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Bacteriology Lecture 25 Respiratory pathogens

Specific learning objectives- 25 •

Describe bacterial respiratory tract pathogens and diseases.

Gram positive rods: - Corynebacterium, Mycobacterium

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Understand the principles of the TB skin test and vaccine.

Gram negative coccobacilli/cocci: - Pasteurella, Haemophilus, Bordetella, Francisella, Moraxella, Neisseria, Legionella

1. Respiratory tract pathogens Gram positive rods •

Aerobes

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No endospores

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Filaments

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Corynebacterium

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Actinomycetales (ORDER)

- Actinomyces (not acid-fast) - Mycobacterium (acid-fast)

CORYNEBACTERIUM Pleomorphic, Gram positive rods, stain irregularly, many species

Clinical disease •

Occ. infection with “animal” coryneforms

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e.g. Corynebacterium equi in AIDS patients: pneumonia

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C. ulcerans: pharangitis etc

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C. diptheriae (discovered in 1884) - localised pharangitis - generalised toxaemia Not all strains are toxigenic (phagemediated) - present in developing countries - many carriers - vaccination

MYCOBACTERIUM •

aerobic non-motile, capsulated rods

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thick waxy wall (extra layer of polysaccharides): very resistant

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survive intracellularly

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“acid fast” (stain red) in the Ziehl Neelsen (ZN) stain

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grow slowly, many species

Granulomatous infections •

Persistent bacterial infections characterised by a strong local host immune response causing localised tissue damage

Tuberculosis complex M. tuberculosis* M. africanum M. bovis* M. avium (avian tuberculosis) M. intracellulare M. scrofulaceum Are all occasional pathogens of humans and animals Cause “tuberculosis” in humans, esp. immunosuppressed Also cause M. bovis + M. tuberculosis cross-reactions that make interpretation of tests more difficult

Human tuberculosis (TB) M. tuberculosis (occasionally M. bovis) 15 X 106 people infected 3 x 106 die each year Common in HIV patients Multidrug resistant strains = XDR-TB (extensive drug resistant); rapidly fatal in eg HIV patients Mantoux testing: chest x-rays BCG vaccine (Bacille Calmette-Guerin) Chronic infections Stress can lead to breakdown Ø

“Miliary”-lung, liver spleen

Ø

tiny spots like millet seeds

Treatment: 6 months +

Tuberculosis Lesions depend on: •

Infective dose

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Route of infection

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Virulence

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Resistance of host

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Delayed type hypersensitivity immune response (DTH)

The bacteria multiply within phagocytic cells causing granulomatous lesions, necrosis, caseation, calcification.

2. TB Diagnosis •

Chest X-rays

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Ziehl-Neelson stain

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Culture

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Animal inoculation

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Tuberculin testing

TB skin test •

Mantoux tuberculin skin test (TST)

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Determine infection with M. tuberculosis

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Inject 0.1ml tuberculin purified protein derivative (PPD) intradermally into skin to produce a wheal

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48-72 hours later test is read

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Reaction is measured

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(palpable, raised, hardened area or swelling)

Tuberculosis vaccine •

BCG vaccine (Bacille Calmette-Guerin, named after Dr Calmette and Dr Guerin who developed the vaccine in 1921)

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Live attenuated vaccine (M. bovis) stimulates cellmediated immunity

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Strain is similar to Mycobacterium tuberculosis

TB Treatment •

Prolonged 6 month treatment with multiple antibiotic drugs (emerging drug resistant strains)

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Australian rapper in quarantine:180 days

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https://www.youtube.com/watch?v=MqGLHluDo e0

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3% of cases in Australia are MDR-TB!

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TB epidemics in other countries (eg PNG)

Mycobacteria causing skin lesions M. ulcerans “Buruli ulcer” M. marinum: fish and “swimming pool ulcers” M. leprae M. lepraemurium

Bentley and Sebaihia Nature Reviews Microbiology 5, 170–171 (March 2007) | doi:10.1038/nrmicro1633

Leprosy •

Multiplies slowly in Schwann cells > anaesthesia (nerve damage) + muscle paralysis

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Common disease

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Bacterial infiltrates: skin lesions

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- spectrum of activity: Granulomas > little response

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Granulomas of nerves, respiratory tract, skin and eyes

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Transmission? Contact or respiratory route or insects

• Curable: Dapsone + rifampicin + clofazimine Symptoms can take 20 years to appear!

Ancient disease Biblical reference 40 times Laws about leprosy (people and garments) Leviticus 13: 1-59 Skin eruptions, (boils) hair turns white on spots, raw flesh in the swellings, covered head to toe… Priest pronounced clean >wash clothes, examine, burn Shut up for seven days or if unclean (leper)> torn clothes Live alone, let hair grow long, dwell outside the camp

3. Gram negative coccobacilli/cocci Pasteurella Haemophilus Bordetella Francisella Moraxella Neisseria Legionella All are - shape intermediate between a cocci and bacilli (very short rods) Respiratory (or genital) carriage (obligate) Commensals and opportunists “Stress” > disease, respiratory disease and septicaemias

PASTEURELLA P. multocida: antigenic types based on capsular (5 types) and O antigens (11 somatic {cell} serotypes) in numerous combinations non-motile, catalase +, oxidase + Penicillin sensitive Zoonotic (pet bites- cats, dogs rabbits)> bite wounds > cellulitis, adenitis, osteomyelitis Occ. pleurisy/pneumonia

Bacterin vaccines widely used

HAEMOPHILUS

Gram negative coccobaccilli Prefer CO2 enrichment Aerobic or Facultative anaerobic Chocolate agar growth

Require blood factor X (hemin) and/or V (nicotinamide adenine dinucleotide) (will not grow on blood agar plates)

Haemophilus influenzae - several capsular types; type “b” most invasive - non-capsulated strains also common Localised respiratory and middle ear infections, usually secondary to viral infections -

Causes sepsis and bacterial meningitis in young children

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Serogroup b vaccines available (Hib vaccine)

Biogroup aegyptius - conjunctivitis + septicaemia = “Brazilian purpuric fever”

Streptococcus pneumoniae “The pneumococcus” •

No group

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Capsulated: 90 + capsular serotypes therefore polyvalent vaccines (23 capsular types)

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Multi-resistant strains emerging Middle ear infections Sinuses Lungs Joints/meninges

BORDETELLA Gram negative encapsulated coccobacilli Obligate aerobes Difficult to culture (fastidious) Bordetella pertussis: “Whooping cough” Local infection, no septicaemia Transmitted by direct contact or via aerosol droplets or fomites Adhere to trachea/bronchiolar epithelium (ciliated epithelial cells) using adhesins, release cytotoxin (peptide derived from peptidoglycan) > cilia stop beating: mucus builds up (paralysis of the mucociliary escalator, inhibits DNA synthesis, cells die), another toxin kills phagocytes (evades immune response) Bouts of paroxysmal coughing: tongue out, sucking in air, cyanotic

Whooping cough Whooping cough still a common cause of death in developing countries esp. > 1 year old https://www.youtube.com/watch?v=S3oZrMGDMMw

Epidemics ~ 4 years as “herd immunity” wains Bacterin vaccines – 3 antigenic types. Babies at 2 – 3 months. Occasional adverse reactions. Disease is common when no vaccine used Many mild cases in older children/incompletely vaccinated > chronic and infectious to other children B. parapertussis > milder form of whooping cough

FRANCISELLA Gram negative coccobacilli Invade macrophages Strict aerobes, non-motile Difficult to culture (fastidious), slow growth but survives several weeks in environment F. tularensis > Tularaemia (rabbit fever): zoonosis Cycles in wildlife: USA, Asia, parts of Europe (different subspecies, with different pathogenic potentials) Acute septicaemia eg after tick bites Can be inhaled (= lawnmower disease) Swollen lymph nodes, granulomas in liver + spleen “Biological warfare select agent”

MORAXELLA Gram negative coccobacilli, commensals of mucosal surfaces Catalase and oxidase positive M. lacunata: conjunctivitis in humans (guinea pigs) zoonotic Attaches to conjunctiva via pili, releases dermonecrotic protease toxins Ø

conjunctivitis > keratitis > cornea opaque > rupture > photophobia > blindness; weeks to recover even from milder forms

Vaccines are used, but many antigenic types M. catarrhalis -

commensal in humans (diplococci)

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chronic chest infections, otitis media,sinusitis

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produces beta-lactamases

NEISSERIA 11 species colonise humans but only 2 are pathogenic: N. meningitidis = the ‘meningococcus’ Septicaemia

N. gonorrhoeae = the ‘gonococcus’ Gonorrhea

Pairs of cocci- diplococci

N. Meningitidis: capsular types A, B, C, W, Y •

Gram negative diplococci, grow best at 37°C, pili

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Invades neutrophils, avoids phagocytosis and complement cytotoxicity, and can vary surface antigens (immune evasion)

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Carriers in resp. tract (5%)

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Can cause acute septicaemic disease (purpuric rash) and/or acute purulent meningitis

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Also pneumonia/arthritis etc

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Highest incidence in < 5 yrs and 15-19 yrs

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Low antibody titres

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Epidemics occur + sporadic cases (military personnel, athletes)

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Previously only group C vaccines were available (= 1/3 of cases), now also group A, B, W and Y vaccines

LEGIONELLA Gram negative environmental organism causing Legionellosis -

Legionnaire’s disease (pneumonia type illness)

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Pontiac fever (mild flu-like illness)

L. pneumophila - esp. air conditioners, water spas etc L. longbeachae – soil, potting compost etc Saprophytic organisms: • obtains nutrients from dead organic matter • preys on protozoa > if inhaled can cause acute and severe pneumonia; often fatal in the elderly • bacteria survive in monocytes • occasional renal + nervous involvement • Ideal growth range 32-42°C

Legionellosis •

Airborne via respiratory droplets (travels 6 km in wind)

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Inhaled bacteria can live in alveolar macrophages

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Flu-like illness at onset

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Advanced stage involves GIT and nervous system, diarrhea and nausea, pneumonia

Sources of Legionella: •

Swimming pools, cooling towers (airconditioning systems for shopping centres, hotels), domestic showers, ice making machines, spas, hot springs, fountains, industrial coolant, windscreen washer fluid. No vaccine

Summary •

Respiratory pathogens - corynebacteria - mycobacteria

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Granulomatous infections (TB and leprosy)

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Tuberculosis (TB) - diagnostic tests - vaccines - treatments

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pasteurella, haemophilus, strep pneumoniae, bordetella, francisella, moraxella, neisseria, legionella

Bacteriology Lecture 26 Mycoplasmas

Specific learning objectives- 26 •

Describe features of mycoplasmas and their associated diseases.

Mycoplasmas • Prokaryotes (separate to bacteria) • Smallest known prokaryotes (3001000nm) • Multiply by binary fission • Lack the cell wall characteristic of bacteria and are bounded instead by a bi-laminar membrane similar to animal cells (thus resistant to most antibiotics, eg penicillin) • Contain intracellular bacterial-like organelles • Can not synthesise peptidoglycan precursors

Mycoplasmas

• Pleomorphic - roughly spherical but tendency to form filaments • Characteristic small "fried egg" colony on agar • Central dense core embedded in agar and a lighter peripheral zone on the surface • Smallest genome of any of the independently replicating organisms

Kingdom Prokaryote Division III Mollicutes •

Latin: “soft skinned”

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Considered a virus until early 1960s or fungus

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Growth in media

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PCR >800,000 nt (689 coding genes)

UTIs

Mycoplasmas cont. • Mycoplasma is a common name for an entire group of organisms in the class Mollicutes. • However, one genera is designated Mycoplasma. • Difficult to culture. • Ubiquitous; found in animals, plants, soil, compost. • About 10% of cell cultures are contaminated with mycoplasmas: difficult to detect and eliminate.

1-4 weeks incubation period People at risk: in schools, universities, college dorms, military barracks, nursing homes, hospitals, those recovering from respiratory illnesses, weakened immune system, asthma. >2 million people infected with mycoplasma/year in USA.

(anaemia and jaundice)

Severe complicationsSerious pneumonia, encephalitis, hemolytic anaemia, renal dysfunction, skin disorders

Primary atypical pneumonia Mycoplasma pneumoniae Smallest bacterium discovered 0.31µm Common in children and young adults Cause of up to 30% of all pneumonia cases “Walking pneumonia”

Nutritional requirements •

Not easy to culture as they have limited genetic information and fastidious nutritional requirements

•

The media for culture needs to contain an extensive range of vitamins and co-factors Nucleic acid precursors are required as mycoplasmas lack the orotic acid pathway for pyrimidine synthesis and the enzymatic pathways for de novo synthesis of purine base. Long chain fatty acids must also be supplied (cannot be synthesised)

Two basic energy-yielding mechanisms produce toxic metabolites Fermentative Fermentation of sugars via a glycolytic pathway and during glycolysis. End products (which may be toxic to cells) include lactate, acetate, pyruvate, acetyl methyl carbinol.

Non-fermentative Enables mycoplasma to derive most of their energy (ATP) requirements from arginine. Arginine

Citrulline + NH3

Citrulline

Carbamoyl phosphat

Carbamoyl phosphate

ATP + NH3

Ureaplasma species Require urea for growth - hydrolysed to NH3 and carbon dioxide

Urea

NH 3

CO 2

Extracellular toxic products important in pathogenesisVirulence factors •

Ammonia

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Mycoplasmas lack catalase, which leads to an accumulation of H2O2, although most of it would be degraded by host peroxidases.

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Acids are produced during fermentative metabolism.

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Some mycoplasmas have a glycoprotein capsule which can have toxic properties.

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Haemolysins (including H2O2)

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Nucleases and other enzymes

Intimate association with cell surface Mycoplasmas are extracellular parasites but are closely associated with the external membrane of cells.

Laboratory Diagnosis Isolation of mycoplasma •

Specimens are initially inoculated into broth medium, then later transferred from broth to agar where they produce the typical fried egg type colony after several days.

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Final identification of species is accomplished by biochemical and serological tests.

PCR – an emerging technology Serological tests for antibody •

Presence of antibody as a guide to whether current infection.

Antibiotics •

Mycoplasmas are resistant to antibiotics inhibiting cell wall synthesis.

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Tetracyclines (4 carbon rings) are most commonly used and inhibit protein synthesis (bacteriostatic). (Also used for prophylactic treatment for anthrax, bubonic plague and malaria)

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After infection, animals commonly excrete (shed) mycoplasmas for long periods and this is not prevented by antibiotic therapy. While antibiotics may cause remission of clinical signs, the mycoplasmas continue to be shed.

Superhuman- microbiome

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https://edutv.informit.com.au/watchscreen.php?videoID=737758

Summary •

Mycoplasmas

- features -

infections

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diagnosis

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treatments

Bacteriology Lecture 27 Symbionts and pathogens

Specific learning objectives- 27 •

Explain the different types of symbiosis.

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Understand the importance of the microbiome.

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Describe bacterial pathogenicity.

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Discuss the host innate immune responses against bacterial infection.

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Describe the mechanisms of immune evasion by bacteria.

Hosts are colonised by symbionts •

The host and its microbes coexist in a state of dynamic cooperation.

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In most symbioses the partners are of different size. - the larger partner is the host - the smaller partner is the symbiont gut bacteria

E. Coli on skin

Colonisation of the body •

In healthy people, the internal tissues eg. blood, brain, muscle etc. are normally free of microorganisms (but now controversial).

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The body surfaces are colonised by a complex community of microorganisms.

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There is a range of interactions between different types of symbiosis.

4

1. Types of Symbiosis •

Commensalism - symbiont benefits, no harm or benefit to the host

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Mutualism- both host and symbiont benefit an obligatory relationship

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Parasitism- the symbiont benefits at the expense of the host 5

Normal Bacterial Flora

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The benefits for a symbiotic life may involve nutrient exchange between the host and bacterial symbiont.

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Host cannot: - synthesise some amino acids & vitamins - break down some complex molecules

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The bacterial partner adds value.

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Together both partners have advantages over a single species.

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Bacteria in large intestine •

More than 800 bacterial species (>7,000 strains) are estimated to inhabit the colon, most of these are anaerobes.

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The majority cannot be cultured by conventional methods.

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Numbers and variety have been estimated by extracting DNA and looking at 16S rRNA sequence variation.

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16S ribosomal RNA (rRNA) •

16S rRNA is a component of prokaryote ribosomes

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~1500 bases in the 16S rRNA gene

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This highly conserved gene is used for phylogenetic studies of bacteria