Lect. 3 Systematic Microbiology PDF

Systematic Microbiology Lecture 3 Elshaymaa Abdel-sattar Lecturer of Medical Microbiology and Immunology Faculty of Pharmacy-SVU  Gram positive bacilli (rods) Gram Positive Bacilli are classified into 1- Aerobic Gram positive No...

Systematic Microbiology Lecture 3 Elshaymaa Abdel-sattar Lecturer of Medical Microbiology and Immunology Faculty of Pharmacy-SVU  Gram positive bacilli (rods) Gram Positive Bacilli are classified into 1- Aerobic Gram positive Non spore forming bacilli Gram positive 2- Aerobic Gram positive bacilli spore forming bacilli 3- Anaerobic Gram positive spore forming bacilli  Treatment and prevention of Anthrax: Antimicrobial agent according to antimicrobial sensitivity test. Penicillin Additional antibiotic options Ciprofloxacin or Doxycycline Course of treatment: 60 days as spores may be able to remain dormant in the lungs and then germinate. ** Prevention: applying protective measures (handling animals, working in susceptible places). Vaccination available: Cell-free filtrate (AVA – Anthrax vaccine adsorbed), five IM injections over 18 weeks & annual booster.  2- Aerobic Gram positive spore forming bacilli b) B. cereus causes opportunistic and causes two distinct forms of food poisoning: 1. The emetic type by a heat-stable enterotoxin. 2. The diarrhoeal type by a heat-labile enterotoxin. c) Bacillus stearothermophilus (Sterilization test bacilli) B. stearothermophilus spores withstand 121°C for up to 12 min. They are used for testing the efficiency of autoclaves by including strips containing the organism within the material being autoclaved and then culturing for surviving spores. B. subtilis and its variant B. globigi have been used to test ethylene oxide sterilizers. Characteristics Anthrax Bacilli Anthracoid Bacilli Motility Non-Motile Generally Motile *Capsule Capsulated Non-Capsulated Shape Grow in long chains Grow in short chains Medusa Head Colony Rough irregular *Nutrient agar colonies Hemolysis absent or Hemolysis Usually well marked weak. Slow Gelatin *Gelatin Rapid Gelatin Liquefaction Liquefaction Liquefaction Inverted tree Disease Anthrax Food poisoning *Ascoli’s test positive Negative Pathogenic to Pathogenesis Nonpathogenic laboratory animals  2- Anaerobic Gram positive spore forming bacilli Clostrida (Clostridium) These are Gram-positive rods, anaerobic, spore forming bacteria. They are found in the environment (particularly soil). 1- Clostridium tetani 1- Microorganism: C. tetani, a gram-positive rod that forms a terminal spore "drum-stick appearance". Commonly found in the soil, dust and animal feces.  2- Mode of transmission: Contamination of wounds, which provide anaerobic conditions, can lead to spore germination, a relatively rare (in western countries) but frequently fatal disease. In third world countries, many (about half) of tetanus cases are in neonates where the unhealed umbilical stump becomes infected, often as a result of cutting the umbilical cord with a contaminated knife. 3- Toxin Production:Cl. tetani produces two types of toxins : 1. Tetanolysin (haemolysin): Lysis blood cells. 2. Tetanospasmin: A potent polypeptide neurotoxin of one antigenic type. Mechanism of action:  The toxin is released, diffuses via the bloodstream to the peripheral nervous system, and reaches the CNS.  The toxin binds to ganglioside receptors and blocks the release of inhibitory mediators.  Motor neurons undergo sustained excitatory discharge, causing the characteristic motor spasms of tetanus.  The toxin exerts its effect on all CNS sites leading to, generalized muscular and spastic paralysis spasms. 4- Clinical significance: Tetanus disease is characterized by convulsive tonic contractions of voluntary muscles including: Jaw muscles lead to trismus (lockjaw). Facial spasm (sardonic grin). Muscles of the back lead to pronounced arching of the back (opisthotonos). 5- Diseases and symptoms: Lockjaw (trismus): This is a stiffness of the jaw muscles that results in inability to open the mouth or swallow. The patient often experiences a fever (a rise of 2 to 4 degrees) with sweating, elevated heart rate, and blood pressure. Continued severe muscle contractions, which can even cause broken bones, and resulting spasms, often lasting for minutes over a period of weeks, can be fatal (spastic paralysis).  6- Diagnosis: Clinical manifestations/ Microscopy/ Serology/ Genetically (PCR). 7- Treatment : 1. Neutralization of unbound toxin with Human tetanus immunoglobulin (antitoxin). 2. Prevention of further toxin production by antibiotics (Metronidazole). 3. Control of spasm: Nursing in a quiet environment, avoid unnecessary stimuli, muscle relaxants, sedation and assisted ventilation. 4. Supportive care: Adequate hydration, Nutrition, Treatment of secondary infection, Prevention of bed sores. 8- Prevention: By DPT (DTP or DTwP) vaccine: Diphtheria and Tetanus toxoids and killed whole cells of the organism that causes pertussis (wP). Older teenagers and adults who have sustained injuries, especially puncture-type wounds, should receive booster immunization for tetanus if more than 10 years have passed since the last booster. Clinical tetanus does not produce immunity to further attacks. Since even a lethal dose of tetanospasmin is insufficient to induce an immune response. 2- Clostridium perfringens (Welchii) 1- Microorganism: Clostridium perfringens, a gram-positive capsulated rod that forms a central or sub-terminal spore. Commonly found in the soil & dust. 2- Mode of transmission: Clostridium perfringens causes wound colonization leading to gas gangrene (Myonecrosis) and to a lesser extent intestinal tract contamination. The term gas gangrene: refers to swelling of tissues due to release of gas, as fermentation products, of clostridia. 3- Important virulence factors: Alpha toxin is a lecithinase that causes the destruction of the cell membrane, cell death, and necrosis. Enterotoxin produced by some strains causes a type of food poisoning that follows ingestion of contaminated warmed meat dishes. The organism produces several tissue-degrading enzymes (including lecithinase, proteolytic, and saccharolytic enzymes). 4- Diseases and symptoms: Necrosis and destruction of blood vessels and the surrounding tissue, especially muscles. This creates an anaerobic environment in adjacent tissue and the organism spreads systemically. Death can occur within 2 days. 5- Diagnosis: Clinical manifestations/ Microscopy/ Serology/ Genetically (PCR). X-rays of the wound may reveal gas formation in tissues. 6- Treatment & Prevention: Anti-toxin. Antibiotic therapy. Debridement of necrotic tissues. 3- Clostridium botulinum 1- Microorganism: Clostridium botulinum, a gram-positive rod that forms a terminal or sub-terminal spore. Commonly found in the soil, dust, and canned food. 2- Mode of transmission: Human acquires botulism most frequently by ingestion of preformed exotoxin in canned food that contains spores of C. botulinum and has not been properly sterilized. 3- Diseases and symptoms: After eating contaminated food, the symptoms of botulism occur usually within a day including: Vision and swallowing are affected, and the patient may become nauseated and constipated. Muscle paralysis usually starts at the head and, when the respiratory muscles are affected, death can result. 4- Pathogenesis and Important virulence factors: Botulism is caused by a potent exotoxin (botulinum toxin – the most powerful toxin known). This toxin acts on the neuromuscular junction by blocking the release of the transmitter acetylcholine (responsible for muscle contraction). This results in flaccid paralysis and often death results (from respiratory and/or cardiac failure). 5- Diagnosis: Clinical manifestations/ Microscopy/ Serology/ Genetically (PCR). 6- Treatment: Enema to clear the gastrointestinal tract from the toxin. Injection of anti-toxin. 7- Prevention: by proper canning of food. Proper sterilization of food to inactivate the heat-labile botulinum exotoxins. 4- Clostridium difficile 1- Microorganism: Clostridium difficile is a gram-positive rod that forms a central or sub-terminal spore. Commonly found in the soil, dust, and animal feces. 2- Mode of transmission: The organism is part of the flora of gastrointestinal tract in approximately 3% of people. Up to 30% of hospitalized patients become colonized. It is transmitted by fecal-oral means. The hands of hospital personnel are important intermediaries.  3- Diseases and symptoms: C. difficile causes antibiotic-associated pseudo-membranous colitis (PMC) due to the misuse of antimicrobials. Watery but non-bloody diarrhea. Pseudomembrane (mucin, fibrin, sloughed epithelial cells, and acute inflammatory cells) on colonic mucosa. PMCs are seen in about half of cases. C. difficile is rarely invading blood. Clindamycin and Ampicillin are 2 of many antibiotics that cause this disease. 4- Pathogenesis & Important virulence factors: Antibiotics suppress drug-sensitive normal flora, allowing C. difficile to multiply and produce exotoxins A and B. Exotoxin A is an enterotoxin that causes watery diarrhea. Exotoxin B is a cytotoxin that causes damage to colonic mucosa leading to the formation of pseudomembrane. 5- Diagnosis:  Clinical manifestations/ Microscopy/ Serology/ PCR.  One of the laboratory diagnostic tests is that enterotoxin B can kill tissue culture. 6- Treatment: Withdrawal of the causative antibiotic. Fluid replacement. Metronidazole should be given. ** Prevention and control: Antibiotics should be prescribed only when necessary.  Gram negative bacilli (rods) Enteric Gram-negative Gram negative rods. bacilli Non-Enteric Gram- negative rods.  A- Enteric Gram-negative rods 1- Enterobacteriaceae Small Gram-negative non-spore- forming scattered single rods. Mostly motile with peritrichous flagella, but Shigella and Klebsiella are nonmotile. Some have prominent capsules (klebsiella) while most strains have loose slime layers.  Classification is based on biochemical properties, antigenic structure, DNA-DNA hybridization, and 16S RNA sequencing.  Cultural characters:  Facultative anaerobes.  Oxidase-ve, but most of them are catalase +ve  All genera ferment glucose but vary in the fermentation of other carbohydrates.  Enterobacteriaceae species differ in their ability to ferment lactose. Some ferment lactose rapidly, some do it slowly and others (e.g., Salmonella and Shigella) do not ferment lactose at all.  Some strains are opportunistic pathogens, and some strains are true pathogens.  MacConkey agar is a selective and differential medium for Enterobacteriaceae. Antigenic Structures:  Lipopolysaccharide is the major cell wall antigen. Serological classification is based on O antigen (somatic), K antigen (Capsular) and H antigen (flagellar).  Endotoxin: Lipopolysaccharide (LPS) consists of polysaccharide (O) and lipid A. The activity of the toxin depends on Lipid A Activation of complement; release of cytokines Leucocytosis, thrombocytopenia, disseminated intravascular coagulation, Shock and Death  Capsule: Encapsulated Enterobacteriaceae are protected from phagocytosis. Capsular polysaccharide is a poor immunogen. It interferes with the binding of antibodies to bacteria.  Lactose Fermenter Enterobacteriaceae 1- Escherichia coli Normal inhabitant of the G.I. tract. - Motile(Peritrichous flagella), indole positive, urease & citrate negative. Virulence factors of E. coli: A. structural: 1. Pili: (adhesions) that mediate adherence to mucosal surfaces. Genes for pili are carried on plasmids. 2. K (CPS antigen) in some strains which is antiphagocytic. 3. LPS: responsible for the endotoxin manifestations. B. Toxin production: 1. Enterotoxins: a) Enterotoxins produced by Enterotoxigenic strains of E. coli (ETEC): - They are genetically determined by a plasmid. - They are of 2 types; heat labile toxin (LT) and heat stable toxin (ST). b) Verotoxins or Shiga like toxin-producing Escherichia coli (STEC): - Produced by Enterohaemorrhagic E.coli (EHEC). - Known as Vero toxins (type 1 and 2) because of their cytotoxic effects on African green monkey kidney Vero cells in culture. - They have some properties similar to Shiga toxin, but both are antigenically and genetically distinct. c) Enteroaggregative heat-stable toxin (EAST):  Produced by enteroaggregative E. coli. It is related to the heat-stable toxin of ETEC that leads to increased fluid secretion. 2. Type III secretion system (injectosome): is a needle-like structure to deliver effector molecules secreted by bacteria (like toxins) into host cells. 3. hemolysin: causes β-hemolysis on blood agar and is important in the pathogenesis of UTI. 4. Colicins: are a type of bacteriocin released into the environment to reduce competition from other bacteria. Clinical significance of E. coli: I- Intestinal diseases by: Enterotoxigenic E. coli (ETEC). Enteropathogenic E. coli (EPEC). Enteroinvasive E. coli (EIEC). Enteroaggregative E. coli (EAEC). Enterohemorrhagic E. coli (EHEC). II- Extra-intestinal diseases: a) ETEC Enterotoxigenic E. coli ETEC is a common cause of traveler’s diarrhea. Transmission occurs through contaminated food and water. Symptoms include diarrhea without fever. The diseases vary from minor discomfort to severe cholera-like syndrome. ETEC colonizes the intestinal mucosa via fimbriae and then liberates enterotoxin. Enterotoxins produced by ETEC include heat-stable toxin (ST) and heat-labile toxin (LT). LT enterotoxin is very similar to cholera toxin in both structure and mode of action. LT enterotoxin is a protein composed of an enzymatically active (A) subunit surrounded by 5 identical binding (B) subunits.  The ST enterotoxin is a family of toxins that are peptides of low molecular weight. It leads to the secretion of fluid and electrolytes resulting in diarrhea. b. EPEC Enteropathogenic E. coli.  EPEC is an important cause of diarrhea in infants (Infantile gastroenteritis).  They use an adhesin known as intimin (an outer membrane protein) to bind host intestinal cells.  EPEC strains do not produce toxins and are not invasive. They produce lesions in the small intestine – Attachment and destruction of microvilli.  Infection occurs through person-to-person spread.  Acute watery diarrhea dehydration c. Enteroinvasive E. coli (EIEC). Non-motile, non-lactose fermenter. EIEC closely resembles Shigella in its pathogenic mechanisms. The clinical syndrome is identical to Shigella dysentery and includes dysentery-like diarrhea (blood and mucous) with high fever. EIEC penetrates and multiplies within epithelial cells of the colon causing widespread cell destruction. They do not produce LT or ST toxin, and, unlike Shigella, they do not produce the Shiga toxin. d. EAEC Enteroaggregative E. coli. EAEC also causes traveler’s diarrhea and persistent diarrhea in young children. Adherence to the small intestine is mediated by aggregative adherence fimbriae. The adherent rods resemble stacked bricks and result in the shortening of microvilli. EAEC strains produce enteroaggregative heat-stable toxin (EAST) plasmid-encoded. Symptoms include watery diarrhea, vomiting, dehydration, and occasional abdominal pain. e. EHEC Enterohaemorrhagic E. coli or Shiga Toxin-producing E. coli (STEC). EHEC binds to cells in the large intestine and produces one of two exotoxins (Shiga-like toxins 1 or 2), resulting in a severe form of copious, bloody diarrhea (hemorrhagic colitis). Serotype O157:H7 is the most common strain of E. coli that produces Shiga-like toxins. A frequent life-threatening situation is its toxic effects on the kidneys (hemolytic uremia) / Acute renal failure. EHEC strains are often associated with the ingestion of inadequately cooked hamburger meat. Treatment of intestinal infection: Treatment of non-STEC: Treatment of urgent cases includes rehydration therapy, Ciprofloxacin, or azithromycin. Antidiarrheal drugs SHOULD NOT be given. Treatment of STEC: I.V. fluids will reduce the risk of Hemolytic-uremic syndrome (HUS) and renal failure. Eculizumab (Soliris) is used in prophylaxis for neurological symptoms. Antibiotics may be required where sepsis or lung infections complicate HUS. (ciprofloxacin is contraindicated) Clinical significance of E. coli: I- Intestinal diseases: II- Extra-intestinal diseases: 1- Urinary tract infection (in pregnant females. E. coli known as uropathogenic strains colonize the vagina and periurethral regions from which they ascend to the bladder or the kidney causing cystitis and pyelonephritis. 2- Wound infection. 3- Septicemia. 4- Neonatal meningitis. E. coli is a major cause of this disease occurring within the first month of life. Strain Syndrome Therapy ETEC Watery diarrhea Antibiotic therapy may be useful. EPEC Watery diarrhea of long Antibiotic therapy may be duration mostly of infants in useful. developing countries. EHEC Bloody diarrhea Avoid antibiotic therapy (hemorrhagic colitis) and because it increases the hemolytic uremic syndrome. risk of HUS. EIEC Bloody diarrhea Rehydration and correction of electrolyte disturbances. EAEC Persistent watery diarrhea in Rehydration and children and patients infected correction of electrolyte with HIV. disturbances. Diagnosis of E. coli infections 1. Samples: According to the site of infection e.g. pus, urine, stool, CSF…etc. 2. Film: Gram negative bacilli, motile, some strains are capsulated. 3. Culture: On MacConkey’s agar plate they produce rose pink colored colonies due to lactose fermentation. On EMB (Eosin Methylene Blue) they produce small dry colonies with a green metallic sheen. Biochemical reactions: They ferment glucose, lactose, maltose, mannite, and sucrose with the production of acid and gas so on TSI medium they produce yellow (acid) butt and yellow slant. IMVC test (indole-methyl red- vogus proskauer, citrate): ++-- Oxidase, urease and H2S negative, reduce nitrate to nitrite.  Some microorganisms normally occur in the stools and if isolated from a water sample this means that the water is contaminated with stools. E. coli, Enterococcus faecalis and Clostridium perfringens.  2. Klebsiella  Morphology: Gram negative bacilli, non-motile, and have large polysaccharide capsules (K antigen). Cultural characters: 1. on MacConkey’s agar: rose pink colored colonies due to lactose fermentation. 2. on EMB: large, mucoid pink colonies due to the production of extracellular slime. Biochemical reactions: 1. on TSI: yellow butt and yellow slant. 2. IMVC test: --++. 3. Variable results with urease test (from strong positive to negative). Serology: Slide agglutination test, capsular swelling test. Diseases: 1. K. pneumoniae (Fried Lander’s bacillus): It is highly pathogenic to mice causing death within 24 hours if injected IP. In humans, it is present as normal commensal in GIT and RT. 2. K. rhinoscleromatis: causes rhinoscleroma (granuloma of nose and throat) 3. K. oxytoca: causes nosocomial infections. 4. K. ozaenae: causes atrophic rhinitis. Treatment: Strains of hospital-acquired infections show marked resistance to B-lactam antibiotics due to the production of ESBL (Extended Spectrum B-Lactamases). In this case, the drug of choice is Imipenem and Meropenem. Polymyxins if k. Pneuminae is resistant to cabapenems.  3- Citrobacter  It is a genus of Gram-negative coliform bacteria in the Enterobacteriaceae family.  Similar to E. coli (+ +- -)in their laboratory identification criteria except their IMVC results are - + - +.  They are rarely the source of illnesses and can be found almost everywhere, except for infections of the urinary tract infection, meningitis, and sepsis.  Some strains have inducible ampC genes encoding resistance to ampicillin and first-generation cephalosporins. In addition, isolates of Citrobacter may be resistant to many other antibiotics because of plasmid-encoded resistance genes.  4. Enterobacter Characters:  Gram-negative, facultative anaerobic bacilli.  Several strains are pathogenic in immunocompromised (usually hospitalized) hosts and in those who are on mechanical ventilation.  The urinary and respiratory tracts are the most common sites of infection. Diagnosis  Its laboratory characteristics are similar to k. Pneuminae except it is motile, IMVC test --++ and urease negative. Treatment 1. Imipenem is of choice in enterobacter treatment. 2. Fourth generation of cephalosporin (Cefepime). 3. Aminoglycosides such as amikacin.  5. Serratia Characters: Gram-negative, facultative anaerobic motile bacilli. The most common species in this genus, S. marcescens, which usually causes nosocomial infections. Members of this genus produce characteristic red pigment, prodigiosin. Distinguished from other members of the family Enterobacteriaceae by their unique production of three enzymes; DNase, lipase, and gelatinase. Infection of humans: Nosocomial infections of the bloodstream, lower respiratory tract, urinary tract, and surgical wounds in adult patients. Treatment: Usually resistant to aminoglycosides and penicillins. Treated by third-generation cephalosporins.  Citrobacte Enterobact Serrati E. coli Klebsiella r er a Motility + - + + + IMVC ++-- --++ -+-+ --++ --++ Urease - Variable - - - Gelatina se, - - - - + DNAse AA AA TSI AA +G AA +G AA +G +G no G Thank you