccine levofloxacin
-prevention
Vaccination
Probiotic diet
Skin infection
Pseudomonas aeruginosa
50% mortality, 4th commonly-isolated nosocomial pathogen
-gram-negative, rod-shaped, motile + long monoflagellum
-Single cell size 1-5 µm long and 0.5-1.0 µm wide
- P. aeruginosa often identified by the production of pyocyanin and fluorescein and ability to grow at 42°C.
- pollution control - break down aromatic hydrocarbons
- intrinsic resistance to antibiotics
P. aeruginosa genome (PA01 strain)
: circular form, 6.2 Mb size with
~65% GC content
Pseudomonas aeruginosa – biofilm formation
reversible attachment
irreversible attachment
microcolony formation
development of 3-dimensional structure
Dispersal
Biofilm formation + motility
Swarming
Swimming
Twitching
Pseudomonas aeruginosa – biofilm formation and quorum sensing
N-acyl homoserine lactone
Pseudomonas aeruginosa – treatment
-low permeability to antibiotics = resistant
- biofilm
-s ome antibiotics have shown to be active against P. aeruginosa
: aminoglycosides (gentamicin, amikacin, tobramycin)
: quinolones (ciprofloxacin, levofloxacin, moxifloxacin)
: carbapenems (meropenem, imipenem, doripenem)
: polymyxins (polymyxinB, colistin)
: monobactams (aztreonam)
: cephalosporines (ceftazidime, cefepime, cefoperazone, cefpirome)
: antipseudomonal penicillins (ureidopenicillins, carboxypenicillins)
- phage therapy combined with antibiotics
Enterococcus species
- nosocomial infections - UTI
-genus of lactic acid bacteria
-tolerant of wide range of environmental conditions 10-45 °C), pH (4.5-10) and high sodium chloride concentrations
-common in intestines : Enterococcus faecalis (90-95%), Enterococcus faecium (5-10%) - pathogenicity : cytolysin, a toxin causes rupture of a variety of target membranes, including
bacterial cells, erythrocytes, and other mammalian cells - vancomycin resistant enterococcus (VRE)
Vancomycin Resistant Enterococcus (VRE)
-1st VRE genome sequence from VanB-type Enterococcus faecalis V583 strain
: 1 circular chromosome with 3.2 Mb long (3337 genes), 3 plasmids
: more than 25% of total genes are exogenously acquired
- 7 integrated phage regions
- 38 insertion elements
- conjugative and composite transposons
- pathogenicity island
- vancomycin resistance genes encoded within a previously unknown mobile element
in genomic DNA (similar to Tn1549 in other VanB-type E. faecalis)
- some other VanA-type VRE carry van resistance allele in plasmid
: pIP816 in E. faecium BM4147
- 11 kb long, 9 encoding vancomycin resistance cluster
- orf1, orf2, vanR, vanS, vanHAX, vanY, vanZ
Staphylococccus species
Greek = bunch of grapes + granule
-gram-positive (thicker peptidoglycan cell wall)+ immotile
- 40 species
-yellow colonies - S. aureus
White colonies - S. epidermidis
-first genome sequence
S. aureus strain NCTC8325
: circular genomic DNA with 2.8 Mb long with
2900 genes.
: 33% GC content
Staphylococcus aureus
Most common
-nosocomial infections, post surgical wound infections
-successsful pathogen - immunodeficiency-evasive strategies
-emergence of MRSA (Methicillin-Resistant S. aureus)
: resistant to almost all clinically available β-lactam antibiotics including penicilins and
cephalosporins
: vancomycin became the primary antibiotic used to combat MRSA
: due to mecA encodes a modified PBP
MRSA
1.27 million (direct) 4.95 million (associated)
Drug resistance in six pathogens 929,000 (direct) + 3.57 million (associated)
Escherichia coli
Staphylococcus aureus
Klebsiella pneumoniae
Streptococcus pneumoniae
Acinetobacter baumannii
Pseudomonas aeruginosa
MRSA itself > 100,000
Pharmageddon?!
Staphylococcus aureus :VRSE
Resistant to vancomycin + teicoplanin
-acquired VanA type vancomycin resistance genes from VanA-type VRE via horizontal gene - transfer mediated by transposon
-antibiotics
Trimethoprim/sulfamethoxazole, quinuprisitin/dalfopristin, tigecycline, linezolid and daptomycin
- novel antibiotics against VRSA is urgently required
Vancomycin resistant MRSA = VRSA (carrying mecA and vanA genes)
