Enterobacteriaceae & Vibrios. Stijn van der Veen

Transcription

1 Enterobacteriaceae & Vibrios Stijn van der Veen

2 Enterobacteriaceae Enterobacteriaceae are a family of gram-negative rod-shaped (bacilli) enteric bacteria that cause a wide variety of diseases, including many disease in the gastrointestinal tract, such as inflammatory or invasive gastroenteritis. These diseases are often the result of foodborne or waterborne transmission.

3 Bacilli A bacillus is a rod-shaped bacteria Most Enterobacteriaceae appear as single rod Escherichia coli Salmonella sp. Shigella sp. Yersinia sp Klebsiella sp.

4 Overview of bacterial infections Gastroenteritis

5 Enterobacteriaceae Escherichia coli Salmonella sp. Shigella sp. Yersinia sp. Enterobacteriaceae are the dominant cause of GI-tract diseases. Klebsiella sp. Proteus sp. Enterobacter sp. Serratia sp.

6 Bacterial identification flowchart Cell morphology + Gram stain - Cell morphology Cocci Rods Cocci Rods Catalase + - Micrococcus # (or) Streptococcus Staphylococcus Coagulase + - Sta. aureus Sta. epidermidis Optichin sensitive + - Str. pneumonia Str. viridans Atmospheres: Anaerobic: Clostridia Aerobic: Bacillus Facultative anaerobes: Corynebacteria Lactobacillus Oxidase + - Neisseria Not a pathogen Indole test + - Grows with bile salts + - Ferments lactose + - Pseudomonas Haemolysis Alpha (green) Beta (clear) Gamma (not) Not a pathogen Oxidase + - Proteus E. coli Klebsiella Lancefield typing (check for capsule) (can confirm D with growth on bile salts) Haemophilus Urease + - H 2 S + - Salmonella Shigella # Micrococcus has larger cells and looks more yellow. This is a simplified version!!!

7 Bile Bile is secreted by the liver, stored in the gall bladder and ejected into the small intestine Bile is severely antimicrobial and only specialized bacteria are able to survive exposure Enteric pathogens are able to grow or survive in the presence of bile Bile salt hydrolase

8 Lactose fermentation test Carbohydrate fermentation tests with ph indicator can show production of acids (color shift). Bacteria that are able to ferment lactose are acidify the growth medium, which turns red

9 Tryptophan conversion (indole test) Some bacteria are able to convert the amino acid tryptophan using the tryptophanase enzyme. Cleavage of tryptophan results in the production of indole. Indole reacts with para-dimethylamino benzaldehyde from Kovacs reagent and produces a red-violet color.

10 Urea conversion Urea agar slants are used to test whether bacteria can convert urea into ammonia and carbon dioxide. Production of ammonia results in alkalization of the medium which is indicated with phenol red. Urease activity is important for bacteria that pass through the gastro-intestinal tract and need to survive the acid environment

11 Cysteine / methionine conversion Some bacteria are able to convert the sulfide containing amino acids methionine and cysteine. Cleavage of these amino acids results in the production of hydrogen sulfide (H 2 S). The hydrogen sulfide combines with ferrous sulfide (Fe 2 S) in the triple sugar iron (TSI) agar to form a black to dark insoluble precipitate.

12 MacConkey s Agar Combination of selective and differential medium. It is selective because it contains bile salts that inhibit growth of most bacteria, except for the bacteria that colonize the gut and have adapted to bile salts (such as Enteric bacteria that contain long LPS). It is a differential medium because it contains lactose as sole carbohydrate and the ph indicator neutral red. Acid production during lactose fermentation results in pink-red colonies.

13 Gastrointestinal tract Pathogens gain entrance of the gastrointestinal (G.I.) tract through contaminated food or water, or dirty hands. Most microbes that enter the G.I. tract are generally eliminated by the gastric acid, bile, and antibacterial enzymes in the small intestine.

14 Fecal - Oral diseases Pathogens enter the G.I. tract at one end and exit at the other end. Spread by contaminated hands, food, and water. Poor personal hygiene.

15 Bacterial gastroenteritis Gastroenteritis is a general term for infection or irritation of the gastrointestinal tract Noninflammatory gastroenteritis Food poisoning Inflammatory gastroenteritis Cholera ETEC, EPEC Invasive gastroenteritis Typhoid fever Shigellosis EHEC, EIEC

16 Different E. coli s Enteropathogenic E. coli (EPEC) Enterotoxigenic E. coli (ETEC) Enteroinvasive E. coli (EIEC) Enterohemorrhagic E. coli (EHEC)

17 Noninflammatory gastroenteritis Foodborne intoxications caused by bacterial toxins Bacteria secrete preformed toxins that when present in the food result in food poisoning Generally involves a brief incubation period and quick resolution Characterized by diarrhea and/or vomiting

18 Food poisoning The causative agent Enterobacteriaceae Sign and symptoms Diarrhea, vomiting Toxin involved Enterotoxin Transmission Foodborne Treatment Recovery without treatment Prevention and control Avoiding suspected foods

19 Inflammatory gastroenteritis Bacterial gastrointestinal infection Generally has a longer incubation period than intoxications because bacteria first have to establish themselves in the body after ingestion Characterized by diarrhea and/or vomiting, usually fever, but NO blood in the stool Dehydration is a common complication of inflammatory gastroenteritis

20 ETEC, EPEC The causative agent Escherichia coli Sign and symptoms Diarrhea, nausea, vomiting, cramps, and low-grade fever Toxin involved Enterotoxin (ETEC) Transmission Foodborne or waterborne Treatment Illness usually resolves without treatment Prevention and control Avoiding suspect foods and untreated water

21 ETEC vs EPEC Enterotoxigenic E. coli Adheres to epithelial cells, but does not invade Enteropathogenic E. coli Adheres to epithelial cells, but does not invade Produces Cholera-like enterotoxin that destroys the water and electrolyte balance in the intestine leading to a very severe watery diarrhea. Does not produce toxin but instead secretes enzymes that rearrange the actin in the microvilli of the epithelial cells, leading to pedestal formation at the apical surface. This prevents readsorption of water into the cells, which leads to severe watery diarrhea.

22 Invasive gastroenteritis Bacterial gastrointestinal infection Generally has a longer incubation period than intoxications and inflammatory gastroenteritis because bacteria first have to establish themselves in the body after ingestion and pass or damage the intestinal lumen Characterized by diarrhea and/or vomiting, fever, and dysentery (passage of blood and mucus in the stool)

23 Typhoid fever The causative agent Salmonella Typhi Sign and symptoms Bloody stools, abdominal pain, fever, lethargy, delirium Toxin involved Not established Transmission Foodborne from person shedding S. Typhi, or waterborne from contaminated sewage Treatment Antibiotics Prevention and control Avoiding risky foods and drinks and getting vaccinated

24 Salmonella Typhi infection Salmonella Typhi infects the spleen, bone marrow, liver, kidneys and gallbladder Salmonella Typhi can reside in the gallbladder of asymptomatic people, these people are carriers of the disease as they constantly shed the bacteria into the environment

25 Salmonellosis The causative agent Salmonella sp. e.g. Salmonella enteritidis Sign and symptoms Fever, diarrhea, vomiting, and abdominal cramps Toxin involved Not established Transmission Foodborne in a broad variety of foods Treatment Antibiotics and fluid replacement Prevention and control Practicing good hand hygiene and food preparation

26 Shigellosis The causative agent Shigella sp. (dysenteriae, flexneri, boydii, sonnei) Sign and symptoms Diarrhea, dysentery (most common cause) Toxin involved Exotoxin (Shiga toxin) Transmission Foodborne and waterborne Treatment Antibiotics, fluid and salt replacement Prevention and control Practicing good hand hygiene

27 EIEC The causative agent Escherichia coli Sign and symptoms Severe bloody diarrhea, with mucus containing PMN s Toxin involved No toxin, but the intestinal wall is severely damaged through mechanical cell destruction Transmission Foodborne through ingestion of contaminated food Treatment Antibiotics is optional Prevention and control Practicing good hygiene while handling food

28 EHEC The causative agent Escherichia coli O157:H7 Sign and symptoms Severe bloody diarrhea Toxin involved Enterotoxin (Shiga-like toxin) Transmission Foodborne through ingestion of undercooked meat or contaminated fruits and vegetables Treatment For HUS: red blood cell and platelet transfusions, kidney dialysis Prevention and control Practicing good hand hygiene, cooking foods properly, washing fruits and vegetables, avoiding unpasteurized milk

29 EHEC infections EHEC attaches to colonic epithelial cells and causes actin rearrangements that lead to pedestal formation and effacement of the brush border (causes diarrhea) They produce a Shiga-like toxin that enters and kills cells causing a massive immune response at site where bacteria are present which together with the cell s death leads to a bloody, pus filled diarrhea (hemorrhagic colitis) The Shiga -like toxin can further travel in the bloodstream and reach the kidneys The toxin can kill kidney epithelial cells which plug up the small vessels in the kidneys leading to HUS (hemolytic uremic syndrome) This can lead to kidney (renal) failure

30 Yersiniosis The causative agent Yersinia enterocolitica Sign and symptoms Diarrhea, fever, abdominal pain Toxin involved Not established Transmission Foodborne from contaminated food, mainly pork Treatment None, antibiotics for severe or prolonged illness Prevention and control Practicing good hand hygiene and avoiding raw or undercooked pork

31 Enterobacteriaceae & other diseases Enterobacteriaceae are also causing a number of non- GI-tract diseases Septicemia/ bacteremia Pneumonia Meningitis Urinary tract infections Plague

32 Plague Black Death The causative agent Yersinia pestis Sign and symptoms Bubobic: Sudden onset of fever and chils, headache, fatugue, muscle aches, and buboes Septicemic: High fever, abdominal pain, diarrhea Pneumonic: Headache, malaise, extensive coughing Transmission Infected flea bite or inhaled infectious droplets from person or animal Treatment Streptomycin and gentamycin given intravenously or intramuscularly Prevention and control Avoiding contact with sick or dead animals, flea control

33 Plague Black Death The Black Death was probably the greatest catastrophe ever to strike Europe!! Quote by Agnolo di Tura (describing bubonic plague in his chronicle The Plague of Siena in 1348) Father abandoned child, wife husband, one brother another And I, Agnolo di Tura buried my five children with my own hands So many died that all believed that it was the end of the world.

34 Plague Black Death By 1348, two-thirds of the European population was stricken and half of the sick had died!! The sick died too quick for the living to properly bury them, so they were often buried in Plague Pits Before the end of the century, at least five more epidemics occurred Painting: The Plague Pit An estimated 40 million people died in the 1300s from the Black Death, which was one-third of the total population

35 Plague Bubonic plague Bacteria enter by flee bites, spread lymphatic or systemic and localize in the lymph nodes, particularly of the armpits, neck and groin. Hemorrhaging in the lymph nodes causes painful and substantial swelling, called buboes. From these buboes, bacteria may spread to the bloodstream. Dark purplish splotches from hemorrhages are visible through the skin. Septicemic plague Multiplication in the bloodstream, septicemia, meningitis Pneumonic plague Human-to-human transmission Lung symptoms are similar to pneumonia Hemorrhaging and fluid accumulation are common.

36 Klebsiella pneumoniae Opportunistic and nosocomial infections Pneumonia Urinary tract infections (particularly in elderly) Sign and symptoms Pneumonia: chills, high fever, sweating, shortness of breath, chest pain, cough with thick greenish or yellow sputum Transmission Respiratory droplets, oral-fecal Treatment Antibiotics, but resistance is major problem Prevention and control Practicing good hand hygiene

37 Proteus mirabilis Opportunistic and nosocomial infections Urinary tract infections Sign and symptoms Painful urinary tract infection, elevation of ph of urine (crystals) Transmission Oral-fecal, indwelling devices in hospitals Treatment Antibiotics Prevention and control Practicing good hygiene Highly motile!!

38 Serratia marcescens Opportunistic nosocomial infections 2% of nosocomial infections of the bloodstream, lower respiratory tract, urinary tract, surgical wounds, and skin and soft tissues in adult patients Sign and symptoms Variety of symptoms depending on the type of infection Transmission Hand-to-hand Treatment Antibiotics Prevention and control Practicing good hygiene Strong biofilm former!!

39 Virulence factors Attachment to host tissues Production and delivery of various factors Replication and evasion of immunity Damage to host tissues

40 Lipopolysaccharide (LPS) LPS is an endotoxin because it is poisonous to mammalian cells. Exert their effects when gram negative bacteria die and the LPS is released. Free lipid A may trigger pyrogenic responses, including fever, inflammation, and septic shock

41 Capsule & slime layer Enterobacteriaceae produce a wide variety of different capsule structures and slime layers (EPS: extracellular polymeric substances), which contributes to virulence of these pathogens

42 Pili / Fimbriae Salmonella sp., Escherichia sp., and many other Enterobacteriaceae have ligands on the tip of pili / fimbriae that are able to directly adhere to structures on the surface of epithelial cells. Fimbriae: can occur at the poles of the bacterial cell or entires surface and have the tendency to adhere to eachother and to surfaces Pili: are usually longer than fimbriae, but shorter than flagella, and are also involved in motility and DNA transfer

43 Type III secretion system syringe-like multi-protein structure able to penetrate host cells and deliver effectors (mostly small proteins or toxins) directly into the host cytosol. The effectors modulate host functions or stimulate uptake into non-phagocytic cells. Similar in structure to type II secretion systems and type IV pili. Found in some Enterobacteriaceae

44 Actin polymerization Some bacteria recruit actin to provide the force for intracellular movement. Means to escape autophagy, which is an intracellular antimicrobial resistance mechanism. Means to move between cells. Example Shigella sp.

45 Hemolysins Hemolysins combine with the membranes of red blood cells, causing it to lyse. Lysing of red blood cells provide pathogens with the iron from hemoglobin, which is required for many metabolic processes. Example: Escherichia sp. Proteus mirabilis

46 Enterotoxins Enterotoxins effect particularly the GI-tract, causing vomiting, diarrhea and abdominal pain Enterotoxins cause increased chloride ion permeability of the apical membrane of intestinal mucosal cells and activation of membrane pores through camp or by increased intracellular calcium Increased chloride permeability leads to leakage into the lumen followed by sodium and water movement This leads to a secretory diarrhea within a few hours of ingesting enterotoxin

47 Enterotoxins Examples: ETEC Heat labile toxins (LT), an adenyl cyclase which catalyze ATP to camp and result in increased secretion of water and ions Heat stable toxins (ST), a guanyl cyclase that catalyzes GTP to cgmp, which inhibits ionic uptake. EHEC Verotoxin (VT), bacteriophage-mediated exotoxin that inhibits protein synthesis and directly damages renal and endothelial cells and may lead to hemolytic uremic syndrome Shigella sp. Shiga toxin, similar to VT

48 Vibrios Vibrios look like curved rods Vibrio cholerae Vibrio parahaemolyticus Vibrio vulnificus

49 Thiosulfate-citrate-bile salts-sucrose agar Vibrio selective and differential agar Bile selects for enteric bacteria Thiosulfate and citrate inhibit growth of enterobacteriaeceae Thiosulfate and ferric citrate allows for detection of H 2 S Sucrose is the fermentable carbohydrate The medium has an alkaline ph, which enhances recovery of V. cholerae Thymol blue and bromothymol blue are ph indicators

50 Cholera The causative agent Vibrio cholerae Sign and symptoms Severe, watery diarrhea, nausea, vomiting, muscle cramps, and dehydration Toxin involved Enterotoxin Transmission Waterborne Treatment Oral rehydration therapy, antibiotics Prevention and control Practicing good hand hygiene and avoiding contaminated water

51 Vibrio enterotoxin Vibrio enterotoxin This toxin alters the water and electrolyte balance in the intestine leading to a very severe, life threatening, watery diarrhea.

52 Cholera epidemics Killed tens of millions of people in epidemics in the 19 th and 20 th century. Still affects 3-5 million people annually, with an estimated 100,000 deaths Cholera outbreak following the Haiti earthquake in 2010 Killed 9,000 people Hospitalized 420,000 people Infected 740,000 people

53 Vibriosis The causative agent Vibrio parahaemolyticus, Vibrio vulnificus Sign and symptoms Acute severe abdominal cramps or pain, vomiting, watery diarrhea, fever, nausea Toxin involved None Transmission Foodborne in contaminated seafood such as oysters and raw shellfish Treatment Antibiotics Prevention and control Cooking seafood thoroughly, especially oysters and clams

54 Next lecture Mycobacterium, Helicobacter & Corynebacterium