1. Gram Negative Infections Chijioke Onejeme chijiokeonejeme@gmail.com 1

2. For each pathogen we study, know its: Biological Characteristics What is the pathogen? Pathogenic Mechanisms How does it cause disease? Pathological Effects What disease does it cause? Laboratory Identification How do we detect it? Treatment How do we treat these diseases? OBJECTIVES 2

3.  1. Distinguish between lactose and nonlactose fermenting enterics.  2. Identify the unique biochemical properties of enteric pathogen  3. Identify the usual route of transmission for enteric infections  4. To identify the symptomatic phases of a Salmonella infection  5. To identify the symptomatic phases of a Shigella infection  6. To explain the mechanism of Shiga toxin  7. Identify the different E. coli pathogenic strains  8. Compare and contrast EHEC with EPEC  9. Distinguish Vibrio from Enterobacterace  10. Identify the pathological symptoms associated with V. cholerae infections  11. Compare Cholerae toxin mechanism to ETEC toxins  12 Compare and contrast EIEC and EHEC pathogenic mechanisms with Salmonella and Shigella  13. To identify mechanisms utilized by ETEC, EPEC, EAEC, EIEC, and EHEC OBJECTIVES 3

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5. Enterobacteriaceae General Characteristics What is the pathogen?  Gram negative bacilli  Biochemical properties  Oxidase negative  Catalase positive  Ferment glucose  Reduces nitrates to nitrites  Faculative anerobes  Ubiquitous  Comprised of the following bacterial genera  Salmonella  Shigella  Klebsiella  Escherichia  Proteus  Yersinia 5

6. Entry  Contaminated medical devices, surgical site infections, IV, urinary catheters  Colonization of the colon, perineum, urethra  Pneumonias (i.e. respiratory spread)  Contaminated food  Large numbers usually needed* Evasion  Capsule  Anti phagocytic  Antigenic phase variation  Altered expression of capsule/flagella  Plasmids  Anti-microbial resistance Enterobacteriaceae General Characteristics What is the pathogen? 6

7. Salmonella sp. General Characteristics What is the pathogen?  Gram (–) bacilli  Lactose (–)  H2S (+) Two species of interest  S. typhi  S. enteritidis 7

8.  Endotoxin  Cellular invasion Salmonella sp. General Characteristics How does it cause disease? 8

9. Salmonella sp. Pathological Effects What disease does it cause? Diseases  Enteritis  Septicemia  Enteric Fever  Carrier State Symptoms  Fever  Abdominal cramps,  Dysentery (Bloody Diarrhea) Gastroenteritis  Adhesion to the microvilli  Cellular Invasion -> Survival in vesicle  Multiplication -> Escape Septicemia (all above plus)  Silent Septicemia -> Uptake by macrophages  Survival in macrophage vesicle -> Systemic Enteric Fever (all above plus)  Multiplication in lymph node, spleen, liver macrophages  Bacteremia Release from macrophages to blood  High Fever (Endotoxin)  Re-Invasion of Intestine via gall bladder (Round 2) Carrier State  – Established gall bladder infection 9

10.  Self-limiting  Usually resolves in 5-7 days  Hydration  Systemic Infection  Ampicillin  Sulfamethoxazole- Trimethoprim (Bactrim©)  Ciprofloxacin Salmonella sp. Treatment How do we treat these diseases? 10

11. Shigella sp. General Characteristics What is the pathogen?  Gram (–) bacilli  Lactose (–)  H2S (–)  Non-motile Three species of interest  S. dysenteriae  S. sonnei  S. flexneri 11

12. Shigella sp. Pathologenic Mechanisms How does it cause disease? Entry  Fecal-oral transmission  Acid Resistant - Small numbers needed Evasion  Cellular invasion – escape from endocytic vesicle, multiplication in cytoplasm  spread to adjacent cells 12

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14. Shigella sp. Pathogenic Mechanisms How does it cause disease? Evasion - Shiga Toxin  AB toxin  N-glycosidase: cleaves an adenine base from the 28S of the 60S rRNA subunit  inhibits protein synthesis  Kills epithelial cells  Associated with hemolytic uremia  Produces local ulcer formation  Non-motile; rarely systemic 14

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16. Shigella sp. Pathological Effects What disease does it cause? Treatment How do we treat these diseases? Shigellosis  Symptoms  Fever, abdominal cramps  Dysentery Self-limiting  Usually resolves in 5-7 days  Hydration  Ampicillin, Bactrim, Ciprofloxacin 16

17. Escherichia coli Biological Characteristics  Gram (-) bacilli  Natural inhabitant of the GI tract  Glucose (+), Lactose (+) with gas  Indole (+)  Green metallic sheen on EMB agar  Motile 17

18. Escherichia coli Pathological Effect What disease does it cause?  Extraintestinal Infections  Urinary Tract Infection  Pulmonary infection  Bacteremia  Meningitis  Intestinal Infections  Diarrhea  Hemolytic-uremic syndrome  Dysentery  Groups – based on how they cause illness  ETEC - Enterotoxigenic  EPEC - Enteropathogenic  EHEC -Enterohemorrhagic  EIEC - Enteroinvasive 18

19. Enterotoxigenic E. coli Pathological Effects What disease does it cause? Enterotoxigenic E. coli - Traveler’s Diarrhea  Symptoms  Nausea, Vomiting, Watery Diarrhea  Virulence Factors  Colonization Factor Antigens CFA/1, CFA/II  Facilitates bacteria adhesion  Enterotoxins  Heat Labile  Heat Stable 19

20. Enterotoxigenic E. coli Pathogenic Mechanisms How does it cause disease?  Heat labile  Two subunits “AB Toxin”  B portion binds to cell membranes  A portion (active) is transported into cells  ADP ribosylation of G protein by A subunit  cAMP cellular levels increase -> increased Cli- on excretion  Results in increased water secretion from cells  Heat stable  Induces an increase in cellular cGMP -> increased Cl- ion excretion  Results in increased water secretion fromcells 20

21. General AB Toxin Cholera Toxin Mechanism Is Similar To ETEC Heat Labile Toxin 21

22. Enteropathogenic E. coli Pathogenic Mechanisms How does it cause disease? Pathology in small intestine  Attaching and effacing lesions  Microbes bind to the epithelial cells via BFP (bundle-forming pili)  Loose, then tight binding  Effacement lesion  Associated pedestal formation 22

23. Enteropathogenic E. coli Pathological Effects What disease does it cause?  Disease  Pediatric Diarrhea  Symptoms  Fever, Nausea, Vomiting, Watery Diarrhea 23

24. Enteroinvasive E. coli Pathogenic Mechanisms How does it cause disease?  Invades intestinal epithelial cells  Lyses the phagosomal vacuole  Moves through the cytoplasm  Spreads to adjacent cells 24

25. Enterohemorrhagic E. coli Pathogenic Mechanisms How does it cause disease?  Virulence Factors  Vero Toxin / Shiga- like toxin  AB Toxin - inhibits protein synthesis (Similar to Shiga toxin)  Acid Resistant  Low infectious dose 25

26. Enterohemorrhagic E. coli Pathological Effects What disease does it cause? Disease  Hemorrhagic colitis  Dysentery  Hemolytic Uremic Syndrome (HUS)  Acute renal failure, hemolytic anemia  Associated wih E. coli O157:H7 Symptoms  Diarrhea  Bloody Diarrhea 26

27. Escherichia coli Treatment How do we treat these diseases? GI infections  Rehydration therapy  Usually do not use antibiotics (no added benefit)  Do not use anti-diarrheal products such as Immodium© Urinary Tract Infections and Systemic Infections  Sulfamethoxazole-Trimethoprim  Nitrofurantoin 27

28. Vibrio cholerae  Gram - curved rod  Not a member of the Enterobacteriaceae  Etiological agent of cholera  Severe diarrhea, rice water stools (thin mucus flexs)  Dehydration and shock --> death  Treatment  Water and ion replacement  Toxin based, similar to the heat labile toxin of E. coli  1A5B Toxin  Cellular cAMP increase, while cellular Cl- ion and water decrease  Oral Fecal Transmission 28

29. Vibrio cholerae Treatment  Most patients can be treated with oral fluid/ electrolyte replacement  IV fluids used for severe cases  Antibiotics usually unnecessary, but…  Doxycycline  Azithromycin 29

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31. Neisseria gonorrhoeae General Characteristics What is the pathogen?  Gram(-) diplococci  Aerobic  Cytochrome oxidase(+)  Maltose (-) 31

32. Neisseria gonorrhea Pathogenic Mechanisms How does it cause disease?  Entry  Infects mucosal surfaces  Ex. cervix, urethra, rectum, oropharynx, nasopharynx, conjunctiva  Adherence  Pilin – initial adhesion  Opa – firm adhesion  Evasion POR  Pathological Effects  LOS  Lipooligosaccharide (endotoxin)  IgA Protease  Destroys soluble IgA  Beta-lactamase  Inactivates penicillins 32

33. Neisseria gonorrhea Pathological Effects What disease doe it cause?  Gonorrhea  Symptomatic or asymptomatic  Transmission  Sexual contact  Newborns (via birth canal)  May co-exist with other STDs 33

34. Neisseria gonorrhoeae Pathological Effects What disease does it cause? Men  Asymptomatic infection  Acute urethritis  Purulent exudate w/ dysuria, polyuria, HA, fever  Anterior to posterior urethral infection  Glands, ducts, and vessicles may become infected  Chronic infection  Prostate, seminal vesicles, and epididymides may become infected Women  Asymptomatic carriers  Acute gonorrhea infection (lower tract)  Endocervix (traditional site of infection)  Acute symptoms  Ab/pelvic pain, vaginal discharge/dysuria  Chronic Gonorrhea infection  Tenderness in lower ab, inflammatoion or urinary tract  Pelvic Inflammatory Disease (PID) 34

35. Neisseria gonorrhoeae Pathological Effects What disease does it cause?  Bacterial infection of the upper genital tract  Infection ascends from vagina and endocervix to the upper genital areas (uterus, fallopian tubes, ovaries)  Symptoms  Fever, lower ab pain, increased risk of ectopic pregnancy  Sites of infection  Endometrium (endometritis), fallopian tubes (salpingitis)  Extragenital infections  Local infections determined by site of contact  Conjunctivitis  Opthalmia neonatorum (newborn)  Pharyngitis or Proctitis  Either may be transmitted sexually 35

36. Neisseria gonorrhoeae Laboratory Diagnosis  Smear of urethral pus with gram (-) diplococcus  Carbohydrate utilization (glucose fermenter)  Direct Fluorescence Antibody Test  ELISA 36

37. Neisseria gonorrhoeae Treatment  Cephalosporins (Ceftriaxone)  Preferred agent for most N. gonorrhoeae infections  Fluoroquinolones (Ciprofloxacin, levofloxacin)  Resistance to antibiotics exists and is rapidly emerging  PPNG – penicillinase producing neisseria gonorrhoeae  Increasing FQ resistance limits use 37

38. Neisseria meningitidis General Characteristics What is the pathogen?  Gram(-) diplococci  Aerobic  Maltose (+) 38

39. Neisseria meningitidis Pathogenic Mechanisms How does it cause disease?  Evasion  Invasion of epithelial cell  followed by invasion of blood stream and systemic spread  Anti-phagocytic capsule  Facultative intracellular parasite  Adherence  Pilin – initial adhesion  Opa – firm adhesion  Pathological Effects  Lipooligosaccharide (LOS)  endotoxin  Sequestration of iron from host  IgA protease  Destroys soluble IgA  Beta-lactamase  Inactivates beta-lactam antibiotics 39

40. Neisseria meningitidis Pathological Effects What disease does it cause?  Meningococcal meningitis  Symptoms – non-specific  High fever, severe HA, neck pain/stiffness  n/v  Sequelae  Deafness, mental retardation, behavior defects 40

41. Neisseria meningitidis Laboratory Identification  Gram (-) diplococci  Cytochrome oxidase (+)  Ferments glucose and maltose  Grows on “chocolate agar” with CO2 production  Serological (antibody test)  serotypes A, B, Y, W-135 etc…  DNA probes  Examination of cloudy CSF, organism in the cytoplasm on neutrophils, or increase WBCs 41

42. Neisseria meningitidis Treatment  Empiric  3rd generation cephalosporins (ex. Ceftriaxone, Cefotaxime)  Targeted  Penicillin, Ampicillin, or Ceftriaxone  Prophylaxis  Ciprofloxacin  Immunization  Covers 4 serotypes of N. meningitidis  Does not protect against other serotypes 42

43. Special thanks to Paula Ingram Darasaw for compiling this Powerpoint 43