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Medical Microbiology Enterobacteriaceae and other gram negative bacilli William H. Benjamin PhD Clinical Pathology.

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Presentation on theme: "Medical Microbiology Enterobacteriaceae and other gram negative bacilli William H. Benjamin PhD Clinical Pathology."— Presentation transcript:

1 Medical Microbiology Enterobacteriaceae and other gram negative bacilli William H. Benjamin PhD Clinical Pathology

2 E. Coli O157:H7 spinach u 204 cases, 8/2006 – 10/2006 103 hospitalizations, 31 HUS, 3 deaths u 26 states u Since 1995, there have been 19 outbreaks of foodborne E. coli O157:H7 for which lettuce or leafy greens were implicated u Found in cattle 1 mile away u Found in feral swine in the area

3 Salmonella on restaurant tomatoes u Sept. 14 – Oct. 2/2008 u 21 states reported 183 cases of illnesses 22 hospitalized v AL (1), AR (4), CT (28), GA (1), IN (1), KY (19), MA (50), ME (8), MI (2), MN (14), NC (4), NE (1), NH (14), OH (4), PA (3), RI (6), TN (9), VA (3), VT (8), WA (1), and WI (2) u Salmonella enterica Typhimurium u raw serrano and jalapeño peppers

4 Cadbury Chocolate Salmonella enterica Montevideo u 65 cases of S. enterica Montevideo in Great Britain u 6 months before cases occurred Cadbury had a S. Montevideo isolate from an ingredient u Leaking pipe at one of Cadbury’s main factories u Cadbury’s rational for not acting on the original isolate is that the numbers identified did not reach a recall “threshold” u Cases matched the PFGE pattern of the original Cadbury isolate and prompted an investigation that lead back to Cadbury

5 Common Characteristics of Enterobacteriaceae u Gram negative Rods u Rounded ends and straight parallel sides u Colony morphology on sheep blood agar –~2 mm colonies, usually dull gray –Other characteristics sometimes present v hemolysis v mucoid v swarming v pigmented colonies

6 Sputum Gram Stain Gram Negative Rods

7 E. coli

8 Gram positive cell wall vs Gram negative cell wall and outer membrane

9 LPS B-cells T-cells Stem cells Vascular cells Macrophages/ monocytes Granulocytes ? Proliferation INFγ IL-2 Proliferation Immunoglobulin IL-1 IL-6 Adhesion molecules TNF-α IL-1 IL-6 IL-8 PAF O 2 - - Radicals Priming for O 2 - - Radicals Adhesion molecules phagocytosis Direct actions Stimulation of additional cells Recruitment of additional mediators (e.g. complement factors, clotting cascade) Fever Hypotension Tachycardia Tachypnea Neutropenia etc. Multi-organ failure Death

10 Endotoxin resistant (lps d ) Mice  lps d mice are not affected by up to 1 mg of LPS, usual lethal dose 50  g u Paradoxically the mice are more susceptible to infection by Salmonella, dying after <100 organisms vs 10,000 or 100,000 for non- lps d mice u Two different spontaneous mutations, one a single bp, one a large deletion. u The lps d mutation is in tlr4 an Endotoxin receptor gene in mice

11 Antigenic Structure of Enterobacteriaceae S. typhi O antigen side chain Pili (Fimbriae)

12 Antigenic Formulae of Enterobacteriaceae u Escherichia coli –O157:H7 –O75:K100:H5 u Salmonella enterica serovar Typhi –O9,12(Vi):Hd:- u Salmonella enterica serovar Typhimurium –O1,4,5,12:Hi:1,2

13 Physiology of Enterobacteriaceae u Facultative anaerobes - ferment glucose u Oxidase negative u Nitrate positive - NO 3 -- > NO 2 u Catalase Positive u Motility is by peritrichous flagella

14 Oxidation Fermentation Test

15 Identification of Species and Genera u Mostly biochemical reactions –Fermentation of carbohydrates pH change –decarboxylation of amino acids –deaminization of amino acids –etc. u Genetic relatedness –16s RNA –DNA hybridization

16 Microscan Walkaway

17 Gram Negative Microscan Panel

18 Enterobacteriaceae - host interactions u Normal flora - beneficial –nutritionally –competition –Immunological stimulation u Opportunistic infections u Infections in otherwise healthy individuals –Non-inflammatory diarrhea –Inflammatory (invasive diarrhea) –Invasive systemic infections (typhoid, sepsis)

19 Escherichia coli u Normal flora of mammals and birds u Most common Enterobacteriaceae in gut u Index of fecal pollution for food or water –coliform count u Lactose positive, indole positive

20 MacConkey Agar plate Lactose fermentation

21 Predisposing factors for UTI u Female anatomy u Honeymoon cystitis in the female u Pregnancy and childbirth u Male prostatic hypertrophy u Catheterization or other mechanical manipulations u Failure to empty the bladder

22 Other Opportunistic Infections u Peritonitis from ruptured gut u Septicemia secondary to UTI or pneumonia u Wound infections

23 Escherichia coli Diarrhea u Estimated 4% of diarrhea in US u Even higher numbers in the developing world u Traveler’s diarrhea - 30-40% of visitors to Mexico

24 E. coli Enteric Pathogens u EPEC Enteropathogenic E. coli –Infantile diarrhea u EAECEnteroaggregative E. coli –Travelers diarrhea u ETECEnterotoxigenic E. coli –Travelers diarrhea u EHECEnterohemorrhagic E. coli –Hemolytic Uremic Syndrome u Enteroinvasive E. coli –Bacillary dysentery

25 Klebsiella pneumoniae u Lactose positive u Nonmotile u Large capsule (77 K antigens)   -lactamase –resistant to ampicillin and carbenicillin –sensitive to cephalosporins

26 Lactose positive Klebsiella sp

27 Klebsiella pneumoniae pneumonia

28 Predisposing Factors for Enterobacteriaceae Pneumonia u Hospitalization - nosocomial u Respirator u Increased age u Aspiration of oral secretions u Alcoholism u Diabetes mellitus u Chronic bronchopulmonary disease

29 Klebsiella pneumoniae disease u Pneumonia –25-50% mortality –Thick non-purulent bloody sputum –Necrosis and abscess formation u Septicemia u Urinary tract infection u Meningitis

30 Enterobacter cloacae E. aerogenes u Lactose positive u Nonmotile u UTI often nosocomial u Cephalosporinase - ampC –Ampicillin resistant –Cephalothin resistant

31 Disc Diffusion Antimicrobial Susceptibility Testing

32 Proteus mirabilis, P. vulgaris u Urease positive u Highly motile, swarm on plate u H 2 S positive u Infections –UTI - community acquired –Wound infections –Pneumonia –Septicemia u Tetracycline resistant u Ampicillin and Cephalosporin resistant

33 Urease test

34 Food borne illness

35 Normal Adult u 9.0 liters of fluid enters the duodenum u 1.5 liters crosses the ileocecal valve u 0.1 liters is passed by the anal sphincter

36 Osmotic Diarrhea u Increased intra-luminal osmotic pressure u Decreased absorption of fluid and solutes u Causes of osmotic diarrhea –Lactose intolerance, Giardia lamblia u Fasting stops diarrhea

37 Secretory Diarrhea u Malfunction of Na + absorption mechanisms u Increased Cl - secretion u Mucosal cAMP increases the above two problems u Bacterial toxins cause increases of cyclic nucleotides in mucosal cells u Fasting does not stop diarrhea (quickly)

38 Vibrio cholerae u 1-5 day incubation period u Profuse watery diarrhea u Spread by contaminated water u Stomach acid offers protection –Infectious dose is 10 7 CFU u Cholera Toxin –B subunit binds intestinal cells –A subunit irreversibly activates adenyl cyclase –Results in water and electrolyte secretion u Treatment - oral hydration and electrolyte replacement which is sufficient

39 Cholera cot Lusaka Zambia

40 Enterotoxigenic E. coli (ETEC) u Watery diarrhea - Secretory diarrhea u Labile toxin related to Cholera toxin –Activates adenylate cyclase (cAMP) –Increased permeability to Na + and Cl - u Stable toxin –Activates guanylate cyclase (cGMP) –Decreases absorption of Na + and Cl - u Toxins are coded by plasmid DNA

41 Bacillary Dysentery u Caused by bacterial invasion of colonic mucosa u Frequent small volume stools –Often containing blood and mucus u Severe abdominal cramps

42 Incidence of Food Borne Illness in the US u Estimated 76 million cases of food- borne illness annually u 80,000 hospitalizations u Estimated 5,000 deaths annually u 7 to 17 billion dollars in medical and social costs

43 The Risk of Food-borne Disease per meal for selected animal- derived foods –FoodIllness episodes/meal u Seafood 1/5,000,000 u Beef 1/200,000 u Poultry 1/20,000 u Raw molluscan shellfish 1/250

44 68%72%73%69%63% 62% 32 28 2731 37 38

45 outbreakscases Salmonella67850%2039245.3% Escherichia coli15711.5455010.1 Clostridium perfringens14210.4841718.7 Staphylococcus aureus1198.731327.0 Shigella816.0510511.3 Campylobacter755.516813.7 Bacillus cereus392.96211.4 Vibrio parahaemolyticus261.95841.3 Listeria monocytogenes131.02710.6 Clostridium botulinum131.0840.2 Yersinia enterocolitica90.7820.2 Vibrio cholerae40.3140.0 multiple40.31030.2 Streptococcus group A10.140.0 1361100%45040100% Bacterial Foodborne outbreaks US 1997 - 2003

46 Bacterial food poisoning u Invasive causes of food poisoning –Salmonella enterica Serotypes –Salmonella typhi –Campylobacter jejuni –Shigella sp –Enteroinvasive E. Coli –Yersinia enterocolitica –Enterohemorrhagic E. coli (HUS) (O157:H7) –Listeria monocytogenes

47 Salmonella enterica u There are 2400 named Serotypes of Salmonella enterica u Salmonella causes three syndromes –Enteric feverTyphoid fever - systemic –EnterocolitisFood poisoning - self limiting –BacteremiaComplication of enterocolitis u Lactose negative u Most H 2 S positive

48 Salmonella enterocolitis u 0.5 to 2 million cases /year in US u 2000 deaths/year u Cost to US economy 0.5 to 1 billion $ u Common serotypes –S. enterica Typhimurium, S. enterica Enteritidis, S. enterica Newport u Vomiting and profuse diarrhea 8-48 hr. u Resolves in 3 to 7 - 10 days u Source of infection Water, meat, dairy products, eggs and human carriers working as food handlers

49 XLD (Xylose Lysine Decarboxylase) plate Black shows H 2 S Clear colonies shows Lactose negative

50 Enteric Fever (Typhoid Fever) u Salmonella enterica Typhi, S. Paratyphi u Humans are the only hosts u 200 - 500 cases/year in US u Millions of cases/year world wide u Mortality –<5% in US –10% in the third world. Antibiotic resistance is common in some countries

51 Typhoid Vaccine u Killed whole S. typhi –Side effects are localized and systemic symptoms mostly because of LPS –50 to 94% effective depending on exposure and personal history u Live vaccine Ty21a –chemically mutagenized strain –mixed results 60-70% protection –some studies no protection

52 Campylobacter jejuni and C. coli u Gram negative curved rods u 1 to 7 day incubation u Fever, bloody diarrhea, abdominal pain u More common/less severe than Salmonella u Carried by animals - food animals and pets u Most human infections are from contaminated food or water u Raw milk and eggs have been implicated u Treatment is rehydration u Cooking and cleanliness are the best defense

53 Enterohemorrhagic E. coli (EHEC) u Diarrhea usually mild –5-10% have hemorrhagic colitis –hemolytic uremic syndrome (HUS) u The most common outbreak strain –O157:H7 (sorbitol negative) u At least 100 other strains have the toxin but seem to be less virulent

54 Hemolytic Uremic Syndrome (HUS) u Shiga like toxin AB 5 type of toxin –A subunit enzymatic activity that removes an adenosine from 28s rRNA –B subunit targets Gb3, or Pk antigen CD77 v Activates apoptosis in target cells, endothelial cells, B cells u renal glomeruli contain a high density of Gb3 in infants u Acute renal failure u hemolytic anemia u Thrombocytopenia u 3% mortality

55 Shigella species u Closely related to E. coli u Four named “species” u Lactose negative u Nonmotile u No gas formed from glucose

56 Bacillary Dysentery u Oral inoculation 1-3 days high numbers in small bowel u high volume watery diarrhea u invade colon epithelium u Dysentery - small volume bloody- mucoid stool u Resolves in 2 - 7 days in healthy adults u Treatment - supportive –Ampicillin, Bactrim

57 Shigella Epidemiology u Humans are the only normal host u 200 organisms are an infectious dose u 10 8 CFU/gram of infected stool u 4 F of Shigella transmission –feces, fingers, food, flies, water u Modern sanitary facilities and hand-washing u Epidemics where sanitation breaks down –wars, day care centers, institutions, cruise ships u 15% of pediatric diarrhea in US

58 Incidence of Shigella species – US Mexico  Shigella dysenterygrpA <2%#1  Shigella flexnerigrpB 18%#3  Shigella boydiigrpC <2%#2  Shigella sonneigrpD 80%#4

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