GI Pathogens: Bacteria and Viruses PDF

GI Pathogens: Bacteria and Viruses Michelle L. Steinauer, Ph.D. 1  Conflict of interest Disclosure In relation to this presentation, Dr. Steinauer has no financial interests or other conflicts that need to be disclosed....

GI Pathogens: Bacteria and Viruses Michelle L. Steinauer, Ph.D. 1  Conflict of interest Disclosure In relation to this presentation, Dr. Steinauer has no financial interests or other conflicts that need to be disclosed. 2  Learning Objectives With the pathogens discussed in this lecture, understand – How the pathogen is transmitted including Reservoir hosts Infectivity (number of pathogens to establish an infection) Risk factors that make a person more likely to be infected – The course of infection: Where the pathogen establishes in the body Does it remain localized or disseminate? What virulence factors does it use to establish, avoid immune clearance, and cause disease – Clinical Course Range of signs and symptoms Most common presentations General timeline – Diagnostics: Shape, Gram stain or other stain characteristics, defining biochemical characteristics Culture techniques that are used for diagnostics Alternative methods of diagnostics – Treatment – Epidemiology Risk factors for infection including geography Prevention 3  Suggested Textbook reading: Harrison’s Ch 128: Acute infectious diarrheal diseases Levinson Review of Medical Microbiology and Immunology – Chapters on relevant microbes 4  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio paraheamolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 5  Transmission Fecal-oral or vomitus-oral – Contamination of food, water, fomites Insufficient handwashing Insufficient sanitation Animal reservoirs – Domestic livestock, wildlife, contaminate food or water – Direct contact with animals (pets and livestock) – Salmonella, Campylobacter, Yersinia enterocolitica, E. coli (EHEC,EAEC) – Shellfish (Vibrio parahaemolyticus, Vibrio vulnificus) Environmental reservoir (water) – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus Spore formers – Clostridioides difficile, Clostridium perfringens, Bacillus cereus Some pathogens can establish more easily than others – Stomach acid 6 7 8  Infectious “Syndromes” Toxemia food poisoning—ingesting preformed toxins in food (not infection) – Rapid incubation (2-8 hours), vomiting, diarrhea of short duration Gastritis, Ulcers→ Stomach cancers Watery diarrhea – Watery, liquid stools. Sometimes cramping and vomiting. Not invasive. Inflammatory diarrhea – Invasive, large bowel, fever, blood in stool, sometimes ulceration of mucosa, painful, Fecal leukocytes present Chronic GI symptoms – Long course—greater than a week – Parasites—often travel history to developing country – Does not rule out bacterial infections as some may form chronic infections (i.e. Yersinia, EAEC and Salmonella-particularly in children). Immunocompromised population – Some evidence that GI infection can lead to IBS 9  Infectious “Syndromes” Toxemia food poisoning—ingesting preformed toxins in food (not infection) – Staphylococcus aureus, Clostridium perfringens, Bacillus cereus Ulcers → Stomach cancers – Helicobacter pylori Watery diarrhea – Viruses, Vibrio cholera, ETEC, EPEC, EAEC, EHEC. Inflammatory diarrhea – Shigella, EIEC, Salmonella, Campylobacter, Yersinia entericolitica, Clostridioides difficile, Listeria monocytogenes, and Entamoeba histolytica Chronic GI symptoms – Diarrhea: Giardia, Cryptosporidium, Cyclospora, Entamoeba histolytica – General (diarrhea, constipation, bloating, pain, malnutrition, growth stunting): Ascaris, Ancylostoma, Necator, Strongyloides, other roundworms – Pruritus: Enterobius vermicularis 10  Diagnosis of GI Pathogens Culture based diagnosis PCR diagnosis 11 Pathogen panel diagnostics 12  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio paraheamolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 13  “Food Poisoning”—Preformed toxins Toxemia, not infections, short incubation and short duration Staphylococcus aureus (G+ coccus, catalase+, coagulase +, beta hemolytic) – Bacteria in food that stored at elevated temperatures→proliferation and toxin production. Moist foods with starches or proteins. – Many enterotoxins (A-E) – 4-6 hours, vomiting, diarrhea, no fever – Resolution in 8-24 hours Clostridium perfringens (G+ rod, anaerobe, spores) – Spores survive cooking, germinate in food kept warm – Watery diarrhea and cramping (some vomiting) – Resolution in 24 hours Bacillus cereus (G+ rod, spores) – Spores – Shorter incubation (1-6 hours)—vomiting RICE – Longer incubation (8-16 hours)—diarrhea, hemorrhagic diarrhea. 14  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 15  Helicobacter pylori Cause of chronic gastritis, peptic ulcers, and can cause gastric adenocarcinoma and MALT lymphoma Pathogenic in small proportion of carriers http://www.ppdictionary.com/bacteria/gnbac/pylori.htm – Chronic development of symptoms – Other factors contribute to disease probability Bacterial Characteristics: – Gram negative – Spiral shaped – Oxidase positive – Catalase positive – Urease positive – Motile (flagella)  Helicobacter pylori invasion and virulence Mucinase helps penetration Urease helps neutralize acidic through mucous layer environment Some strains produce vacuolating cytotoxin  H. pylori Virulence Factors Flagella—motility Mucinase: Penetration of mucous layer Urease—neutralizes acidic environment – Hydrolyzes urea into carbon dioxide and Ammonia – (NH2)2CO + H2O → CO2 + 2NH3 Cytotoxins (several) – CagA: exotoxin associated with carcinogenic phenotype. Occurs on a pathogenicity island with ~30 genes, each involved in pathogenicity. – VacA: All H. pylori have this exotoxin, but it is variable. Subtype S1 and m1 is associated with carcinogenic phenotype.  H. pylori Diagnosis and Treatment Diagnosis Treatment Urea breath test – Radiolabeled urea ingested Best to base on susceptibility – Radioactive CO2 detected in testing breath – Culture based on biopsy Stool antigen test Triple/quadruple therapy Test for IgG antibodies in 14 days serum – Active v. past infection PCR (stool or biopsy) Endoscopic tests – Biopsy and culture – Rapid urease test  Case Report https://www.cureus.com/articles/45651-persistent- helicobacter-pylori-infection-an-insight-to-the-limitations-of- current-clinical-practice 64-year-old woman 4 month history of epigastric pain, bloating, and early satiety. No weightloss or GI bleeding. Upper GI endoscopy biopsy showed H. pylori. 14 day course of clarithromycin triple therapy. One year later returned with similar symptoms but 10 pound weightloss. Endoscopy and urea breath test revealed H.pylori infection. Several treatment regimens tried due to drug resistance of pathogen. Figure 1: Endoscopic image revealing patchy erythematous antral mucosa 20 21  Helicobacter pylori Summary Gram negative curved Gastritis rod Ulcers Flagella—mobility Carcinomas Oxidase positive Urease positive Urea breath test Triple/quadruple therapy  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio paraheamolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 23  Vibrio Environmental organisms G negative, curved rods (comma shaped) Motile—single polar flagellum Species: – Vibrio parahaemolyticus Raw undercooked seafood Gastroenteritis – Mild watery diarrhea to severe diarrhea, cramping, nausea and vomiting fever (3 days) – Vibrio vulnificus Raw undercooked seafood Open wound contact with salt water Gastroenteritis and rapidly progressing cellulitis Disseminates rapidly--septicemia – Vibrio cholerae Cholera http://remf.dartmouth.edu/Cholera_SEM/images/01_Cholera%200395%20area1%205kX.jpg  Vibrio parahaemolyticus and V, vulnificus Found in warm coastal waters – Coastal areas esp: Gulf of Mexico – Warming waters = spread GI infection – Exposure: ingestion, particularly through raw oysters Wound infection – Exposure: open wound Vibrio vulnificus is more invasive – GI and wound infections more likely to disseminate and cause septicemia – Risk increases with advanced age, diabetes, autoimmune disorders, kidney and liver diseases. 25  Vibrio parahaemolyticus and V. vulnificus Treatment – V. parahaemolyticus GI symptoms typically self limiting and do not require treatment Vulnerable patients – V. vulnificus 20-50% mortality rate GI infections disseminate Wound infections progress rapidly – Debride necrotic tissue CDC recommends combination therapy with intravenous ceftazidime with either a quinolone (ciprofloxacin) or a tetracycline (doxycycline) – Drug resistance in V. vulnificus is an increasing problem 26  Vibrio vulnificus Summary 27 https://jamanetwork.com/journals/jama/fullarticle/2801603  Vibrio cholerae: Cholera Sept-Nov 2024 Since 1 January 2024 and as of 25 November 2024, 490 700 cholera cases, including 3 693 deaths, have been reported worldwide. In comparison, between 1 January 2023 and 25 November 2023, 822 344 cholera cases, including 4 776 deaths, were reported worldwide. 28 https://www.ecdc.europa.eu/en/all-topics-z/cholera/surveillance-and-disease-data/cholera-monthly  Vibrio cholerae Globally important diarrheal disease Transmission through fecal contamination of water or food Large dose required—(>107), sensitive to stomach acid Environmental reservoirs – Environmental biofilms – Plankton – Shellfish Humans may continue to carry pathogens months after symptoms end Trends in Microbiology, September 2019, Vol. 27, No. 9  V. cholerae Virulence Factors Toxin co-regulated pili: Attach to epithelial cells, attach to each other to aggregate bacteria into microcolonies – Protection from host defenses – Concentrate the toxin Cholera toxin 30 Cholera Toxin (coded by bacteriophage) A-B toxin Enters cytoplasm The mechanism is Catalyzes ADP- similar to LT toxin of ribosylation of G protein Enterotoxigenic E. Activation of G protein coli increases adenylate cyclase which cleaves ATP to cAMP Activates ion channel: cystic fibrosis transmembrane regulator (CFTR) Cl- and HCO3- secretion, block absorption of Na+ Efflux of water https://commons.wikimedia.org/wiki/File:Cholera_toxin.jpg 31  Vibrio cholerae Clinical Course Deadly dehydrating diarrhea – Cholera toxin = watery diarrhea in large volumes (10-20 Liters/day) – when symptoms start, can progress rapidly (within hours) – as more fluid lost, the stool becomes colorless/odorless and speckled with mucous (rice-water stools) Non-bloody – Hypovolemic shock Disease is often self-limiting – Antibiotics shorten course and reduce fluid loss, may reduce carriage after symptoms resolve. https ://ridingric ksha w.wordpress.c om /2011/08/21/cholera-cots-bewa re-of-g ra phic-pictures/ https://ridingrickshaw.wordpress.com/2011/08/21/cholera-cots-beware-of-graphic-pictures/ V. cholerae: Diagnostics and Treatment Microbiological Diagnosis – TCBS agar (Thiosulfate citrate bile salt sucrose agar) Selective due to thiosulfate, sodium citrate, and alkalinity which inhibit the Enterobacteriaceae Bile salts inhibit gram-positives. Can somewhat diagnose Vibrios by color changes, but imperfect Vibrio cholerae turns agar yellow due to sucrose fermentation Vibrio parahaemolyticus and Vibrio vulnificus appears green to blue – OX+ – Microscopy Shape and movement in darkfield – Comma with polar flagella Rapid antigen test – Immunoassay for some strains Nucleic acid amplification tests Treatment – Rehydration therapy – Antibiotics shorten course Ciprofloxacin Doxycycline V. vulnificus typically looks green. TCBS agar is also used to diagnose Vp and Vv Strains that have genes to33 ferment sucrose will turn yellow  V. cholerae Prevention Vaccines available – 4 oral vaccines available – One available in the U.S. that is 80-90% effective for at least 6 months (unknown how long after) Reducing fecal contamination in the environment and in food and water – With the relatively high dose needed for infection this is typically the result of large and wide-spread contamination of environments. 34  Vibrio Summary Gram-negative, curved rods, oxidase positive Environmental microbes 3 species – Vibrio cholerae: gastroenteritis – Vibrio parahemolyticus: gastroenteritis – Vibrio vulnificus: cellulitis and gastroenteritis Cholera – Profuse watery diarrhea: Cholera toxin (ADP-ribosylation ADP- ribosylating toxin that constitutively activates adenylate cyclase in host cells) – Rice water stools – Cholera toxin, toxin co-regulated pilus – Dehydration – Vaccine available 35  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio paraheamolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 36  Enterobacteriaeceae Member of the Major Pathogenic Enterobacteriaceae Members of – Gram negative rods Enterobacteriaceae – Oxidase negative – E. coli – Reduce nitrates to nitrites – Klebsiella – Catalase positive – Salmonella – Serotyping: O,H,K antigens – Shigella Diagnose using agars that – Enterobacter differentiate based on – Citrobacter lactose fermentation – Serratia – Proteus 37  Gram Negative Rods Oxidase Test Oxidase Positive Oxidase Negative Enterobacteriaceae Rod Curved Rod Lactose fermentation Pseudomonas Campylobacter (grows at 42C) Vibrio (TCBS agar) Lactose positive Lactose negative E. coli Klebsiella H2S test Enterobacter H2S positive H2S negative Yersinia Proteus (swarmer, urease positive) Shigella Salmonella (urease negative) From Kaplan COMLEX USA Immunology and Microbiology Lecture Notes 2020 38  Enterobacteriaceae Diagnostics Gram negative rod Oxidase negative Nucleic Acid Amplification tests – Pathogen panels that test for multiple pathogens at once Selective and indicator media for lactose fermentation – MacConkey Agar Lactose + Pink Many are commensals in colon of – EMB Agar humans and other animals E. coli green metallic Lactose + purple Test for EHEC – Sorbitol MacConkey Agar – Shiga toxin tests ELISA Nucleic Acid amplification 39  “Pathotypes” of E. coli Gene clusters transferred Within these pathotypes there is a lot of variation. Multiple serotypes ETEC UPEC EPEC EIEC/Shigella 40 EHEC  GI Pathotypes of E. coli EPEC enteropathogenic EHEC enterohemmorhagic or STEC shiga-toxin producing ETEC enterotoxigenic EAEC enteroaggregative EIEC enteroinvasive DAEC diffusely adherent AIEC adherent invasive 41 Kaper et al. 2004. Nature Reviews Microbiology 2:123 42  Enteropathogenic E. coli (EPEC) Attaching and effacing lesions – Attach to intestinal epithelial cells and M cells – T3SS injects factors to rearrange actin create pedestal formation – Form microcolonies. Damage brush border of microvilli http://www.annauniv.edu/biotech/epec.jpg Animation: http://www.youtube.com/watch?v=gnNFNI9_pT0 http://www.hhmi.org/biointeractive/disease/ecoli.html 43  Enteropathogenic E. coli (EPEC) During the early stages of infection, E. coli rapidly removes microvilli on the surface of small intestinal cells The microvilli (grey) are lost both underneath and around the bacteria. http://www.staff.ncl.ac.uk/p.dean/Images E.coli on the surface of Caco-2 cells /body_images.html causing a increasing zone of peripheral effacement around the dividing bacteria 44  Enteropathogenic E. coli (EPEC) Distribution: occurs where the infrastructure is inadequate to prevent contamination of the environment with human waste. – High mortality in infants due to dehydration In these areas primarily infects infants – < 6 months Watery diarrhea 5-15 days, but may be persistent Travelers of all ages are susceptible – Acquired resistance especially to symptomatic infection 45 Kaper et al. 2004. Nature Reviews Microbiology 2:123 ”Pediatric diarrhea” Attaching and effacing lesions, pedestal formation Watery diarrhea 5-15 days. Sanitation. 46  Enterotoxigenic E. coli (ETEC) Distribution: Occurs where the infrastructure is inadequate to prevent contamination of the environment with human waste. “Traveler’s diarrhea”, pediatric diarrhea in endemic areas ~80,000 cases in US travelers/year Watery diarrhea that is self limiting ~1-3 days Enterotoxins 47  Enterotoxins heat labile toxin (LT) – similar to cholera toxin http://www.sumanasinc.com/webcontent/animations/content/diphtheria.ht ml binds to epithelial cells of small intestine ADP ribosylation of G protein, activates adenyl cyclase → cAMP Blocks Na+ transport into cell, water moves to lumen by osmosis. Also causes secretion of Cl- and HCO3- into lumen. heat stabile toxin (ST) increases cGMP = effects signaling pathways leading to loss of electrolytes and water from intestinal cells. 48 Kaper et al. 2004. Nature Reviews Microbiology 2:123 ”Pediatric diarrhea” Attaching and effacing Traveler’s diarrhea lesions, pedestal formation Watery diarrhea 1-3 days. Watery diarrhea 5-15 days. Enterotoxins Sanitation. Sanitation. 49  Enteroaggregative E. coli (EAEC ) Watery diarrhea that can be persistent All age groups—clinically a common cause of acute diarrhea of patients reporting to the ED. > 14 days (especially infants and young children) HIV patients Common cause of traveler’s diarrhea Distributed worldwide Food and water borne infection Pili or AAF (Aggregative adherence fimbriae) allow for bacteria to stack up on each other “Stacked brick” Mucosal damage, some inflammation Enterotoxins (not LT and ST) Cytotoxins Biofilm Persistence Drug resistance 50 Kaper et al. 2004. Nature Reviews Microbiology 2:123 ”Pediatric diarrhea” Attaching and effacing Traveler’s diarrhea lesions, pedestal formation Watery diarrhea 1-3 days. Watery diarrhea 5-15 days. Enterotoxins Sanitation. Sanitation. Worldwide distribution Traveler’s diarrhea but also present in “high resource” countries Persistant watery diarrhea >14 days. Aggregative biofilms on surface 51 Kaper et al. 2004. Nature Reviews Microbiology 2:123 ”Pediatric diarrhea” Attaching and effacing Traveler’s diarrhea lesions, pedestal formation Watery diarrhea 1-3 days. Watery diarrhea 5-15 days. Enterotoxins Sanitation. Sanitation. Worldwide distribution Traveler’s diarrhea but also Traveler’s diarrhea present in “high resource” Watery diarrhea 1-3 days. countries Enterotoxins Persistant watery diarrhea >14 Sanitation. days. Aggregative biofilms on surface 52 Kaper et al. 2004. Nature Reviews Microbiology 2:123 53  EHEC = STEC Enterohemorrhagic E. coli Shiga toxin producing E. coli Hemorrhagic colitis Hemolytic Uremic Syndrome (HUS) 54  EHEC/STEC Watery diarrhea that can become bloody due to action of shiga toxin – Shiga toxin can lead to hemolytic uremic syndrome Distribution: worldwide Most common E. coli enteric pathotype in US – ~265,000 STEC infections occur each year in the United States. Many serotypes O157:H7, O26, O104:H4 Serotyping: O antigen is lipopolysaccharide; K antigen is Capsular; H is Flagella Antigen 55  EHEC/STEC Transmission Reservoir host: ruminants and other wild mammals Foodborne Human to human contact Animal contact Low infectious dose < 100 cells 56 CDC: Outbreak of O157:H7 57  EHEC O26 Outbreak France 2019 16 total cases 14 cases of pediatric HUS Linked to consumption of soft raw cow milk cheese 58 https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2019.24.22.1900305 59  EHEC/STEC Virulence Factors Shiga toxin – Shiga-like toxin (verotoxin) – Interferes with 60S subunit of ribosomes → stops protein synthesis Damages cells, bloody diarrhea Hemolytic Uremic Syndrome (HUS) – Triad of thrombocytopenia, thrombatic microangiopathy, and hemolytic anemia – Toxin binds to renal endothelium where there is high receptor density (GB3) » Microthrombi—destruction of RBC’s, hemolytic anemia » Destruction of renal tissue/renal failure 60  EHEC/STEC Clinical Course Diarrhea May progress to hemorrhagic colitis due to shiga toxin – RBC in stool – Typically no PMNs in stool May progress to HUS – More common in children – Triad of thrombocytopenia, thrombatic microangiopathy, and hemolytic anemia 61  EHEC/STEC Diagnostics Test for Shiga toxin: Enzyme immunoassay (EIA or ELISA) or PCR for toxin genes Serotype to look for common shiga toxin producing strains – O157:H7 – O26 – O45 – O103 – O111 – O121 – O145 62  EHEC/STEC Treatment NO ANTIBIOTICS Supportive therapy Monitor for HUS 63 Kaper et al. 2004. Nature Reviews Microbiology 2:123 Hemorrhagic colitis→ ”Pediatric diarrhea” HUS Attaching and effacing Common in US Traveler’s diarrhea lesions, pedestal formation Watery diarrhea 1-3 days. Shiga toxin Watery diarrhea 5-15 days. No antibiotic treatment Enterotoxins Sanitation. Sorbitol MacConkey Agar Sanitation. Diarrhea in 1-5 year olds, Worldwide distribution exacerbation of diarrhea UC Traveler’s diarrhea but also and IBD present in “high resource” Disrupts cell signaling and countries generate growth of long Persistant watery diarrhea >14 finger-like cellular days. projections, which wrap Aggregative biofilms on surface around the bacteria 64 Kaper et al. 2004. Nature Reviews Microbiology 2:123 65  EIEC Enteroinvasive E. coli Inflammatory diarrhea/dysentery Rare! Bacteria invade cells Multiply within cells Form actin rocket tails to move to adjacent cells Cause ulcers in mucosa Clinically: – Starts as watery diarrhea – Progress to inflammatory diarrhea Blood and PMN in stool Fever and cramping 66  Several 4-year-old children who attend the same preschool are hospitalized for severe, bloody diarrhea. All of the children ate at the same fast-food restaurant during class field trip. In the second week of the illness, one of the children additionally develops thrombocytopenia, hemolytic anemia, and acute renal insufficiency. The most likely causative agent is: A. EAEC ⑧ B. EHEC C. EIEC D. EPEC E. ETEC F. Campylobacter jejuni Levinson Review of Medical Microbiology and Immunology 67  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus Watery Diarrhea – Helicobacter pylori – Sapovirus – Vibrio cholerae, Vibrio paraheamolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica Diarrhea – Campylobacter jejuni – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 68  Shigella CDC estimates ~450,000 infections in the US each year – Highly contagious – Multidrug resistant strains circulating in 2023 Eneterobacteriaceae, non-lactose fermenting 4 main species, different serotypes within each species (47 serotypes) – S. dysenteriae (Group A) – most pathogenic “Type 1” shiga toxin Rare in the U.S. – S. flexneri (Group B) – S. boydii (Group C) – Rare in the US – S. sonnei (Group D) – Most common in U.S. mildest 69  Shigella Course of Infection Highly infectious—fewer than 10 cells could cause infection Invade cells Actin Rockets (Like EIEC) Shiga toxin: Some strains produce shiga toxin https://doi.org/10.1016/j.micres.2015.08.006 70  Shigella Clinical Course Begins as watery diarrhea Can progresses to bloody diarrhea – Lower abdominal cramps – Tenesmus – Abundant RBCs and PMNs in stool – Fever Diarrhea lasts about 7 days Hemolytic Uremic Syndrome – From infections with strains that carry genes for shiga toxin 71  Gram Negative Rods Oxidase Test Oxidase Positive Oxidase Negative Enterobacteriaceae Rod Curved Rod Lactose fermentation Pseudomonas Campylobacter (grows at 42C) Vibrio (TCBS agar) Lactose positive Lactose negative E. coli Klebsiella H2S test H2S positive H2S negative Yersinia Proteus (swarmer, urease positive) Shigella Salmonella (urease negative) From Kaplan COMLEX USA Immunology and Microbiology Lecture Notes 2020 72  Shigella Diagnosis and Treatment Microbiological diagnosis – Gram negative rod – Oxidase negative – Lactose negative – Hektoin’s Agar (Shigella-Salmonella agar) H2S negative Hektoins turns red if lactose positive Nucleic acid amplification Salmonella (and Rapid point of care tests Shigella and Yersinia are Proteus) is – Lateral flow immune assay – Test for pathogen and shiga toxin colorless on the black on the green-blue agar green-blue agar Treatment – Self resolution – Antibiotics shorten course and severity and may reduce the spread of infection Ciprofloxacin Azithromycin 73  Shigella Summary Highly contagious – Fewer than 10 cells required to establish infection Disease course – Watery diarrhea – Progresses to inflammatory diarrhea Pain and tenesmus Fever, RBC and PMNs in stool Ciprofloxacin, azithromycin 74  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 75  Salmonella—What do I call it? Salmonella enterica – 6 major types—2 clinically relevant Gastroenteritis Typhoid fever 2400+serovars Salmonella enterica Typhi Salmonella enterica Typhimurium Salmonella enterica Paratyphi A Salmonella enterica Paratyphi B 76  Salmonella Gastroenteritis CDC says annually: – 1.35 Million illnesses – 26,500 hospitalizations – 420 deaths Transmission – Sensitive to stomach acid: Larger inoculum needed ~105 – Contaminated Food Several animal reservoirs (chickens and eggs) Outbreaks from peanut butter, and vegetables and fruits (fruit juices) – exposure to pets (especially reptiles and turtles) ~90% of reptiles carry the bacteria birds, rodents, dogs, and cats are also potential reservoirs 77 78 79  Salmonella: Invasion of Cells Invades M-cells and macrophages—replicates within them. Some strains disseminate readily. 80  Salmonella Clinical Course Nausea, vomiting, watery diarrhea that can progress to inflammatory diarrhea – Pain, fever, RBCs and PMNs in stool ~4-7 days Some strains disseminate and cause infections elsewhere in the body – Usually young or elderly or immunocompromised Osteomyelitis in those with sickle cell disease Can cause reactive arthritis post infection 81 Salmonella Diagnosis Enterobacteriaciae – G- rod – Oxidase negative – Lactose negative Motile (Shigella is not) Produces H2S Hektoen agar or Shigella- salmonella agar = black colonies – Differentiate from Shigella Nucleic acid amplification 82  Salmonella Treatment CDC: Antibiotics not recommended unless symptoms are severe or patient is vulnerable – Fluoroquinolones for adults – Azithromycin for children 83  Salmonella summary Enteric disease v. Typhoid fever Animal reservoirs, food borne infection Inflammatory diarrhea with nausea, vomiting and abdominal pain Non-lactose fermenting Produce H2S—black colonies on SS agar or Hektoen’s agar Antibiotics only in severe cases or vulnerable patient – Fluoroquinolones for adults – Azithromycin for children 84  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 85  Yersinia enterocolitica and Yersinia pseudotubuculosis Yersinia enterocolitica most common US : 117,000 illnesses, 640 hospitalizations, 35 deaths per year (CDC) – Zoonotic Reservoir (pigs, cattle, deer, birds, dogs, aquatic organisms) – Contaminated water and food Meat—especially pork Milk Some reports of contaminated vegetables Can grow in cold environments (refrigerator) 86 87  Yersinia Clinical Course Watery diarrhea→Inflammatory diarrhea with fever, abdominal pain (esp. right lower quadrant) and bloody diarrhea – Pseudo-appendicitis Symptoms last 1-3 weeks or longer Mesenteric lymphadenitis—palpable enlarged lymph nodes Postinfectious syndromes – Erythema nodosum – Reactive arthritis can follow infection http://onlinelibrary.wiley.com/doi/10.1111/j.1346-8138.2010.00892.x/full 88 Yersinia Diagnosis Gram-negative rod Non-lactose fermenter, No production of H2S Isolate on highly selective agar – YSA (Yersinia Selective Agar) Selects for all species of Yersinia including Y. entericolitica and Y. pseudotuberculosis – CIN broth/agar Selects for Yersinia entericolitica specifically Cefsulodin, Irgasan, Novobicin – Grows better at cooler temps 25C v 37C Grows inside refrigerator at 4C 89  Gram Negative Rods Oxidase Test Oxidase Positive Oxidase Negative Enterobacteriaceae Rod Curved Rod Lactose fermentation Pseudomonas Campylobacter (grows at 42C) Vibrio (TCBS agar) Lactose positive Lactose negative E. coli Klebsiella H2S test H2S positive H2S negative Yersinia Proteus (swarmer, urease positive) Shigella Salmonella (urease negative) From Kaplan COMLEX USA Immunology and Microbiology Lecture Notes 2020 90  Yersinia Treatment Self resolution Severe or complicated infections: – Trimethoprim-sulfamethoxazole – Aminoglycosides – Third generation cephalosporins – Fluoroquinolone – Typically resistant to: First generation cephalosporins Most penicillins 91  Yersinia entericolitica A 74-year-old patient presented to an internal medicine clinic with fever, vomiting and diarrhea lasting for 4 days. She was diagnosed with acute gastroenteritis and was sent home with gastrointestinal medication. The symptoms progressed 5 days later when she attended for a follow up, and she was admitted to hospital. On the 4th day of hospitalization, she developed skin lesions on the knees that were tender, relatively well-demarcated, erythematous nodules. Each nodule was approximately 1–2 cm in diameter. Fecal sample was first cultured 24 h in Salmonella Shigella agar (SS agar), and colonies colorless colonies were identified. To confirm diagnosis, we then incubated the sample at 25 and 37°C for 48 h with the use of CIN broth (Y. enterocolitica-selective agar). The growth rates were faster at 25°C than 37°C, and therefore, we diagnosed the patient with EN associated with Y. enterocolitica. The patient was treated for 11 days with bedrest and levofloxacin 400 mg/day oral medication. Yotsu et al 2010 The Journal of Dermatology: 92 http://onlinelibrary.wiley.com/doi/10.1111/j.1346-8138.2010.00892.x/full  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio paraheamolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 93  Campylobacter jejuni ~1.5 million illnesses in the US per year. CDC estimates it is the most common intestinal infection diagnosed in returning travelers Bacterial characteristics – Gram negative, spiral/curved rods, motile, bipolar flagella – Oxidase positive Transmission – Foodborne Poultry and other meats – Contact with animals (several— chickens, cows, sheep…) Petting zoos and farms – Drinking contaminated water 94  Campylobacter Clinical Course Nausea and vomiting Watery diarrhea→Inflammatory diarrhea – Abdominal pain, fever, diarrhea that may turn bloody – Self limiting ~1 week – Ulceration of mucosa Cytotoxins – Confused with ulcerative colitis/Crohns disease Last about 5 days-1 week Post infectious syndromes: – Associated with Guillan-Barre syndrome, – Post infectious IBS – Reactive arthritis 95  Campylobacter Diagnosis Microbiological diagnosis – Media = “Campy” or Skirrow agar Inhibit normal flora – Growth at 42 C (high temp) – Microaerophillic (low oxygen , high Carbon dioxide) – Comma or S-shaped, motile “seagull shaped” Nucleic amplification tests 96  Gram Negative Rods Oxidase Test Oxidase Positive Oxidase Negative Enterobacteriaceae Rod Curved Rod Lactose fermentation Pseudomonas Campylobacter (grows at 42C) Vibrio (TCBS agar) Lactose positive Lactose negative E. coli Klebsiella H2S test H2S positive H2S negative Yersinia Proteus (swarmer, urease positive) Shigella Salmonella (urease negative) From Kaplan COMLEX USA Immunology and Microbiology Lecture Notes 2020 97  Campylobacter Treatment Treatment – Self resolution – Azithromycin and fluoroquinolones used for treatment in vulnerable patients Antibiotic resistance to fluoroquinolones is becoming common 98  Campylobacter Summary Major cause of diarrhea worldwide Transmission via food – Poultry – Meat – Milk Contact with animals Diagnosis – Gram negative curved rods – Media = “Campy” or Skirrow agar Inhibit normal flora – Growth at 42C – Microaerophillic (high Carbon dioxide) – Comma or S-shaped – Azithromycin and fluoroquinolones used for treatment 99  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 100  Clostridioides difficile ~500,000 infections per year in the US Antibiotic associated inflammatory diarrhea and colitis (5-10 days after antibiotic use) Gram positive rod, spore-former, obligate anaerobes 101  C. difficile Transmission Normal flora Obligate anaerobe SPORES! Contaminate environments – Higher risk in hospitals and long-term care settings People are susceptible to infection particularly after antibiotic treatment. – Kills normal flora – Kills vegetative C. dif – Does not kill spores—they germinate and C. dif overgrows when there is no competition for resources – With no normal flora, spores in the environment more easily colonize Any antibiotic treatment but strongly associated with: clindamycin, cephalosporin, ampicillin, amoxicillin Can also get overgrowth without antibiotic treatment 102 Course of Infection and Virulence Factors toxin A enterotoxin Inflammation Disrupts tight junctions toxin B cytotoxin Rupnik et al 2009. Nature Reviews 103 Microbiology 7: 526-36.  Pseudomembranous colitis (PMC) www.cfpc.ca PMC is due to bacteria and inflammatory damage of colon epithelia 104  C. difficile Clinical Course Inflammatory diarrhea Pseudomembranous colitis – Necro-inflammatory reaction Recurrent infections common 105  C. dif Diagnosis and Treatment Microbiological Diagnosis – Is “difficile” and normal flora (anaerobe) Detect toxins with ELISA or PCR tests Treatment: Use antibiotic that will kill C. diff: vancomycin, fidaxomycin – Fecal microbiota transplants in clinical studies Prevention: – disinfection of hospital rooms/long term care facilities – avoid giving antibiotics when not necessary 106  Clostridioides difficile summary Pseudomembranous colitis – Adherent inflammatory membrane overlaying injury – Toxin A and B Exotoxins Used for diagnosis G+ Spore formers Anaerobes Antibiotic treatment leads to C. diff overgrowth – Spores remain in patient – Spores in environment establish more easily when native fauna is disturbed Diagnosis with Nucleic acid amplification, Toxin ELISA (no culture) Treatment with vancomycin or fidaxomycin 107  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica – Campylobacter jejuni Diarrhea – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 108  Listeria monocytogenes ~1500-2000 cases in the US per year according to the CDC Found worldwide in soil and animals Typically obtained by ingesting contaminated food – Unpasteurized milk and cheese – Deli meats/hotdogs – Vegetables and fruits – Undercooked meat – Need large inoculum to get infected—bacteria continue to Gram-positive rod replicate at refrigeration temperatures Most likely to affect – Pregnant women and newborns https://www.studyblue.com/notes/note/n/unit-5/deck/9768332 – People over 65 – People with weakened immune systems Listeria Infection and Virulence Factors Intracellular – Invades intestinal epithelial cells – Highly motile Polymerizes actin Actin rocket (like Shigella and EIEC) – Can invade monocytes and macrophages and be distributed throughout the body. – Can cross blood-brain barrier (meningitis) – Can cross placenta Video of actin rockets: https://www.youtube.com/watch?v=4JWp G8XPku4  Listeria Clinical Course What Does Listeriosis Look Like in a Patient? Spectrum: Asymptomatic-gastroenteritis-febrile illness— meningoencephalitis—death Healthy: Often asymptomatic large inoculum necessary for infection. Mild gastroenteritis with or without fever Children, elderly, immunocompromised: – Febrile gastroenteritis, myalgia, athralgia – Bacteria can disseminate Bacteremia/septicemia Cross blood-brain barrier=Meningoencephalitis, Pregnant Women and Fetus Febrile illness, gastroenteritis, myalgia, athralgia Antibiotic treatment important (Ampicillin) – Crosses the placenta Sepsis and death of fetus or newborn Meningitis 111 Listeria Case Report 28-year-old-woman 29 Weeks gestation – Vaginal bleeding, abdominal pain, tenderness, “fever” 100.6 F. – Deceleration of fetal heart rate forced induction of labor – Birth of Neonate Abscesses on placenta Neonate → NICU – Sepsis, meningitis, pulmonary hemorrhage, hypocalcemia, seizures, respiratory distress, hyperbilirubinemia – Blood culture + for Listeria 112  Listeria Diagnosis and Treatment Microbiological: – Gram positive rods – Culture: Blood or CSF blood agar (or other specialized selective agars) Mildly beta-hemolytic Mobile at 22C not 37C Grows in cold temperatures Nucleic Acid amplification tests Treatment – Ampicillin or amoxicillin for mild symptoms – Invasive infection or immunocompromised patient http://microbe-canvas.com/Bacteria/gram-positive-rods/cells- straight/facultative-anaerobic-4/catalase-positive-1/camp- Ampicillin with gentamycin reaction-4/listeria-monocytogenes.html  Listeria Summary Febrile gastroenteritis, septicemia, meningitis – Children, elderly, immunocompromised: Cross blood-brain barrier=Meningoencephalitis, – Pregnant Women and Fetus Food borne – Raw milk, soft cheese, vegetables Intracellular—actin rockets Listeriolysin to escape phagosome Culture on Blood agar Gram positive rod, beta hemolytic  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus – Helicobacter pylori Watery Diarrhea – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC DAEC AIEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica Diarrhea – Campylobacter jejuni – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei Whipples disease 115  Tropheryma whipplei Gram positive rod – Does not gram stain well Colonizes mucosa of intestine Most people remain asymptomatic, some develop Whipples disease. GI symptoms include gastroenteritis with diarrhea, abdominal pain, steatorrhea, hepatosplenomegaly, anorexia The bacteria can disseminate via macrophages and become a multisystemic disease. – Intermittent arthralgia, diarrhea, weightloss, lymphadenopathy, neural manifestations, bone and joint infection, endocarditis Disease typically develops between 48-54 years old Men are 8 times as likely to have symptoms than women Diagnosis: histopathology of biopsy or PCR Treatment: Ceftriaxone or meropenem 116  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus Watery Diarrhea – Helicobacter pylori – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica Diarrhea – Campylobacter jejuni – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 117  Viral Gastroenteritis Viruses are significant cause of gastroenteritis worldwide – High morbidity for young, elderly, and immunocompromised – Norovirus most significant in the U.S. Viral gastroenteritis typically mild and self limited – Reported and confirmed diagnoses less common – Statistics limited Highly contagious – Person to person contact – Food borne transmission Mattison CP, Dunn M, Wikswo ME, et al. Non-Norovirus Viral Gastroenteritis Outbreaks Reported to the National Outbreak Reporting System, USA, 2009-2018. Emerg Infect Dis. 2021;27(2):560-564. doi:10.3201/eid2702.203943 118 Norovirus 119  Norovirus—Norwalk Virus Leading cause of acute gastroenteritis among all age groups in the United States. – 58% of all food borne illness in the US according to the CDC Very Common Worldwide 19 to 21 million total illnesses per year. It is estimated to cause 56,000- 71,000 hospitalizations and 570-800 deaths annually in the United States Highly infectious – 10 virions = infection Remains viable in the environment for days or weeks and is resistant to disinfection (non-enveloped) https://www.cdc.gov/norovirus/trends-outbreaks/burden-US.html Hall AJ, Lopman BA, Payne DC, Patel MM, Gastañaduy PA, Vinjé J, Parashar UD. Norovirus disease in the United States. Emerg Infect Dis. 2013 Aug;19(8):1198-205. [PMC free article] [PubMed] [Reference list] 120  Norovirus: Viral Characteristics Calciviridae Icosahedral Nonenveloped + ssRNA genome 121  Norovirus: Transmission https://www.cdc.gov/norovirus/trends-outbreaks/burden-US.html 122  Norovirus: Clinical Course Nausea and vomiting, watery diarrhea Sometimes stomach cramps, chills, muscle aches ~24-72 hrs Virus present in feces – Before and after symptoms – Patients should remain isolated for 48 hours after symptoms resolve Highly contagious – Outbreaks Daycare settings Cruise ships Restaurants Healthcare settings 123  Norovirus: Summary Acute gastroenteritis vomiting and watery diarrhea Short duration All age groups Highly contagious – Non-enveloped – Small infectious dose Transmission peaks in winter Calciviridae, icosahedral, +ssRNA genome 124 Reported non-noroviral gastroenteritis outbreaks https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853577/ 125  Rotavirus Severe watery diarrhea and vomiting – Infants and young children Clinically less common in the U.S. – Vaccine available Prior to vaccine availability (2006) rotavirus was the leading cause of severe diarrhea among infants and young children in the U.S. Worldwide significant source of mortality of infants and young children 126  Rotavirus: Viral Characteristics Reoviridae Ds RNA genome – Segmented – Recombination leads https://www.cdc.gov/dotw/rotavirus/index.html to diversity Non-enveloped Icosahedral “wheel-like appearance” 127  Rotavirus: Clinical Course Vomiting and watery diarrhea – Sometimes: fever and abdominal pain 3-8 days Multiple infections throughout life – First episode usually most severe – Partial immunity gained with infection – Vaccine does not prevent all infections 128  Rotavirus: Transmission Transmission peaks January-June (U.S.) – Winter and spring Young children and infants, but adults can be infected Outbreaks – Especially in unvaccinated populations – Daycare settings – Adults in assisted living 129  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus Watery Diarrhea – Helicobacter pylori – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica Diarrhea – Campylobacter jejuni – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 130  Viral Gastroenteritis Astrovirus Sapovirus Adenovirus ~2-9% of cases ~3-17% of cases ~2-10% of cases worldwide worldwide worldwide Young children Young children Children < 24 months Astroviridae Calciviridae Adenoviridae Icosahedral Icosahedral Icosahedral + ss RNA genome + ss RNA genome DS DNA genome Nonenveloped Nonenveloped Nonenveloped Star shaped Types 40 and 41 cause enteritis with watery diarrhea and vomiting. (others typically cause respiratory illness or 131 conjunctivitis)  Norovirus Rotavirus Adenovirus Astrovirus Sapovirus Family Calciviridae Reoviridae Adenovirida Astroviridae Calciviridae e Genome Ss + RNA Ds RNA Ds DNA Ss + RNA Ss + RNA Envelope Non- Non- Non- Non- Non- enveloped enveloped enveloped enveloped enveloped Capsid Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral shape Features Wheel Star shaped shaped Segmented genome Recombinati on Vaccine No Yes No No No 132  Pathogens Bacteria Viruses – Preformed toxin “food poisoning” – Norovirus Staphylococcus aureus – Rotavirus Bacillus cereus – Adenovirus Clostridium perfringens – Astrovirus Watery Diarrhea – Helicobacter pylori – Sapovirus – Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus – Escherichia coli ETEC EPEC EAEC EHEC EIEC – Shigella Inflammatory – Salmonella enterica – Yersinia enterocolitica Diarrhea – Campylobacter jejuni – Clostridioides difficile – Listeria monocytogenes – Tropheryma whipplei 133  Your patient is a 40-year-old patient with diarrhea that began on the airplane while she was returning from a vacation in the Middle East. She had had 5 episodes of watery non-bloody diarrhea and little vomiting. She is afebrile. A stool culture reveals only lactose-fermenting colonies on EMB agar. Which is the most likely cause? A. Shigella sonnei B. Salmonella typhi C. Vibrio cholerae D. ETEC E. EHEC Levinson Review of Medical Microbiology and Immunology 134  Your patient is a 56-year-old woman who was treated with ampicillin for cellulitis caused by Streptococcus pyogenes. Several days later she developed bloody diarrhea and a colonoscopy indicated pseudomembranous plaques. Which of the following describes the most likely pathogen? A. It is an anaerobic Gram-positive rod that produces exotoxins B. It is a comma shaped Gram- negative rod that grows best at 41C C. It is a facultative Gram-negative rod that forms spores D. It is an obligate intracellular parasite that grows in cell culture but not blood agar E. It is a parasite that produces cysts with four nuclei Levinson Review of Medical Microbiology and Immunology 135  Your patient is a 56-year-old woman who was treated with ampicillin for cellulitis caused by Streptococcus pyogenes. Several days later she developed bloody diarrhea and a colonoscopy indicated pseudomembranous plaques. Which of the following describes the most likely pathogen? A. It is an anaerobic Gram-positive rod that produces exotoxins B. It is a comma shaped Gram- negative rod that grows best at 41C C. It is a facultative Gram-negative rod that forms spores D. It is an obligate intracellular parasite that grows in cell culture but not blood agar E. It is a parasite that produces cysts with four nuclei Levinson Review of Medical Microbiology and Immunology 136  A 3-year-old boy who attends a large daycare facility suddenly develops a fever, vomiting, diarrhea, and abdominal pain. When he is brought to the ED three days later, he has more than 10 bowel movements in a day, and mucus, blood, and pus are seen in the stool. He has developed weight loss and severe dehydration. His temperature is 38C, blood pressure is 90/60 mm Hg, pulse is 130 min, and respirations are 20/min. O 2 saturation is 98%. No rash is observed. Which is most likely? A. Norovirus B. Rotavirus C. Salmonella enterica typhi D. Shigella sonnei E. Vibrio cholerae 137  A 4-year-old boy is brought to the ED because of watery diarrhea. He is not up to date on vaccinations. PCR analysis shows a pathogen that has the ability to undergo recombination. Which is most likely? A. Bacteria, Gram negative B. Bacteria Gram positive C. Virus double-stranded RNA genome* D. Virus double stranded linear DNA genome E. Virus, single stranded RNA genome  Rotavirus is a very common cause of watery diarrhea in the developing world and unvaccinated children. Rotavirus has a double stranded RNA genome (Reoviridae). The genome is segmented and recombination occurs. Other viruses with segmented genomes: influenza, bunyaviruses, arenaviruses Review Slides 140  Bacterial Pathogens: Culture Based Methods Enterobacteriaceae Other Gram Negative Rods G- rod Oxidase negative Oxidase positive Curved rods Escherichia coli Vibrio cholerae – ETEC Vibrio paraheamolyticus – EPEC Vibrio vulnificus – EAEC Campylobacter jejuni – EHEC – EIEC Helicobacter pylori Shigella (don’t forget that Pseudomonas Salmonella enterica aeruginosa is also an ox+ G- rod) Yersinia enterocolitica 141  Bacterial Pathogens: Culture Based Methods Enterobacteriaceae Lactose fermentation Oxidase negative Escherichia coli – ETEC – EPEC – EAEC – EHEC No Sorbitol Fermentation – EIEC Shigella Salmonella enterica H2S production Yersinia enterocolitica CIN agar, 25C, cold enrichment 142 First aid for the basic sciences, pg 279. Yersinia 143  Lab identification of the enterics MacConkey Agar Selective – Bile salts/crystal violet inhibit gram positives and most others besides Enterobacteriaceae and Pseudomonas Indicator – Neutral red – Ferment lactose (acid=pink) – No fermentation - colorless www.mc.maricopa.edu 144  Enterobacteriaceae Lactose Fermentation Positive Negative E. coli Shigella Klebsiella Salmonella Enterobacter Yersinia 145  Eosin Methylene Blue Agar Selective—only Gram negatives (mostly)(eosin and methylene blue) Lactose fermenters = dark purple High lactose = green metallic sheen (E. coli) 146  EMB Agar Lactose Positive Lactose Negative Shigella Enterobacter Salmonella Yersinia Klebsiella E. coli 147  Hektoen’s Agar (differentiates Salmonella from Shigella from other enterics) Lactose fermenter → (acidify) red/orange agar Peptone utlization→ (alkalises) green/ blue agar H2S production → black (thiosulfate) Shigella Salmonella E. coli Differential for lactose fermentation Differential for H2S production Red = lactose + Black=Salmonella Green/Blue = lactose - Clear = Shigella 148 www.austincc.edu/microbugz/  Bacterial Pathogens: Culture Based Methods Other Gram Negative Rods Oxidase positive Vibrio cholerae Curved rods Alkaline Medium: TCBS agar Vibrio paraheamolyticus Vibrio vulnificus CAMPY agar or Skirrow Agar Microaerophillic, grows at 42C Campylobacter jejuni Helicobacter pylori No culture, serologic test or breath test 149 First aid for the basic sciences, pg 279. 150 Sample Questions 151  A 30-year-old patient presents with fever, abdominal cramps, and bloody diarrhea. The patient reports recent travel Nicaragua and the day before returning home, several members of the group developed similar symptoms. Which is the LEAST likely cause? A. Shigella dysenteriae B. Salmonella enteritidis C. Vibrio cholerae D. Campylobacter jejuni Levinson Review of Medical Microbiology and Immunology 152  What color would Salmonella appear on MacConkey Agar? A. Pink B. No color 153  What color would EHEC appear on MacConkey Sorbitol Agar? A. Pink 50% 50% B. No Color k lor Pin Co No 154 A 20-year-old man presents with watery diarrhea that has lasted 3 days. He just returned from a trip to Kenya 2 days ago. He has no fever and no fecal leukocytes are detected in a stool sample. The pathogen is an oxidase negative, Gram-negative rod that grows pink colonies on Mac Conkey Agar. Which of the following is most likely? A. Vibrio cholerae B. Shigella sonnei C. Campylobacter jejuni D. ETEC E. EHEC F. EPEC 155  What color would the colonies be on EMB (eosin methylene blue) agar? A. Blue B. Green C. Black D. Yellow E. Pink F. Colorless 156  Cholera toxin is most similar to: A. Enterotoxin of Staphylococcus aureus B. Alpha toxin of Staphylococcus aureus C. Shiga Toxin of Shigella sonnei D. Diphtheria toxin of Corynebacterium diphtheriae E. LT toxin of enterotoxigenic E. coli (ETEC)  Of the following, which is most compatible with cholera? A. Watery diarrhea, no PMN’s in stool, and growth of curved Gram-negative rods on blood culture B. Watery diarrhea, no PMN’s in stool, and no organisms on blood culture C. Bloody diarrhea, PMN’s in stool, and growth of curved Gram- negative rods on blood culture D. Bloody diarrhea, PMN’s in stool, and no growth of organisms on blood culture Levinson Review of Medical Microbiology and Immunology 158  For which one of the following enteric illnesses is a chronic carrier state MOST likely to develop? A. Campylobacter enteritis B. Shigellosis C. Cholera D. Typhoid fever Levinson Review of Medical Microbiology and Immunology 159  Each of the following statements is correct EXCEPT: A. Proteus sp. are highly motile, found in the human colon and can cause UTI. B. Klebsiella pneumoniae is part of the normal flora of the colon and can cause pneumonia. C. E. coli is part of the normal flora of the colon; therefore, it rarely causes diarrhea D. E. coli ferments lactose, whereas Salmonella and Shigella do not. Levinson Review of Medical Microbiology and Immunology 160  Your patient is a 40-year-old woman with diarrhea that began on the airplane while she was returning from a vacation in the Middle East. She had had 5 episodes of watery non-bloody diarrhea and little vomiting. She is afebrile. A stool culture reveals only lactose-fermenting colonies on EMB agar. Which is the most likely cause? A. Shigella sonnei B. Salmonella typhi C. Vibrio cholerae D. ETEC E. EHEC Levinson Review of Medical Microbiology and Immunology 161  Which of the following host defense mechanisms is the MOST important for preventing dysentery caused by Salmonella? A. Gastric acid B. Salivary enzymes C. Normal flora of the mouth D. Alpha interferon Levinson Review of Medical Microbiology and Immunology 162  Which one of the following pathogens has the LOWEST 50% infectious dose (ID50)? A. Shigella sonnei B. Vibrio cholerae C. Salmonella enterica D. Campylobacter jejuni Levinson Review of Medical Microbiology and Immunology 163  Which one of the following pathogens that infects the GI tract is most likely to cause bacteremia? A. Shigella flexneri B. Vibrio cholerae C. Salmonella enterica typhi D. Campylobacter jejuni E. Helicobacter pylori Levinson Review of Medical Microbiology and Immunology 164  A 3-year-old boy who attends a large daycare facility suddenly develops a fever, vomiting, diarrhea, and abdominal pain. When he is brought to the ED three days later, he has more than 10 bowel movements in a day, and mucus, blood, and pus are seen in the stool. He has developed weight loss and severe dehydration. His temperature is 38C, blood pressure is 90/60 mm Hg, pulse is 130 min, and respirations are 20/min. O 2 saturation is 98%. No rash is observed. Which is most likely? A. Norovirus B. Rotavirus C. Salmonella enterica typhi D. Shigella sonnei E. Vibrio cholerae 165  A 56-year-old patient was treated with ampicillin for cellulitis caused by Streptococcus pyogenes. Several days later she developed bloody diarrhea and a colonoscopy indicated pseudomembranous plaques. Which of the following describes the most likely pathogen? A. It is an anaerobic Gram-positive rod that produces exotoxins B. It is a comma shaped Gram- negative rod that grows best at 41C C. It is a facultative Gram-negative rod that forms spores D. It is an obligate intracellular parasite that grows in cell culture but not blood agar E. It is a parasite that produces cysts with four nuclei Levinson Review of Medical Microbiology and Immunology 166 Questions from FastPas USMLE A previously healthy 45-year-old is brought to the emergency room because of fever and bloody diarrhea over the past 3 days following a company barbecue. The man is oriented to person and place, but not time. His temperature is 101F, pulse 105/min, respirations 14/min. WBC count = 13000/mm3, hemoglobin of 10g/dL, hematocrit of 30%, platelets of 75,000/mm3. He has creatinine of 1.8 mg/dL, and elevated lactate dehydrogenase. Which is most likely? A. ETEC B. Salmonella C. Shigella D. Staphylococcus aureus E. Campylobacter jejuni  Shigella Bloody diarrhea—inflammatory He is anemic, thrombocytopenic, with acute kidney injury (creatinine). He also has signs of hemolysis given his elevated lactate dehydrogenase level. These are all signs of hemolytic uremic syndrome HUS. A 20-year-old man is brought to the emergency room because of a 2-day history of bloody diarrhea. He has had fever, nausea, and vomiting that began after he ate at a new restaurant. The causative organism is isolated and Gram stain shows a Gram-negative bacillus that is colorless on MacConkey agar and colorless on Salmonella-Shigella agar. Which is the most likely pathogen? A. Campylobacter jejuni B. Escherichia coli C. Shigella sonnei D. Vibrio cholera E. Vibrio parahaemolyticus  Shigella is the best answer. Inflammatory diarrhea. Non-lactose fermenting, gram negative rod, and is oxidase negative. Campylobacter and Vibrio are oxidase positive  Cholera toxin is most similar to: A. Enterotoxin of Staphylococcus aureus B. Alpha toxin of Staphylococcus aureus C. Shiga Toxin of Shigella sonnei D. Diphtheria toxin of Corynebacterium diphtheriae E. LT toxin of enterotoxigenic E. coli (ETEC)  A 10-year old child is brought to the ED because of diarrhea, fever, nausea and vomiting. He went on a Boy Scout trip several days ago. Analysis of the pathogen indicates a bacterium that does not ferment lactose but produces hydrogen sulfide and expresses flagellar motility. Which is the most likely pathogen? A. Shigella B. Salmonella C. Vibrio cholera D. Campylobacter jejuni E. Yersinia entericolitica  Inflammatory diarrhea and bacteria that does not ferment lactose and produces H2S = Salmonella A 53-year-old patient with hypertension and dyslipidemia comes to the physician because of a 4-month history of dyspepsia, epigastric pain, and unintentional (10 lb) weight loss. Abdominal CT scan shows a mass at the antrum of the stomach. The mass biopsy show CD19 and CD20 positivity. DNA analysis shows translocation t(11;18) Urease breath test is positive. Which of the following treatment options is most likely to eradicate the mass? A. Famotidine and penicillin B. Metoclopramide C. Omeprazole, amoxicillin, and clarithromycin D. Penicillin alone E. Vincristine  The immunohistological staining and DNA translocation indicates a gastric mucosa- associated lymphoid tissue lymphoma (MALToma) Most commonly associated with Helicobacter pylori infection Tumors frequenly go into remission upon bacterial treatment Triple drug therapy including proton-pump inhibitor and two antibiotics. A 4-year-old child is brought to the ED because of watery diarrhea. He is not up to date on vaccinations. PCR analysis shows a pathogen that has the ability to undergo recombination. Which is most likely? A. Bacteria, Gram negative B. Bacteria Gram positive C. Virus double-stranded RNA genome* D. Virus double stranded linear DNA genome E. Virus, single stranded RNA genome  Rotavirus is a very common cause of watery diarrhea in the developing world and unvaccinated children. Rotavirus has a double stranded RNA genome (Reoviridae). The genome is segmented and recombination occurs. Other viruses with segmented genomes: influenza, bunyaviruses, arenaviruses  A 34-year old woman comes to the physician because of greasy, foul-smelling diarrhea over the past 3 weeks. She traveled to Mexico last month. She has nausea and bloating but no fever, chills, or blood in the stool. She has lost 4 lbs over the past 3 weeks. She has no oral ulcer, joint pain, skin rash, or abdominal pain. Examination of fecal materials shows motile, flagellated, binucleated, pearshaped trophozoites. Which is the treatment of choice? A. Chloroquine B. Diloxanide C. Emetine D. Iodoquinol E. Metronidazole  Classic case of giardia Shape of trophozoites Adhere to mucosa of small intestine and cause malabsorption—steatorrhea and weightloss. Metronidazole is the treatment. The other drugs are for treatment of amebiasis  A five year old boy is brought to the physician because of impaired growth. He was in the 54 th percentile for height and weight at birth; however recently he has not been growing despite a nutritious diet. His family lives on a farm. A scotch tape test is negative. Laboratory studies show eosinophilia. Which is the most likely pathogen: A. Ascaris