Lecture 8 Microbiology 29-04-21 PDF

Lecture 8 Vibrio, Campylobacter , Helicobacter 11/3/2022 1  Content Microbiology: Vibrios, Campylobacter, Helicobacter 1. Vibrios The main biological characteristics of Vibrio cholerae Pathoge...

Lecture 8 Vibrio, Campylobacter , Helicobacter 11/3/2022 1  Content Microbiology: Vibrios, Campylobacter, Helicobacter 1. Vibrios The main biological characteristics of Vibrio cholerae Pathogenesis of the diseases induced by Vibrio cholerae and the immunity (Vibrio cholerae enterotoxin and function in the disease) Diagnostic laboratory tests for the diseases induced by Vibrio cholerae Treatment, Epidemiology, Prevention and Control of the diseases induced by the Vibrio cholerae Overview of Vibrio parahaemolyticus & Other Vibrios, Aeromonas and Plesiomonas Campylobacters The main biological characteristics of Campylobacter Diseases caused by Campylobacter, including pathogenesis and pathology Diagnostic laboratory tests for the diseases caused by Campylobacter Epidemiology and control of diseases caused by Campylobacter Helicobacter The main biological characteristics of the Helicobacter species Pathogenesis and pathology of the Helicobacter species-associated diseases Diagnostic laboratory tests for the diseases induced by Helicobacter species 11/3/2022 2 11/3/2022 3  Vibrio sp. general characteristics – Gram negative rods with polar, sheathed flagella in broth, but peritrichous unsheathed flagella on solid media – Rods are “curved” in clinical specimens, but small, straight rods after culture – Facultatively anaerobic 11/3/2022 4  Vibrio cholerae For growth prefers alkaline (pH 8,0-9,5) over acid conditions; ✔ Distinguished from other Vibrios by: Biochemical reactions ✔ Lipopolisaccharide (LPS) O antigenic structure ✔ Production of cholera toxin (CT) 11/3/2022 5  Vibrio cholerae (cont’d) There are over 200 O antigen serotypes, Only O1 and O139 are causing Cholera Vibrio cholera biogroup El Tor and O1 variant, is a biotype of classical strains The O139 strains phenotypically resemble O1 El Tor strains, but also produce polysaccharide capsule. 11/3/2022 6  Vibrio cholerae (cont’d) All strains capable causing cholera produce a colonizing factor known as the toxin-coregulated pilus (TCP) because its expression is regulated together with cholera toxin (CT). In aquatic environments V.cholerae produce polysaccharide biofilms, which contain carbohydrate moieties mediating cell-cell adhesion and attachment to surfaces. 11/3/2022 7  Cholera toxin (CT) CT is an A-B type of ADP-ribosylating toxin Its molecule is an aggregate of multiply polypeptide chains organized in two toxic subunits (A1, A2) and five binding (B) units. 11/3/2022 8  Mechanism of action of cholera toxin The toxin binds gangliosidase receptor found on many types of cells. A1 sub-unit is released from the toxin molecule and enters the cell by translocation. In the cell it exerts its effect on the membrane associated adenilate cyclase system. The target of the toxin A1 sub-unit is a guanine nucleotide (G) protein which regulates activation of adenylate cyclase system. This stimulates the conversion of adenosine triphosphate to cyclic adenosine 3’5’ monophosphate (cAMP). The net effect is excessive accumulation of cAMP at the cell membrane which causes hypersecretion of chloride, potassium, bicarbinates and associated with water molecules out of the cell. 11/3/2022 9 11/3/2022 10 11/3/2022 11  V. cholerae serogroups The V cholerae serogroup O1 antigen has determinants that make possible further typing; the serotypes are Ogawa, Inaba, and Hikojima. Two biotypes of epidemic V cholerae have been defined, classic and El Tor. The El Tor biotype produces a hemolysin, gives positive results on the Voges-Proskauer test, and is resistant to polymyxin B. Molecular techniques can also be used to type V cholerae. Typing is used for epidemiologic studies, and tests generally are done only in reference laboratories. 11/3/2022 12  V cholerae serotypes V cholerae O139 is very similar to V cholerae O1 El Tor biotype. V cholerae O139 does not produce the O1 lipopolysaccharide and does not have all the genes necessary to make this antigen. V cholerae O139 makes a polysaccharide capsule like other non-O1 V cholerae strains, while V cholerae O1 does not make a capsule. 11/3/2022 13  Voges–Proskauer test Voges–Proskauer or VP is a test used to detect acetoin in a bacterial broth culture. The test is performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth which has been inoculated with bacteria This test is used to determine which fermentation pathway is used to utilize glucose. In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids). 11/3/2022 14  Vibrio sp. (cont’d) Best indicators of Vibrio infection is presence of recognized factors – Recent consumption of raw seafood (especially oysters) – Recent immigration or foreign travel – Gastroenteritis with cholera-like or rice-water stools – Accidental trauma during contact with fresh or marine water 11/3/2022 15  Culture TCBS= Thiosulfate-Citrate-Bile-Salt agar 11/3/2022 16  Difference between Vibrios and Aeromonas Vibrio species are susceptible to the compound O/129 (2,4-diamino-6,7-diisopropylpteridine phosphate), which differentiates them from Aeromonas species, which are resistant to O/129. Most Vibrio species are halotolerant, and NaCl often stimulates their growth. Some vibrios are halophilic, requiring the presence of NaCl to grow. Another difference between vibrios and aeromonas is that vibrios grow on media containing 6% NaCl, whereas aeromonas does not. 11/3/2022 17  Vibrio cholerae Clinical Infection – Acute diarrheal disease – Spread through contaminated water, but also improperly preserved foods, including fish and seafood, milk, ice cream, and unpreserved meat – Incubation period 2 days – “Rice Water” stools Caused by cholera toxin or choleragen Dehydration is usual cause of death 11/3/2022 18  Vibrio cholerae About 60% of infections with classic V cholerae are asymptomatic, as are about 75% of infections with the El Tor biotype. The incubation period is 1–4 days for persons who develop symptoms, depending largely upon the size of the innoculum ingested. There is a sudden onset of nausea and vomiting and profuse diarrhea with abdominal cramps. The mortality rate without treatment is between 25% and 50%. 11/3/2022 19  V. cholerae V. cholerae O1 is causative agent of cholera – Also known as Asiatic cholera or epidemic cholera; particularly prevalent in India and Bangladesh – Also seen along the Gulf coast of the U.S. 11/3/2022 20  Cholera symtoms Cholera produces the most dramatic watery diarrhea known. Intestinal fluids pour out in voluminous bowel movements, this eventually leads to dehydration and electrolyte imbalance. The effects come from the action of cholera toxin secreted by V. cholerae in the bowl lumen. Despite of profound psychological effects there is no fever, inflammation or direct injury to the bowl mucus. 11/3/2022 21  Lab diagnostic Specimens: for culture consist of mucus flecks from stools. Smears: The microscopic appearance of smears made from stool samples is not distinctive. Dark-field or phase contrast microscopy may show the rapidly motile vibrios. Culture: Growth is rapid in peptone agar, on blood agar with a pH near 9.0, or on TCBS agar, and typical colonies can be picked in 18 hours Specific tests: V cholerae organisms are further identified by slide agglutination tests using anti-O group 1 or group 139 antisera and by biochemical reaction patterns. 11/3/2022 22  Treatment The most important part of therapy consists of water and electrolyte replacement to correct the severe dehydration and salt depletion. Many antimicrobial agents are effective against V cholerae. Oral tetracycline tends to reduce stool output in cholera and shortens the period of excretion of vibrios. In some endemic areas, tetracycline resistance of V cholerae has emerged; the genes are carried by transmissible plasmids. 11/3/2022 23  (from Greek) The eight pandemic was registered in 2003. More recently, the 2016–19 Yemen cholera outbreak. 11/3/2022 24  Epidemics El Tor serotypes dominated in 20th century 11/3/2022 O139 serotypes is spreading 25  Treatment Oral or intravenous fluid and electrolyte replacement are critical Antibiotic therapy plays a secondary role after fluid replacement, but can reduce duration and severity (azithromycin, doxycycline, trimethoprin). 11/3/2022 26  Vibrio sp. (cont’d) Vibrio species can be isolated from a variety of clinical specimens, including feces, wound, and blood Major species are: V. cholerae, V. parahaemolyticus, V. vulnificus, V. alginolyticus 11/3/2022 27  Vibrio parahaemolyticus Second most common Vibrio species involved in gastroenteritis “Summer diarrhea” in Japan Most cases traced to recent consumption of raw, improperly cooked, or re-contaminated seafood, especially oysters 11/3/2022 28  Vibrio parahaemolyticus (cont’d) Gastrointestinal symptoms are generally self-limiting: watery diarrhea, moderate cramps or vomiting 11/3/2022 29  Vibrio vulnificus Found in marine environments along all coasts in the U.S. Two categories of infections – Primary septicemia following consumption of contaminated shellfish, especially raw oysters; patients with liver dysfunction that results in increased levels of iron are predisposed – Wound infections following traumatic aquatic wound 11/3/2022 30  Vibrio vulnificus About 50% of the patients with bacteremia die. Wound infections may be mild but often proceed rapidly (over a few hours), with development of bullous skin lesions, cellulitis, and myositis with necrosis. 11/3/2022 31  Vibrio vulnificus Because of the rapid progression of the infection, it is often necessary to treat with appropriate antibiotics before culture confirmation of the etiology can be obtained. Diagnosis is by culturing the organism on standard laboratory media; TCBS is the preferred medium for stool cultures, where most strains produce blue-green (sucrose negative) colonies. Tetracycline appears to be the drug of choice for vulnificus infection; ciprofloxacin may be effective also based on in vitro activity. 11/3/2022 32  Other Vibrios Several other vibrios also cause disease in humans: Vibrio mimicus causes diarrhea after ingestion of uncooked seafood, particularly raw oysters. Vibrio hollisae and Vibrio fluvialis also cause diarrhea. Vibrio alginolyticus causes eye, ear, or wound infection after exposure to seawater. Vibrio damsela also causes wound infections. Other vibrios are very uncommon causes of disease in humans. 11/3/2022 33  Aeromonas The taxonomy of the genus Aeromonas is in transition. The genus has been placed in the new family Aeromonadaceae from the family Vibrionaceae. Based on DNA hybridization groups, many genospecies have been recognized; some are renamed species, some are newly named, and some are not yet named. The following three groups are of primary clinical importance in human infections: Aeromonas hydrophila complex, Aeromonas caviae complex, Aeromonas veronii biovar sobria. 11/3/2022 34  Aeromonas (cont’s) Aeromonads are 1–4 μm long and are motile. Their colony morphology is similar to that of enteric gram-negative rods , and they produce large zones of hemolysis on blood agar. Aeromonas species cultured from stool specimens grow readily on the differential media used to culture enteric gram-negative rods and can easily be confused with enteric bacteria. Aeromonas species are distinguished from the enteric gram-negative rods by finding a positive oxidase reaction in growth obtained from a blood agar plate. Aeromonas species are differentiated from vibrios by showing resistance to compound O/129 and lack of growth on media containing 6% NaCl. 11/3/2022 35  Plesiomonas Plesiomonas shigelloides is a gram-negative rod with polar flagella. Plesiomonas is most common in tropical and subtropical areas. It is a water and soil organism and has been isolated from freshwater fish and many animals. Most isolates from humans have been from stool cultures of patients with diarrhea. Plesiomonas grows on the differentia media used to isolate salmonella and shigella from stool specimens. Some plesiomonas strains share antigens with Shigella sonnei, and cross-reactions with shigella antisera occur. Plesiomonas can be distinguished from shigellae in diarrheal stools by the oxidase test: Plesiomonas is oxidase-positive and shigellae are not. Plesiomonas is positive for DNase; this and other biochemical tests distinguish it from aeromonas. 11/3/2022 36  History of Campylobacter First isolated as Vibrio fetus in 1909 from spontaneous abortions in livestock Campylobacter enteritis was not recognized until the mid-1970s when selective isolation media were developed for culturing campylobacters from human feces. Most common form of acute infectious diarrhea in developed countries; Higher incidence than Salmonella & Shigella combined 11/3/2022 37 Incidence of Campylobacter, Salmonella, Shigella and E.coli O157 in 1996 11/3/2022 38  Campylobacter and Campylobacter-like species Campylobacter – Non-spore-forming, curved, gram-negative rods, showing an S-shaped (“seagull-wing”) appearance – Exhibit “darting” motility – Tend to be pleomorphic – Characteristics that facilitate penetration and colonization of mucosal environments (e.g., motile by polar flagella; corkscrew shape) – Microaerophilic atmospheric requirements – Become coccoid when exposed to oxygen or upon prolonged culture – Neither ferment nor oxidize carbohydrates 11/3/2022 39  Campylobacter species (cont’d) Curved, gram-negative rods appearing as long spirals, S shapes, or seagull-wing shapes; stains poorly, so safranin counterstaining should be extended to 2 to 3 minutes Cultured on Campy plates at 42 degrees C in microaerophilic (i.e. requires environments containing lower levels of oxygen than are present in the atmosphere (i.e. 106 CFU Pathogenesis not fully characterized 11/3/2022 50  Putative Virulence Factors Cellular components: Endotoxin Flagellum: Motility Adhesins: Mediate attachment to mucosa Invasins Gullian-Barr Syndrome (GBS) is associated with C. jejuni Serogroup O19 S-layer protein “microcapsule” in C. fetus Extracellular components: Enterotoxins Cytopathic toxins 11/3/2022 51  Treatment, Prevention & Control Gastroenteritis: Self-limiting; Replace fluids and electrolytes Antibiotic treatment can shorten the excretion period; Erythromycin is drug of choice for severe or complicated enteritis & bacteremia; Fluoroquinolones are highly active (e.g., ciprofloxacin was becoming drug of choice) but fluoroquinolones resistance has developed rapidly since the mid-1980s apparently related to unrestricted use and the use of enrofloxacin in poultry Azithromycin was effective in recent human clinical trials Control should be directed at domestic animal reservoirs and interrupting transmission to humans 11/3/2022 52  Complication are rare but occurs occasionally Complications are relatively rare, but infections have been associated with reactive arthritis, hemolytic uremic syndrome (HUS), and following septicemia, infections of nearly any organ. The estimated case/fatality ratio for all C. jejuni infections is 0.1, meaning one death per 1,000 cases. Fatalities are rare in healthy individuals and usually occur in cancer patients or in the otherwise debilitated. 11/3/2022 53  Beginning of Scientific understanding Peptic ulcers have plagued men throughout the centuries, but the exact cause of the condition was uncertain. In 1940, Dr. A. Stone Freedberg of Harvard Medical School identified unusual curved bacteria in the stomachs of ulcer victims; he suspected that they might be responsible for ulcers but abandoned the research when his team was unable to grow the bacteria in the laboratory. 11/3/2022 54  History of H.pylori H.pylori discovered in the stomachs of patients with gastritis & stomach ulcers nearly 25 years ago by Dr Barry J. Marshall and Dr J. Robin Warren of Perth, Western Australia. At the time (1982/83) the conventional thinking was that no bacterium can live in the human stomach as the stomach produced extensive amounts of acid which was similar in strength to the acid found in a car-battery. Marshall & Warren literally “re-wrote” the text-books with reference to what causes gastritis & gastric ulcers. 11/3/2022 55  A Tribute to Warren and Marshall for Discovery of H.pylori 11/3/2022 56  Helicobacter pylori Strongly associated with gastric and duodenal ulcers; low grade inflammatory process The organism does not invade the gastric epithelium, but the host immune antibody response causes inflammation Can be cultured, but it is so strongly urease positive that presumptive identification often made from biopsy specimen by testing for urease production 14C-labeled urea breath test 11/3/2022 57  Helicobacter pylori Helicobacter pylori (H. pylori) is a type of bacteria. Researchers believe that H. pylori is responsible for the majority of peptic ulcers. H. pylori infection is common in the United States. About 20 per cent of people under 40 years old and half of those over 60 years have it. Most infected people, however, do not develop ulcers. Why H. pylori does not cause ulcers in every infected person is not known. Most likely, infection depends on characteristics of the infected person, the type of H. pylori, and other factors yet to be discovered 11/3/2022 58  Helicobacter pylori Helicobacter pylori is the prototype organism in this gastritis group. It is associated with antral gastric ulcers, and gastric carcinoma 11/3/2022 59  Helicobacter pylori Helicobacter pylori is a spiral gram negative bacteria, H pylori is oxidase-positive and catalase-positive. It has a multiple polar flagella above the pole and motile 11/3/2022 60  How is it transmitted? Believed to be transmitted orally due to tainted food or water. Also believed to be passed through belching, or gastro-esophageal reflux, which is when small amount of stomachs contents is forced up the esophagus. After this process, it’s believed that the H. Pylori is passed orally. 11/3/2022 61  Pathogenicity Genes Contribute to Pathogenicity CAG – Cytotoxin associated gene Vac – Vacuolating cytotoxin gene 11/3/2022 62  Pathogenic Mechanism It is well established that urease, Vacuolating cytotoxic VacA, and the pathogenicity island (cag PAI) gene products, are the main factors of virulence of this organism. Thus, individuals infected with strains that express these virulence factors probably develop a severe local inflammation that may induce the development of peptic ulcer and gastric cancer. The way the infection spreads throughout the world suggests the possibility that there are multiple pathways of transmission 11/3/2022 63  Pathogenic Mechanism The mechanisms by which H pylori causes mucosal inflammation and damage are not well defined but probably involve both bacterial and host factors. The bacteria invade the epithelial cell surface to a limited degree. Toxins and lipopolysaccharide may damage the mucosal cells, and the ammonia produced by the urease activity may directly damage the cells also. 11/3/2022 64  Culturing H.pylori H.pylori grows on“Skirrow”s medium with 1Vancomycin, 2 Polymyxin 3 Trimethoprim Grows in 3 -6 days at 370C Colonies appear Translucent 1-2 mm in diameter Optimal growth occurs in Microaerophic environment 11/3/2022 65 11/3/2022 66  Urea Hydrolysis C=O(NH2)2 + H+ + 2H2O HCO3- + 2 (NH4+) Urea Bicarbonate Ammonium ions Urease And then… HCO3 CO2 + OH- 11/3/2022 67  Pathology and Pathogenesis H.pylori is found in the deep mucus layer Grows optimally at pH 6.0 to 7.0 But gastric mucosa has a strong buffering in spite of lower pH on the lumen side of stomach H.pylori also produces a protease that modifies the gastric mucus and further reduces the ability of acid to penetrate through the mucus 11/3/2022 68  Patogenicity 11/3/2022 69  Pathogenesis The potential character of H.pylori lies with production of potent Urease activity which yields production of Ammonia and further buffering acid. H.pylori is quite motile even in mucus finds its way to epithelial surface H.pylori overlies the gastric type but not intestinal epithelial cells 11/3/2022 70  Pathogenesis 11/3/2022 71 11/3/2022 72  Clinical Manifestations Acute infection Upper Gastrointestinal illness Nausea Pain Fever – very occasionally Acute symptoms lasts for < 1 week, May extend up to 2 weeks Infection last for years, decades or even lifetime 11/3/2022 73  Consequences of H.pylori 11/3/2022 74  Consequences of H.pylori Association of Duodenal and Gastric ulcers in H.pylori About 90 % of patients with Duodenal ulcer, and 50- 80 % of gastric ulcers are associated with H.pylori infection H.pylori may have greater role in Gastric carcinoma and Lymphomas 11/3/2022 75  Diagnosis Urea Breath Test H. pylori infection can be detected in the exhaled breath using this special test. This test is positive only if the person has a current infection. Sensitivity and specificity of this test ranges from 94-98% 11/3/2022 76  Urea Breath Test In this test 13C or 14C labeled urea is ingested by patients If H.pylori is present the urease activity generates labeled Co2 that can be detected in the patients exhaled breath 11/3/2022 77  Treatment Triple therapy has prompt response, contain a combination of following drugs 1 Metronidazole 2 Bismuth subsalicylate or Bismuth sub citrate 3 Amoxicillin or Teracycles administered up to 14 days Eradicates H.pylori In 70 – 95 % of patients Acid suppressing agent is supporting 11/3/2022 78  Epidemiology In Developed countries H.pylori are present in