Lecture 7 Microbiology 2022-04-21 PDF

Lecture 7 Enterobacteriaceae: General introduction, Escherichia coli & Shigella, Salmonella, Yersinia  Content Microbiology: Enterobacteriaceae: General introduction, Escherichia coli & Shigella, Samonella 1. General characterization and classificat...

Lecture 7 Enterobacteriaceae: General introduction, Escherichia coli & Shigella, Salmonella, Yersinia  Content Microbiology: Enterobacteriaceae: General introduction, Escherichia coli & Shigella, Samonella 1. General characterization and classification of Enterobacteriaceae 2. Escherichia coli Diseases induced by Escherichia coli, including pathogenesis and Clinical Findings Diagnostic laboratory tests for Escherichia coli -associated diseases Treatment, Epidemiology, Prevention and Control of Enterobacteriaceae- associated diseases 3. Shigella Serological classification of Shigella, pathogenesis and pathology Diagnostic laboratory tests and immunity of Shigella-associated diseases Treatment, Epidemiology, Prevention and Control of the diseases induced by Shigella 4. Salmonella The Morphology , Identification and Classification of Salmonella Pathogenesis and clinical finding for the disease induced by Salmonella Diagnostic laboratory tests for the diseases caused by Salmonella, including bacteriologic methods for isolation of salmonella, serologic methods Treatment, Epidemiology, Prevention and Control, including carriers, source of infection (water, milk and other dairy products, shellfish, dried or frozen eggs, meats and meat products, animal dye and household pets) 5. Yesrinia  Enterobacteriaceae Classification The Enterobacteriaceae are a large, heterogeneous group of Gram-negative rods Resident microflora: whose natural habitat is the intestinal tract of Escherichia – is a part of the normal microbiota and humans and animals. incidentally cause disease Due to nucleic acid hybridization and nucleic acid Klebsiella sequencing technologies that are measuring Proteus evolutionary distances 63 genera have been Enterobacter defined; however, the clinically significant Enterobacteriaceae comprise 20–25 species, and Exist in carrier state: other species are encountered infrequent: Shigella Escherichia Salmonella serovar Typhi Shigella Edwardsiella Strict human pathogens Salmonella with no animal reservoir Citrobacter Klebsiella Enterobacter Hafnia Serratia Proteus Providencia Morganella Yersinia Erwinia Pectinobacterium  Morphology and General Characteristics Gram-negative, no-spore forming, rod shaped bacteria, facultatively anaerobic Catalase positive, oxidase negative (except for Plesiomonas) and reduce nitrate to nitrite(there are a few exceptions) Ferment glucose and may or may not produce gas in the process (aerogenic vs anaerogenic) If motile, motility is peritrichous flagella (except Shigella and Klebsiella)  Identification The traditional methods for organism identification are being replaced by other methods over the past years; The implementation of Matrix-Assisted Laser Desorption Ionization Time Of Flight Mass Spectroscopy (MALDI-TOF MS) for identification of culture isolates is replacing the more traditional panels of biochemicals currently in use in most clinical well equipted microbiology laboratories. MALDI-TOF operating principle MALDI-TOF spectra for E.coli and Shigella  Enterobacteriaceae Are facultative anaerobes. If motile, motility is by peritrichous flagella Many are normal inhabitants of the intestinal tract of man and other animals Some are enteric pathogens and others are urinary or respiratory tract pathogens Differentiation is based on biochemical reactions and differences in antigenic structure Traditional approach of identification  Enterobacteriaceae Most grow well on a variety of lab media including a lot of selective and differential media originally developed for the selective isolation of enteric pathogens. Most of this “differential” media is selective by incorporation of dyes distinguishes lactose-fermenting (coloured) from non-lactose-fermenting colonies (nonpigmented). Incorporation of bile salts into the media inhibit G+ organisms may suppress the growth of nonpathogenic species of Enterobacteriaceae. Differential media may allow rapid presumptive identification of enteric bacteria. Culture on media that contain special dyes and carbohydrates (eg, eosin-methylene blue [EMB], MacConkey, or deoxycholate medium) Many are differential on the basis of whether or not the organisms ferment lactose and/or produce H2S.  Growth of Enterobacteriaceae on selective media On Chocolate Blood Agar (CBA) they all produce similar colonies that are relatively large and dull gray. They may or may not be hemolytic. The three most useful media for screening stool cultures for potential pathogens are: Triple sugar iron agar (TSI), Lysine Iron Agar (LIA), and urea or phenylalanine agar. The antigenic structure is used to differentiate organisms within a genus or species. Triple sugar iron agar (TSI), Many complex media have been devised to help in identification of the enteric bacteria. One such medium is triple sugar iron (TSI) agar, which is often used to help differentiate salmonellae and shigellae from other enteric Gram-negative rods in stool cultures. The Triple Sugar Iron agar (TSIA) test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish them from other gram negative intestinal bacilli. The differentiation is based on fermentation of glucose and lactose or sucrose and hydrogen sulfide (H2S) production  The medium contains 0.1% glucose, 1% sucrose, 1% lactose, ferrous sulfate (for detection of H2 S production), tissue extracts (protein growth substrate), and a pH indicator (phenol red). It is poured into a test tube to produce a slant with a deep butt and is inoculated by stabbing bacterial growth into the butt. S.N Result (slant/butt) Symbol Interpretation. 1 Red/Yellow K/A Glucose fermentation only, peptone catabolized. 2 Yellow/Yellow A/A Glucose and lactose and/or sucrose fermentation. No fermentation, Peptone catabolized under 3 Red/Red K/K aerobic and/or anaerobic conditions. Glucose and lactose and/or sucrose fermentation, 4 Yellow/Yellow with bubbles A/A,G Gas produced. 5 Red/Yellow with bubbles K/A,G Glucose fermentation only, Gas produced. Red/Yellow with bubbles and Glucose fermentation only, Gas produced, H2S 6 black precipitate K/A,G,H2S produced. Yellow/Yellow with bubbles Glucose and lactose and/or sucrose fermentation, 7 and black precipitate A/A,G,H2S Gas produced, H2S produced. Red/Yellow with black 8 precipitate K/A,H2S Glucose fermentation only, H2S produced.  Lysine Iron Agar (LIA) The medium has an aerobic slant and an anaerobic butt. Lysine iron agar (LIA) slants tests organisms for the ability to deaminate lysine or decarboxylate lysine. Lysine deamination is an aerobic process which occurs on the slant of the media. Lysine decarboxylation is an anaerobic process which occurs in the butt of the media. When glucose is fermented, the butt of the medium becomes acidic (yellow). If the organism produces lysine decarboxylase, cadaverine is Slant formed. Cadaverine neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts to the alkaline state (purple). If the decarboxylase is not produced, the butt remains acidic (yellow). If oxidative deamination Butt of lysine occurs, this forms a burgundy color on the slant. If deamination does not occur, the LIA slant remains purple. Bromocresol purple, the pH indicator, is yellow at or below pH 5.2 and purple at or above pH 6.8. Lysine –iron agar (LIA) Phenylalanine test The phenylalanine agar is designed to test for the ability of some specific species to convert amino acid phenylalanine to phenylpyruvic acid; an important reaction in the differentiation of Enterobacteriaceae. The phenylpyruvic acid is detected by adding a few drops of 10% ferric chloride which acts as a chelating agent ; a green colored complex is formed between these two compounds indicating a positive test. If the medium remains a straw color, the organism is negative for phenylalanine deaminase production, due to the color of ferric chloride.  Three major classes of antigens are found: Somatic O antigens – are the most external part of the cell wall lipopolysaccharide and consist of repeating units of polysaccharide. Some O-specific polysaccharides contain unique sugars. O antigens are the heat and alcohol resistant polysaccharide part of the LPS. Variation from smooth to rough colonial forms is accompanied by progressive loss of smooth O Antigen. They are detected by bacterial agglutination tests. Antibodies to O antigens are predominantly IgM. Flagellar H antigens – are located on flagella and are denatured or removed by heat or alcohol. They are preserved by treating motile bacterial variants with formalin. Such H antigens agglutinate with anti-H antibodies mainly IgG. Envelope or capsule K antigens – overlay the surface O antigen and may block agglutination by O specific antisera. Boiling for 15 minutes will destroy the K antigen and unmask O antigens. K antigens are of polysaccharide or protein nature. They can be identified by capsular swelling tests with specific antisera The K antigen is called the Vi (virulence) antigen in Salmonella.  Antigens Enterobacteriaceae have a complex antigenic structure. >150 different heat-stable somatic O (lipopolysaccharide) antigens, > 100 heat-labile K (capsular) antigens > 50 H (flagellar) antigens  Enteric bacteria The enteric bacteria generally do not cause disease, and in the intestine, they may even contribute to normal function and nutrition. When clinically important infections occur, they are usually caused by E. coli, but the other enteric bacteria are causes of hospital-acquired infections and occasionally cause community-acquired infections. The bacteria become pathogenic only when they reach tissues outside of their normal intestinal or other less common normal microbiota sites. The most frequent sites of clinically important infection are the urinary tract, biliary tract, and other sites in the abdominal cavity, but any anatomic site (eg, bloodstream, prostate gland, lung, bone, and meninges) can be the site of disease.  Escherichia coli Normal inhabitant of the G.I. tract. Some strains cause various forms of gastroenteritis. Is a major cause of urinary tract infection and neonatal meningitis and septicemia. May have a capsule. Biochemistry – lactose fermenting Most are motile. Selective agar - ENDO E.Coli on ENDO agar  E. coli-associated diarrheal diseases E. coli that cause diarrhea are extremely common worldwide. These E. coli are classified by the characteristics of their virulence properties, and each group causes disease by a different mechanism. At least six of which have been characterized. The small or large bowel epithelial cell adherence properties are encoded by genes on plasmids. Similarly, the toxins often are plasmid or phage mediated.  Antigenic structure Has O, H, and K antigens. K1 has a strong association with virulence, particularly meningitis in neonates. Virulence factors Toxins: Enterotoxins – produced by enterotoxigenic strains of E. coli (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea.  E. coli toxins There are two types of enterotoxins: LT – is heat labile and binds to specific Gm1 (monosialotetrahexosylganglioside) gangliosides on the epithelial cells of the small intestine where it ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP (cyclic adenine mono phosphate). Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.  E.coli toxins ST – is heat stable and binds to specific receptors to stimulate the production of cGMP (cyclic guanine mono phosphate) with the same results as with LT. Composition of subunits of enterotoxins : Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity).  E. coli : E. coli Shiga-type toxin – also called the verotoxin -produced by enterohemorrhagic strains of E. coli (EHEC) – is cytotoxic, enterotoxic, neurotoxic, and may cause diarrhea and ulceration of the G.I. tract. There are two types: shiga-like toxin 1 shiga-like toxin 2. Enteroaggregative ST-like toxin – produced by enteroaggregative strains of E. coli (EAEC) – causes watery diarrhea. Hemolysins – two different types may be found: cell bound secreted. They lyse RBCs and leukocytes and may help to inhibit phagocytosis when cell bound. Endotoxin Type III secretion system to deliver effector molecules directly into the host cells. Involved in inducing uptake of enteroinvasive E.coli (EIEC) into intestinal cells. Eneterotoxigenic E.coli Enteroaggregative E.coli Cause gastroenteritis Enteropathogenic E.coli Enterohemmoragic E.coli Enteroinvasive E.coli  Enteropathogenic E.coli Enterohemmoragic E.coli Eneterotoxigenic E.coli Enteroaggregative E.coli Enteroinvasive E.coli Diffusely adherent E.coli  E. coli: Adhesions Adhesions are also called colonization factors and include both pili or fimbriae and non-fimbrial factors involved in attachment. There are at least 21 different types of adhesions. Antibodies to these may protect one from colonization. Virulence factors that protect the bacteria from host defenses: Capsule Iron capturing ability (enterochelin) Outer membrane proteins - are involved in helping the organism to invade by helping in attachment and in initiating endocytosis.  E. coli infections : Neonatal meningitis – is the leading cause of neonatal meningitis and septicemia with a high mortality rate. Usually caused by strains with the K1 capsular antigen. Gastroenteritis – there are several distinct types of E. coli that are involved in different types of gastroenteritis: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC) , enteroaggregative E. coli (EAEC), and enterohemorrhagic E. coli (EHEC).  E. coli gastroenteritis EPEC EPEC –Enteropathogenic E.coli bundle forming pili are involved in attachment to the intestinal mucosa. This leads to changes in signal transduction in the cells, effacement of the microvilli, and to intimate attachment via a non-fimbrial adhesion called intimin. EPEC adhere to the mucosal cells of the small bowel. Pathogenicity requires two important factors, the bundle forming pilus encoded by a plasmid EPEC adherence factor (EAF) and the chromosomal locus of enterocyte effacement (LEE) pathogenicity island that promote the tight adherence characteristic of EPEC (attachment and effacement). After attachment, there is loss of microvilli (effacement); formation of filamentous actin pedestals or cuplike structures; and, occasionally, entry of the EPEC into the mucosal cells. The exact mode of pathogenesis is unclear, but diarrhea with large amounts of mucous without blood or pus occurs along with vomiting, malaise and low grade fever. This is a problem mainly in hospitalized infants and in day Adhesion of E.coli to jejunal mucosa Electron micrograph of enteropathogenic Escherichia coli (EPEC) adherent to the jejunal mucosa and demonstrating the attaching and effacing lesion, also called pedestal formation. Tightly adherent E. coli are obliterating the brush border. Pedestal formation  E. Coli gastroenteritis ETEC: ETEC –Eneterotoxigenic E.coli is a common cause of traveler’s diarrhea and diarrhea in children in developing countries. The organism attaches to the intestinal mucosa via colonization factors ((pili known as colonization factor antigens [CFAs]) specific for humans promote adherence of ETEC to epithelial cells of the small bowel and then liberates enterotoxin. Some strains of ETEC produce a heat-labile enterotoxin (LT) that is under the genetic control of a plasmid and is closely related to cholera toxin. Its subunit B attaches to the GM1 ganglioside in the apical membrane of enterocytes and facilitates the entry of subunit A into the cell, where the latter activates adenylyl cyclase. The end result is an intense and prolonged hypersecretion of water and chlorides and inhibition of the reabsorption of sodium. Some strains of ETEC produce the heat-stable enterotoxin STa, which is under the genetic control of a heterogeneous group of plasmids. STa activates guanylyl cyclase in enteric epithelial cells and stimulates fluid secretion. Many STa -positive strains also produce LT. The strains with both toxins produce a more severe diarrhea The disease is characterized by a watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. Transmission is via contaminated food or water.  E. coli gastroenteritis EIEC EIEC –Enteroinvasive E.coli- The organism attaches to the intestinal mucosa via pili and outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa. There is lateral movement of the organism from one cell to adjacent cells. Symptoms include fever, severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous, and pus. EIEC produce a disease very similar to shigellosis. The disease occurs most commonly in children in developing countries and in traveler’s to these countries. Similar to Shigella, EIEC strains are nonlactose or late lactose fermenters and are nonmotile.  E. coli gastroenteritis EAEC: EAEC – Enteroaggregative E.coli - Mucous associated autoagglutinins cause aggregation of the bacteria at the cell surface and result in the formation of a mucous biofilm. The organisms attach via pili and liberate a cytotoxin distinct from, but similar to the ST and LT enterotoxins liberated by ETEC. EAEC causes acute and chronic diarrhea (>14 days in duration) in persons in developing countries. These organisms also are the cause of foodborne illnesses in industrialized countries and have been associated with traveler’s diarrhea and persistent diarrhea in patients with HIV. They are characterized by their specific patterns of adherence to human cells. This group of diarrheagenic E. coli is quite heterogeneous, and the exact pathogenic mechanisms are still not completely elucidated. Some strains of EAEC produce ST-like toxin (see earlier discussion on E. coli O104:H11); others a plasmid-encoded enterotoxin that produces cellular damage; and still others, a hemolysin Symptoms include watery diarrhea, vomiting, dehydration and occasional abdominal pain.  E. coli gastroenteritis : EHEC pr STEC – Enterohemmoragic E.coli - The organism attaches via pili to the intestinal mucosa and liberates the shiga-like toxin. The symptoms start with a watery diarrhea that progresses to bloody diarrhea without pus and crampy abdominal pain with no fever or a low-grade fever. STEC are named for the cytotoxic toxins they produce. There are at least two antigenic forms of the toxin referred to as Shiga-like toxin 1 and Shiga-like toxin 2. STEC has been associated with mild non-bloody diarrhea, hemorrhagic colitis, a severe form of diarrhea, and with hemolytic uremic syndrome, a disease resulting in acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia. A low infectious dose (< 200 CFU) is associated with infection. Of the more than 150 E. coli serotypes that produce Shiga toxin, O157:H7 is the most common and is the one that can be identified most readily in clinical specimens. This may progress to hemolytic-uremic syndrome (HUS) that is characterized by low platelet count, hemolytic anemia, and kidney failure. This is most often caused by serotypes O157:H7. This strain of E. coli can be differentiated from other strains of E. coli by the fact that it does not ferment sorbitol in 48 hours (other strains do). A sorbitol-Mac (SMAC) plate (contains sorbitol instead of lactose) is used to selectively isolate this organism. One must confirm that the isolate is E. coli O157:H7 using serological testing and confirm production of the shiga-like toxin before reporting out results.  Urinary tract infection E. coli is the most common cause of urinary tract infection and accounts for approximately 90% of first urinary tract infections in young women. The symptoms and signs include urinary frequency, dysuria, hematuria, and pyuria. Flank pain is associated with upper tract infection. None of these symptoms or signs is specific for E. coli infection. Urinary tract infection can result in bacteremia with clinical signs of sepsis. Ascending urinary tract infection Most of the urinary tract infections that involve the bladder or kidney in an otherwise healthy host are caused by a small number of O antigen types that have specifically elaborated virulence factors that facilitate colonization and subsequent clinical infections. These organisms are designated as uropathogenic E. coli. Typically, these organisms produce hemolysin, which is cytotoxic and facilitates tissue invasion. Strains that cause pyelonephritis express K antigen and elaborate a specific type of pilus, P fimbriae, which binds to the P blood group antigen.  E.coli: Antimicrobial therapy E. coli is usually susceptible to a variety of chemotherapeutic agents, though drug resistant strains are increasingly prevalent. It is essential to do susceptibility testing.  Shigella species : Shigella Contains four species that differ antigenically and, to a lesser extent, biochemically. S. dysenteriae (Group A) S. flexneri (Group B) S. boydii (Group C) S. sonnei (Group D) Biochemistry: Urea – Motility – All ferment mannitol except S. dysenteriae and S. sonnei may show delayed lactose fermentation  Shigella species: Antigenic structure Differentiation into groups (A, B, C, and D) is based on O antigen serotyping. K antigens may interfere with serotyping, but are heat labile. O antigen is similar to E. coli, so it is important to ID as Shigella before doing serotyping. Virulence factors Shiga toxin – is produced by S. dysenteriae and in smaller amounts by S. flexneri and S. sonnei. Acts to inhibit protein synthesis by inactivating the 60S ribosomal subunit by cleaving a glycosidic bond in one of the rRNA constituents. This plays a role in the ulceration of the intestinal mucosa. Shigella attachment and invasion  Type three secretion system Type three secretion system (often written Type III secretion system and abbreviated TTSS or T3SS, also called Injectisome) is a protein appendage found in several Gram-negative bacteria. This is a needle-like structure is used as a sensory probe to detect the presence of eukaryotic organisms and secrete proteins that help the bacteria infect them. The secreted effector proteins are secreted directly from the bacterial cell into the eukaryotic (host) cell, where they exert a number of effects that help the pathogen to survive and to escape an immune response. Shigella species : Outer membrane and secreted proteins These proteins are expressed at body temperature and upon contact with M cells in the intestinal mucosa they induce phagocytosis of the bacteria into vacuoles. Shigella destroy the vacuoles to escape into the cytoplasm. From there they spread laterally (Polymerization of actin filaments propels them through the cytoplasm) to epithelial cells where they multiply but do not usually disseminate beyond the epithelium.  Shigella: Clinical significance Causes shigellosis or bacillary dysentery. Transmission is via the fecal-oral route. The infective dose required to cause infection is very low (10-200 organisms). There is an incubation of 1-7 days followed by fever, cramping, abdominal pain, and watery diarrhea (due to the toxin)for 1-3 days. This may be followed by frequent, scant stools with blood, mucous, and pus (due to invasion of intestinal mucosa). It is rare for the organism to disseminate. The severity of the disease depends upon the species one is infected with. S. dysenteria is the most pathogenic followed by S. flexneri, S. sonnei and S. boydii.  Shigella: Antimicrobial therapy Sulfonamides are commonly used as are streptomycin, tetracycline, ampicillin, and chloramphenicol. Resistant strains are becoming increasingly common, so sensitivity testing is required.  Salmonella : Classification Salmonella classification has been changing in the last few years. There is now 1 species: S. enteritica, and 7 subspecies: 1, 2,3a, 3b,4,5, and 6. Subgroup 1 causes most human infections Clinically Salmonella isolates are often still reported out as serogroups or serotypes based on the Kauffman-White scheme of classification based on O and H (flagella) antigens The H antigens occur in two phases; 1 and 2 and only 1 phase is expressed at a given time. Polyvalent antisera is used followed by group specific antisera (A, B, C1, C2, D, and E) Salmonella typhi also has a Vi antigen which is a capsular antigen.  Salmonella : Virulence factors: Endotoxin – may play a role in intracellular survival Capsule (for S. typhi and some strains of S. paratyphi) Adhesions – both S. Typhimurium and S. Typhi possess partly fimbrial and overlapping and a partly distinct repertoire of non-fimbrial virulence factors. Both serovars express the type III secretion system, lipopolysaccharide, and other surface polysaccharides, fimbrae, flagellin, and bacterial DNA.  Selective media for Salmonella The most commonly used media selective for Salmonella are SS agar, bismuth sulfite agar, Hektoen enteric (HE) medium, brilliant green agar and xylose-lisine-deoxycholate (XLD) agar. All these media contain both selective and differential ingredients and they are commercially available. Growth of Salmonella (white non lactose Growth of Salmonella Growth of Salmonella on a Hektoen enteric (HE) agar. S. Typhimurium colonies grown on HE agar fermenting) and E.coli (pink- lactose on a XLD agar. Black are blue-green in color indcating that the fermenting) on a McConkey agar. centers indicate on bacterium does not ferment lactose However it does produce hydrogen sulfide, (H2S), as indicated production of H2S by black deposits in the centers of the colonies Salmonella and Shigella on SS agar  Salmonella virulence factors : Salmonella virulence factors Type III secretion systems and effector molecules. 2 different systems may be found: One type is involved in promoting entry into intestinal epithelial cells. The other type - outer membrane proteins - is involved in the ability of Salmonella to survive inside macrophages Flagella – help bacteria to move through intestinal mucous Enterotoxin - may be involved in gastroenteritis Iron capturing ability  Salmonella: Clinical Significance Salmonella causes two different kinds of disease: enteric fevers gastroenteritis. Both types of disease begin in the same way, but with the gastroenteritis the bacteria remains restricted to the intestine and with the enteric fevers, the organism spreads Transmission is via a fecal-oral route, i.e., via ingestion of contaminated food or water.  Salmonella The organism moves through the intestinal mucosa and adheres to intestinal epithelium. Effector proteins of the type III secretion system mediate invasion of enterocytes and M cells via an induced endocytic mechanism. Salmonella multiplies within the endosome. Salmonella invasion of epithelial cells a, Salmonella invasion. Entry into host cells is mediated by the Salmonella pathogenicity island-1 (SPI-1) type III secretion system (TTSS) and its effectors. Fig 7. Graphical illustration of the role of the SPI1-T3SS effectors SopA, SopB, SopE2 and SipA during enterocyte invasion and intraepithelial proliferation in vivo. Zhang K, Riba A, Nietschke M, Torow N, Repnik U, et al. (2018) Minimal SPI1-T3SS effector requirement for Salmonella enterocyte invasion and intracellular proliferation in vivo. PLOS Pathogens 14(3): e1006925. https://doi.org/10.1371/journal.ppat.1006925 https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006925  Salmonella invasion The endosome moves to the basal side of the cell and Salmonella are released and may be phagocytosed by macrophages. For gastroenteritis the Salmonella multiply and their presence induces a strong inflammatory response which causes most of the symptoms seen in gastroenteritis (mild to moderate fever with diarrhea and abdominal cramps). The inflammatory response prevents the spread beyond the GI tract and eventually kills the bacteria. In enteric fevers (typhoid and paratyphoid) the Salmonella disseminate before they multiply to high enough levels to stimulate a strong inflammatory response so the initial symptoms are only a low-grade fever and constipation.  Salmonella Typhi manifestation The bacteria move via the lymphatics and bloodstream to the liver and spleen where phagocytosis and multiplication occurs. The bacteria re-enter the bloodstream to disseminate throughout the body to all organs causing fever, headaches, myalgia, and GI problems. Rose spots (erythematous, muculopapular lesions) are seen on the abdomen. Osteomyelitis, cystitis, and gall bladder infections may occur. Symptoms of paratyphoid fevers (due to S. paratyphi A, B, or C) are similar to but less severe than those that occur with typhoid fever (due to S. typhi)  Diagnosis of typhoid fever Blood cultures are positive during the first week and after the second week Stool cultures and sometimes urine cultures are positive after the second week The Widal test is a serological test for antibodies against Salmonella typhi. One looks for a 4-fold rise in titer between acute and convalescent stages. 10% of those infected become short term carriers and a smaller % become long-term carriers due to persistence of the bacteria in the gallbladder or urinary bladder. Salmonella: Antimicrobial therapy Enteric fevers – use chloramphenicol usually. Resistant strains have emerged making antimicrobial susceptibility testing essential. Gastroenteritis – usually doesn’t require antimicrobic therapy. Replace lost fluids and electrolytes.  Enterobacteriaceae: Citrobacter Are opportunistic pathogens causing urinary tract or respiratory tract infections and occasionally wound infections, osteomyelitis, endocarditis, and meningitis. Enterobacteriaceae Edwardsiella tarda Clinical significance – causes GI disease in tropical and subtropical countries  Enterobacteriaceae: Klebsiella May cause Neurofibromatosis (NF - neurocutaneous syndrome) of GI tract, but potential pathogen in other areas Motility – Has both O and K antigens  Klebsiella: Virulence factors Capsule Adhesions Iron capturing ability Clinical significance Causes pneumonia, mostly in immunocompromised hosts. Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia) A major cause of nosocomial infections such as septicemia and meningitis  Enterobacteriaceae: Enterobacter Normal flora of Gi tract Clinical significance: Nosocomial infections Bacteremia in burn patients  Enterobacteriaceae: Serratia A free-living saprophyte Has been found in respiratory tract (RT) and urinary tract (UT) infections. Is resistant to many antimicrobials  Enterobacteriaceae: Proteus, Providencia, and Morganella Are all part of the NF of the GI tract (except Providencia). All motile Proteus mirabilis Morganella morgani  Proteus, Providencia, and Morganella Virulence factors Urease – the ammonia produced may damage the epithelial cells of the UT Clinical Significance UT infections, as well as pneumonia, septicemia, and wound infections  Yersinia Three species are important pathogens in man Yersinia pestis – causes plague Yersinis enterocolitica – enteropathogenic Yersinia pseudotuberculosis - enteropathogenic  Yersinia Species Identification Y. pestis can be separated from Y. enterocolitica and Y. pseudotuberculosis by the fact that it is non-motile. Y. enterocolitica and Y. pseudotuberculosis are both non-motile at 370 C, and motile at 220 C. Y. pestis is identified based on the following: Non-motile Yersinia pestis bipolar staining Bipolar staining (the ends of the bacilli stain more intensely than the middle) Slow growth of small colonies on ordinary culture media – it grows better at lower temperature (25-300 C) Yersinia pestis Wayson’s stain The Wayson stain is a basic fuchsin-methylene blue, ethyl alcohol-phenol microscopic staining procedure. With this stain, Yersinia pestis appears purple with a characteristic safety-pin appearance, which is due to the presence of a central vacuole.  Yersinia species Yersinia pestis – virulence characteristics: Endotoxin – is responsible for many of the symptoms Murine toxin – causes edema and necrosis in mice and rats, but has not been shown to play a role in human disease  Y. Pestis virulence factors Fraction 1 – a protein component of the antiphagocytic protein capsule V antigen – a secreted protein that controls expression of many of the virulence genes plus it appears to have another unknown function that is essential for virulence Pla – a protease that activates plasminogen activator (acts as a fibrinolysin) and degrades C3b (prevents formation of complement membrane attack complex) and C5a (prevents attraction of phagocytes) Psa – a pilus adhesion for attachment Iron acquisition and sequestering system  Y. pestis – clinical significance In man plague occurs in two forms; bubonic and pneumonic Bubonic plague – transmitted by fleas from an infected rodent. The bacteria travel in the blood to the nearest lymph node where they are engulfed by fixed macrophages. A high fever develops and the lymph nodes in the groin and armpit become enlarged (buboes) as the bacteria proliferate and stimulate an inflammatory response. The bacteria growing in the lymph node leak into the bloodstream. Lysis of the bacteria releases LPS, causing septic shock. Subcutaneous hemorrhages , probably due to LPS causing disseminated intravascular coagulation (DIC) gave the disease the name, the black death, in the middle ages. The untreated mortality rate is quite high.  Buboes Typical bubos St Sebastian lancing a plague bubo. Detail from the St Sebastian murals in the St Sebastian Chapel, Lanslevillard, France. French 15th century, anonymous This old method was both painful and ineffective, draining the pus from sufferers’ sores did more harm than good. First, it could infect the bloodstream and cause septicemia secondly, letting the pus out would make the spread of disease faster.  Y. Pestis –clinical significance Eventually bacteria reach the lungs where they are ingested by lung macrophages to cause pneumonic plague. Pneumonic plague – this can be transmitted directly to others via aerosol. Direct inhalation of aerosols containing the organism produces a form of the disease that progresses much more rapidly and the mortality rate is close to 100%. Treatment for plague - Streptomycin or tetracycline are effective Plague  Yersinia enterocolitica and Yersinia pseudotuberculosis identification Both are motile at 22-250 C, but non-motile 0 at 37 C Both exhibit bipolar staining Both grow better at lower temperatures and 0 produce small colonies at 37 C  Culturing of Yersinia species Cefsulodin-irgasan-novobiocin (CIN) agar is a selective media developed specifically for the isolation of Y. enterocolitica from gastrointestinal specimens. The media also contains mannitol and phenol red to differentiate mannitol from non-mannitol fermenting organisms. The media is incubated at room temperature and Yersinia are the only Enterobacteriaceae that will grow on the media. Aeromonas and Pleisiomonas, both members of the Vibrionaceae will also grow. After 48 hours at RT, Y. enterocolitica and Y. pseudotuberculosis both produce typical pink (from mannitol fermentation) colonies with a bulls-eye appearance.  Y. enterocolitica growth on CIN (Cefsulodin-Irgasan-Novobiocin Agar) Bulls eye colonies Y. enterocolotica – virulence factors Enterotoxin similar to E. coli ST (increases cGMP i.e. cyclic guanosine mono phosphate - leading to watery diarrhea) Adhesions – include both fimbrial and non-fimbrial adhesions. At least four different adhesions have been identified thus far. Antiphagocytic proteins – include both outer membrane and secreted proteins. Some are actually injected directly into the host via a type III secretion mechanism. Some interfere with signal transduction in host cells, thus interfering with the ability of PMNs to respond to signals leading them to the invading bacteria. Others disrupt the action cytoskeleton and lead to death of the PMNs.  Yersinia species virulence factors V antigen - a secreted protein that controls expression of many of the virulence genes plus it appears to have another unknown function that is essential for virulence Iron capturing ability Yad A – an outer membrane protein that interferes with C3b binding to bacteria thus preventing the formation of a membrane attack complex. Endotoxin Y. pseudotuberculosis – virulence factors Has all of the same virulence factors as Y. enterocolitica except the enterotoxin.  Yersinia enterocolitica and Y. pseudotuberculosis – clinical significance Both are acquired by ingestion of contaminated food or water. Y. enterocolitica - a cause of human disease Y. pseudotuberculosis - mainly a disease of other animals Both cause a disease involving fever and abdominal pain. Y. enterocolitica also causes a watery diarrhea. After ingestion, the bacteria invade the intestinal epithelium by invasion of M cells. They are transcytosed through the M cells and released at the basal surface. Once through the intestinal epithelium, the bacteria penetrate into the underlying lymphoid tissue, where they multiply both inside and outside host cells. Medical significance of Yersinia species Multiplication of the bacteria produces an inflammatory response that is responsible for the extreme pain associated with the infections (resembles acute appendicitis) Fever is due to the activity of the LPS endotoxin. Sometimes they drain into adjacent mesenteric lymph nodes, causing mesenteric lymphadenitis. Reactive arthritis may occur in some people following Y. enterocolitica infection. It is thought to be due to cross reacting T cells or antibodies that attack the joints.  Treatment of Yersinia infections Antimicrobic susceptibility - must do antimicrobial susceptibility testing.  Thank you A look of medieval doctor Your task for lecture 7 Your manual Read: CHAPTER 15 Enteric Gram-Negative Rods (Enterobacteriaceae) Chapter 19. Yersinia and Pasteurella 92