Lecture 1 Mycoplasma, Lactic acid bacteria inc Streptococci 2023 PDF

Gram positive bacteria – Topic 1 The Mycoplasmas Graham Stewart BMS2037 Cellular Microbiology and Virology GRAM-POSITIVE BACTERIA Graham Stewart [email protected] Eighties chart topper contracts MRSA after brain haemorrhage By Ian Evans...

Gram positive bacteria – Topic 1 The Mycoplasmas Graham Stewart BMS2037 Cellular Microbiology and Virology GRAM-POSITIVE BACTERIA Graham Stewart [email protected] Eighties chart topper contracts MRSA after brain haemorrhage By Ian Evans Menacing Rampant Superbug Slammed over MRSA superbug Superbug left me hours from death says Hollywood Swank Superbug Scandal Superbug shame Sore toe girl gets superbug Docs and Nurses left superbug mum to die Superbug victims in secret TV mop blitz Kardashian clan 'freaking out’ Marine is killed by new superbug over Khloe’s staph infection… How to beat deadly superbug MRSA hits hundreds of babies Row over high rate of MRSA superbug Hospital superbug killed my husband 2019 The Firmicutes- Actinobacteria – Low GC gram-positive bacteria the high GC gram positive bacteria Non Spore forming bacteria The lactic acid bacteria Streptomyces Streptococcus Lactobacillus Corynebacteria Staphylococcus Mycobacteria Listeria Mycoplasma The spore forming bacteria Clostridium Bacillus  Mycoplasmas Completely lack cell wall - only plasma membrane. Therefore they stain gram negative but sequencing of their genes reveals that they The Mollicutes are clearly phylogenetically related to the low GC gram positives( Lactobacillus- Clostridium branch) Latin mollis, soft; cutis, skin Sensitive to osmotic lysis. Plasma membrane is strengthened by inclusion of sterols. Plasma membrane may also include lipoglycans (heteropolysaccharide linked to the membrane lipids) and lipoproteins – involved in avoiding the immune response. Pleomorphic – big, small, long, branched…… the coccoid cells 0.12µm -0.25µm in diameter are probably the smallest living cells. Easily deformable cells that can squeeze into different shapes. Most mycoplasmas are non-motile, except some human and animal pathogens (eg. M. pneumoniae, M. genitalium) Growth of Mycoplasmas All are parasites of eukaryotes Many can be grown outside of cells in lab media Mycoplasma can have complex growth factor requirements due to their Limited biosynthetic capabilities All require sterols except Acholeplasma, Asteroleplasma and Mesoplasma Many require a variety of vitamins, fatty acids, amino acids, purines and pyrimidines On solid agar they form a fried egg shaped colony and embed into the media  Mycoplasma Genomics GC content approx 25-35 % depending on the species. Mycoplasma genitalium has the smallest known genome of any self-replicating Organism measuring only 580 kbp and encoding 472 genes. Close to the smallest amount of DNA capable of encoding a living cell The genome of Mycoplasma pneumoniae is larger at 816 kbp, containing 679 putative genes. For comparison: Escherichia coli genome = 5440 kbp Human genome = 3,300,000 During the evolution of mycoplasma, multiple reductions in genome size have occurred and the overall rate of evolution uncharacteristically high. WHY?  Overview of the Mollicutes (Mycoplasma-like organisms) Mycoplasmas - require cholesterol. Parasites of animal mucous membranes. Facultative anaerobes. Over 60 species recognised. Ureaplasma (T-Strain Mycoplasmas) - microaerophilic. Require cholesterol and urea for growth. Urease - urea -> ammonia and CO2. Tiny colonies that are not visible with naked eye. May be associated with nongonococcal urethritis but can be isolated from genital tract of 15% asympomatic people. Maybe disease only on initial exposure? However transmitted by sexual contact. Also been associated with arthritis animal models - but not isolated from tissue. Acholeplasma - widely distributed animal parasites. Facultative aerobes. Able to grow in absence of sterols. Common contaminants of cell lines in tissue culture. Anaeroplasma - strict anaerobes. Inhabit bovine or ovine rumen. Spiroplasma - helical, motile. parasites on arthropods and plants, but a few cause disease in animals  Mycoplasma pneumoniae Causes primary atypical pneumonia in humans, PAP, sometimes called "walking pneumonia“ Usually in individuals 5-20 years old Symptoms: Range from mild flu-like disease headache, sore throat, cough to severe pneumonia characterised by fever, chills and malaise Protracted course with gradual resolution (month) Mycoplasma pneumoniae: respiratory pathogen Mycoplasma pneumoniae is communicated through close personal contact via respiratory droplets. May be a prevalence of infections occurring in autumn. The bacterium settles on a respiratory epithelial cell and binds to it. Polar tip organelles - specific adhesion P1 protein, interactive proteins, and adherence- accessory proteins. Result – colonization of mucous membranes and eukaryotic cell surfaces The bacterium produces hydrogen peroxide which is thought to be responsible for much of the initial cell disruption in the respiratory tract. Mycoplasma interacting with an epithelial cell  M. pneumoniae: Diagnosis and treatment Diagnosis Clinical symptoms including a patchy diffuse bronchopneumonia by X-Ray Serological tests (Serum cold agglutination, Complement fixation test, ELISAs) DNA test (PCR) is replacing serological test Microbiological detection: Not that useful because culture is slow. Take sputum, or throat swab. M. pneumoniae can be grown in media containing serum with penicillin to inhibit growth of other bacteria. Beta-hemolytic Fluorescent antibody N.B. Accurate diagnosis is important since organism is resistant to antibiotics that act on the cell wall (penicillins, cephalosporins). Treatment Erythromycin, doxycycline (not children), azithromycin, levofoxacin. Erythromycin is often used as it is also effective against legionella.  GENITAL MYCOPLASMAS Mycoplasma genitalium M. hominis and Ureaplasma urealyticum 50% of normal adults have antibodies Causes of non-gonnococcal (non-chlamydial) urethritis NGU May also be associated with infertility in men Woman – associated with cervicitis and endometritis and serologically with tubal factor infertility Transmitted by direct venereal contact. Also vertically transmitted from mother to offspring at delivery or directly to the fetus – pneumonia, bacteremia and meningitis. Detection relies on molecular techniques (PCR) due to difficulty in culturing UK recommended Treatment with doxycycline pretreatment then azithromycin (or moxifloxacin) Antibiotic resistance is high! (azithromycin is a macrolide, moxifloxacin is a fluoroquinolone)  Spiroplasma kunkeli (in a phloem cell) Spiroplasma citrii Spiroplasma and plants Citrus stubborn disease leafhopper Corn stunt disease Gram positive bacteria – Topic 2 The Lactic Acid Bacteria Including Streptococcus BMS2037 The Firmicutes- Actinobacteria – Low GC gram-positive bacteria the high GC gram positive bacteria Non Spore forming bacteria The lactic acid bacteria Streptomyces Streptococcus Lactobacillus Corynebacteria Staphylococcus Mycobacteria Listeria Mycoplasma The spore forming bacteria Clostridium Bacillus  The lactic acid bacteria The lactic acid bacteria produce lactic acid as a product of fermentation Non spore forming non motile. They lack cytochromes and obtain energy by substrate level phosphorylation and not by Electron transport and oxidative phosphorylation Normally obtain energy only from sugars Only limited biosynthetic capability and require many vitamins, amino acids purines and pyrimidines Aerotolerant anaerobes. Growth not affected by presence of air. Usually grown on media containing yeast, peptone, supplemented by a fermentable carbohydrate. Even when grown on rich media, the colonies are usually small, rarely pigmented. They are also tolerant of acid - many other bacteria cannot grow at pH 6. The ability to produce and tolerate high concentrations of lactic acid is of great selective value. - eliminates competition - also used in selective media  The main genera Streptococcus - chains of cells - divides in one plane. Lactobacillus - rods Pediococcus – pairs or tetrads Leuconostoc - spheres in chains  Lactic Acid Bacteria can be grouped by their fermentation patterns Homofermentative – produces a single fermentation product – lactic acid Embden- Meyerhof (glycolytic) pathway The fermentation of 1 mole of glucose yields two moles of lactic acid; C6H12O6 2 CH3CHOHCOOH Eg. Strep. Enterrococcus Glucose lactic acid Heterofermentative – produces lactic acid and other products, mainly CO2 and ethanol, uses pentose phosphate pathway The fermentation of 1 mole of glucose yields 1 mole each of lactic acid, ethanol and carbon dioxide; C6H12O6 CH3CHOHCOOH + C2H5OH + CO2 Glucose lactic acid ethanol carbon dioxide Eg. Leuconostoc  Lactic acid bacteria and dairy products Major known Species Product function Lactobacillus bulgaricus Lactobacillus lactis Bulgarian buttermilk, yoghurt, Acid and kefir, koumiss, Swiss, Lactobacillus helveticus flavour Emmental, and Italian cheeses Lactobacillus acidophilus Acid acidophilus buttermilk Emmental, Cheddar, and Streptococcus thermophilus Acid Italian cheeses, and yogurt Sour cream, ripe cream, butter, cheese, buttermilk and Streptococcus diacetilactis Acid starter cultures. Cultured buttermilk, sour Streptococcus lactis cream, cottage cheese, all types of foreign and domestic Streptococcus cremoris Acid cheeses, and starter cultures. Streptococcus durans Acid and Soft Italian, cheddar, and Enterrococcus faecalis flavour some Swiss cheeses. Leuconostoc citrovorum Cultured buttermilk, sourcream, cottage cheese, ripened cream butter, and Leuconostoc dextranicum Flavor starter cultures. Many LAB are Probiotics Go get a yoghurt and enjoy whilst cogitating some of the more unsavoury members of the LAB  Streptococcus Spherical cells divide only in one plain and so form chains Require complex media for growth Unable to make haem group of cytochromes or catalase Grown on blood agar (good source of catalase) Streptococcus Grouping of Streptococci I: Hemolysis Hemolysins: toxins  - complete hemolysis -> clear zone. - incomplete hemolysis of red blood cells to produce a greenish-brown zone  - hemolytic - no hemolysis Grouping of Streptococci II: Antigenically/Serologically Lancefield Groups of ß-hemolytic Streps Named after Rebecca Lancefield A, B, C, D…H and K-W based on the specific carbohydrate antigen extracted by heating cells up to 150°C (About 20 groups) Group A - S. pyogenes Group B - Cattle and humans eg S. agalactiae Group D - Intestinal tract of man and animals Enterococcus Etc etc R. C. Lancefield: The antigenic complex of Streptococcus haemolyticus. I. Demonstration of a type-specific substance in extracts of Streptococcus heamolyticus. Journal of Experimental Medicine, 1928, 47: 481-491. R. C. Lancefield: A serological differentiation of human and other groups of heamolytic streptococci. Journal of Experimental Medicine, New York, 1933, 57: 571-595. Group A Strep. = Streptococcus pyogenes  hemolytic It causes many of the streptococcal diseases of humans. Antigenicity of M-proteins in the cell wall provides sub-classification of group A into over 100 sub-serotypes. Habitat: 5 - 30% of healthy people may be carriers of group A, beta hemolytic Streps in their throat and nasopharynx; however numbers usually low. Bacitracin sensitivity test Strep Throat Impetigo Streptococcal impetigo. Mostly in young children. Small vesicles on the skin to form a thin amber crust.  Scarlet Fever Usually result of Strep sore throat caused by a pyrogenic toxin-producing organism -Superantigen toxin encoded by a phage. Symptoms- Fever, toxic shock, erythematous rash. Three antigenically distinct toxins (A, B and C) - immunity to one does not protect against the others. Resurgence in UK - in 2014/15 a total of 5,746 cases were recorded. 2018 highest rates for 50 years Cellulitis - infection of the deep layers of the skin. Necrotizing fasciitis – highly invasive, toxigenic, infection of soft tissues and fascia LOOK AWAY NOW IF YOU ARE SQUEEMISH  Warning! Skip the next slide if you are squeamish! From Lippincott’s microbiological reviews  Non-suppurative diseases Rheumatic fever. It occurs in a small percentage of individuals, 2 - 3 weeks after an untreated pharyngeal infection that was caused by a  hemolytic group A Strep. Joints and heart affected Recovery occurs without residual injury to the joints but serious damage to heart valves cause rheumatic heart disease – used to be one of the most common heart conditions. Still a major problem in developing countries and in overcrowded conditions. May be a resurgence occurring in USA. May be an immunological reaction - antibody to Strep also binding to heart tissue.  Non-suppurative diseases Glomerulonephritis. Most cases of glomerulonephritis occur about a week after group A Strep infection (skin or throat). Also thought to be an immunolgical reaction in which a Strep-directed antibody reacts with the glomerular basement membrane; or antibody-antigen complexes are deposited onto basement membrane. Leads to loss of protein (blood initially in urine) through kidneys and hypertension. Can lead to chronic glomerulonephritis and kidney failure.  2022 Group B Strep. Prominent cases in this report leading to mortality or disabilities Other Streptococci Group B Streptocci: S. agalactiae is the predominant species in this Lancefield group Present in vaginal flora of 25% of woman. May cause serious infections in newborn - septicaemia, pneumonia, meningitis with high fatality rates Hippurate test Some streps can Hydrolyse sodium hippurate Group D Streptococci - faecal flora - enterococci. Urinary and wound infections. Major problem of vancomycin-resistance. In just ten years resistance has increased from virtually zero to nearly 50% in some communities. α-hemolytic Streptococci Viridans group: Strep. Viridans is not a species but a large group of bacteria include many species that are normal inhabitants of throat and nasopharynx of humans. One example of a viridans species is S.mutans which causes dental caries Viridans Strep are a significant cause of bacterial endocarditis - infection of heart valve - invariably fatal if not treated.  Take another break! Maybe go and inspect your tonsils OR brush that yoghurt off your teeth whilst cogitating Streptococcus viridans!  Streptococcus pneumoniae Lancet shaped organism - usually arranged in pairs. Virulent organisms are encapsulated. -resist phagocytosis. Subdivided into 90 types on the basis of antigenicity of capsular polysaccaride. Habitat: normal commensal of the upper respiratory tract.  Streptococcal pneumonia Acute lung inflammation. Chills, fever and pleural pain. Alveoli fill with exudate. Bacteremia occurs in 25%. Pneumococci may invade other tissues, eg. sinuses, middle ear and meninges. Recovery is abrupt and coincides with appearance of circulating antibodies. [Note: many other causes of pneumonia - Mycoplasma pneumoniae, Legionella, etc.] Accounts for about one million deaths per annum worldwide. Has a 10 - 20% mortality Pneumococcus is also the second most common cause of meningitis in adults. Also cause of middle ear infection – otitis media. Also causes acute exacerbations of chronic bronchitis.  Diagnosis S.pneumoniae Direct smears of sputum are examined for G+, encapsulated cocci. Also perform quellung test - encapsulated bacteria + type-specific antibody -> capsular swelling - capsule becomes more visible and refractile due to antiboby attachment. Growth of organism on blood agar – a-hemolytic mucoid colonies, sunken in center - doughnut shaped (autolytic enzymes). Colonies then tested by: bile solubility test (10% deoxycholate drop onto colony - colony dissolves). Optochin disks - inpregnated with ethylhydrocuprine hydrochloride - are laid on the surface of an agar plate inoculated with unknown organism. Pneumococci are extremely sensitive to compound and will fail to grow in proximity of disk.  Treatment and vaccination Usually treated with penicillin G, except for meningitis which is often treated with chloramphenicol. However, strains of multiple-resistant pneumococci have appeared and are becoming increasingly common in some parts of the world (Spain). These are presently treated with vancomycin but there are fears that resistance will spread from enterococci. Vaccine has been made containing polysaccharide from 23 of the most prevalent types. Gives 75 - 95% protection. Recommended for high-risk groups, eg. those with chronic respiratory disease. Given to all children in UK (since 2006) and elderly age groups.