Gram-Negative Bacteria Lesson 12 PDF

Pathogenic Gram-Negative Bacilli- part 1 Cecilia Garlanda Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • The Enterobacteriaceae: An Overview – Pathogenic Enterobacteriaceae classified into three groups • Coliforms – Rapidly ferment lactose – Normal microbiota but may be opportunisti...

Pathogenic Gram-Negative Bacilli- part 1 Cecilia Garlanda Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • The Enterobacteriaceae: An Overview – Pathogenic Enterobacteriaceae classified into three groups • Coliforms – Rapidly ferment lactose – Normal microbiota but may be opportunistic pathogens • Noncoliform opportunists – Do not ferment lactose • True pathogens Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Aerobic or facultatively anaerobic, Gram-negative, rodshaped bacteria that ferment lactose to form gas in lactose broth – Commonly found in soil, on plants, and on decaying vegetation – Colonize the intestinal tracts of animals and humans – Coliforms in water indicate impure water and poor sewage treatment – Most common opportunistic pathogens are in the genera Escherichia, Klebsiella, Serratia, Enterobacter, Hafnia, Citrobacter Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Escherichia coli • Most common and important of the coliforms • E. coli antigens (O, H, K) used to identify particular strains • Virulent strains have virulence plasmids – Have genes for fimbriae, adhesins, exotoxins Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Escherichia coli • Large numbers of E.c. live in our intestine: opportunistic pathogen if it localizes out of intestine or if it acquires virulence factors • Causes several diseases: septicemia, urinary tract infections (UTI), gastroenteritis, neonatal meningitis, pneumonia, endocarditis. E.c. is the most common agent isolated from patients with sepsis and UTI. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Escherichia coli • Gastroenteritis is most common disease – Caused by specific strains – Caused by consumption of fecally contaminated food or water – Produces secretory diarrhea, cramps, nausea, and vomiting upon 1-2 days incubation, lasting 3-5 days. – Often mediated by enterotoxins coded by transferable plasmids that cause loss of sodium, chloride, potassium, bicarbonate and water from the cell (action similar to cholera toxin). – Major cause of pediatric diarrhea in developing countries (enterotoxigenic strains) and traveller’s diarrhea. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Escherichia coli • Gastroenteritis is most common disease –Other forms with different pathogenesis: »Enteropathogenic strains »Enteroaggregative strains »Enterohemorragic strains (see later) »Enteroinvasive strains Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Escherichia coli : Enterohemorragic strains (O157:H7) – Most prevalent pathogenic E. coli in developed countries – Ingestion of 10 bacteria causes diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (acute renal failure). Can be fatal – Associated with consumption of undercooked ground beef or contaminated milk or fruit juice, rare – Produces type III secretion system (disruption of cell metabolism, attachment of additional bacteria) and Shiga-like toxin (transported by neutrophils, causes inhibition of protein synthesis, cell death; transmitted by lysogenic bacteriophages). Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Escherichia coli • Common cause of non-nosocomial urinary tract infections – Occur more often in women than men – Can ascend from bladder and cause acute pyelonephritis – Uropathogenic strains: adhesins bind epithelial cells lining the bladder and upper urinary tract • Sepsis Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Klebsiella • In digestive and respiratory systems of humans and animals • Capsule protects the bacteria from phagocytosis; mucoid appearance of colonies • Can cause opportunistic infections • K. pneumoniae – Most commonly isolated pathogenic species – Causes pneumonia (destruction of alveoli and bloody sputum) – May be involved in bacteremia, meningitis, wound infections, UTIs – Associated to antimicrobial resistance: carbapenem-resistant Klebsiella pneumoniae The prominent capsule of Klebsiella pneumoniae Capsules Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Serratia: Serratia marcescens • Produce a red pigment when grown at room temperature • Can grow on catheters, in saline solutions, and on other hospital supplies • Can cause life-threatening opportunistic infections in immunocompromised patients • Frequently resistant to antimicrobial drugs Red colonies of Serratia marcescens Color develops in colonies grown at room temperature Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Coliform Opportunistic Enterobacteriaceae – Enterobacter, Hafnia, and Citrobacter Found in soil, water, decaying vegetation, and sewage Reside in the digestive tracts of animals and humans Enterobacter can contaminate dairy products All can be opportunistic pathogens – Cause nosocomial infections in immunocompromised patients: blood, wounds, surgical incisions, urinary tract • Difficult to treat due to resistance to antimicrobial drugs • • • • Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Noncoliform Opportunistic Enterobacteriaceae Do not ferment lactose. Include Proteus, Morganella, Providencia, Edwardsiella. – Proteus • Facultative anaerobe • Two morphologies: Rod-shaped with few polar flagella in broth or elongated with peritrichous flagella in agar • Proteus mirabilis – Most commonly associated with human disease – Often associated with urinary tract infections (UTIs) in patients with long-term urinary catheters – Infection-induced kidney stones can develop: urease, urea broken in carbon dioxide and ammonia, pH increases, ions precipitate to form kidney stones of Mg ammonium phosphate or Ca phosphate. – Resistant to many antimicrobial drugs The wavelike concentric rings of the swarming Proteus microbilis Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Noncoliform Opportunistic Enterobacteriaceae – Morganella, Providencia, and Edwardsiella • Nosocomial infections in immunocompromised patients • Primarily involved in urinary tract infections • Providencia may release urease and cause kidney stones Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Non-lactose fermenting bacteria – Include Salmonella, Shigella, Yersinia – Almost always pathogenic due to numerous virulence factors – Produce type III secretion systems • Introduce proteins into host cells – Inhibit phagocytosis – Rearrange the cytoskeletons of eukaryotic cells – Induce apoptosis Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Salmonella • Motile, peritrichous bacilli • Ferment glucose, produce gas, urease and oxidase negative, produce hydrogen sulfide (H2S) (rotten egg smell!!) • 2000 unique serotypes (strains) belonging to a single species, Salmonella enterica, but previously called S. typhi and S. paratyphi, S. choleraesuis…. • Today they are called Salmonella enterica serovar Typhi or Salmonella Typhi: serotype name capitalized and not in italics. • Pathogenicity is due to clusters of genes, pathogenicity islands, coding for virulence factors and type III secretion system. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Salmonella • Live in the intestines of birds, reptiles, and mammals. Animal-to-animal spread: animal reservoir. • Most human infections due to consuming food contaminated with animal feces (pet reptiles) or in children due to fecal-oral spread. • Poultry and eggs are also common sources of Salmonella • Strains adapted to humans (S. Typhi and Paratyphi) • Can cause salmonellosis and typhoid fever Salmonella 1 Epithelial cell Salmonella attaches to epithelial cells lining the small intestine. The events in salmonellosis Nucleus 2 3 4 Salmonella triggers Endocytosis using type III secretion systems. Salmonella multiplies within food vesicle. Salmonella kills host cell, inducing fever, cramps, and diarrhea. Capillary (blood vessel) 5 Bacteremia: Salmonella moves into bloodstream. Infection with fewer than 1 million cells of most strains are usually asymptomatic in immunocompetent individuals. For S. Typhi the infection dose is low. Larger infective doses can result in salmonellosis: nonbloody diarrhea, nausea, vomiting, fever, myalgia, headache, abdominal cramps. Some strains ( S. Typhi, Paratyphi, Choleraesuis) can cause bacteremia, and localize in the lining of the heart, the bones, the joints. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae: Salmonella • Typhoid fever – Caused by Salmonella enterica serotype Typhi (S. typhi) – A mild form is caused by serotype Paratyphi » Humans are the only host » Carriers are often asymptomatic – Bacteria ingested in contaminated food or water (1000-10000 cells) – Bacteria pass through intestines into the bloodstream » Phagocytic cells ingest the bacteria and carry them to various organs: liver, spleen, bone marrow, gallbladder. From gallbladder, S.T. re-infects the intestine. » Causes increasing fever followed by gastroenteritis, bacteremia, and peritonitis » Chronic colonization for more than 1 year occurs in 1-5% of patients. Gallbladder is the reservoir. The incidences of diseases caused by Salmonella in the United States Typhoid fever worldwide: 20 million cases, with 200,000 deaths per year. In particular in children in developing countries. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Salmonella • Phage use approved in Europe to reduce bacteria in food • Salmonellosis treated with fluid and electrolyte replacement • Typhoid fever treated with antimicrobial drugs • Gallbladder may be removed from carriers to prevent infection of others • Vaccines provide temporary protection to travelers Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Shigella • Oxidase-negative, urease-negative, nonmotile pathogens, do not produce H2S. • Primarily a pathogen of the digestive tract of humans • Produce diarrhea-inducing enterotoxin • Four well-defined species, 50 serogroups – S. dysenteriae – S. flexneri – S. boydii – S. sonnei Shigella 1 Shigella attaches to epithelial cell of colon. The events in shigellosis Epithelial cell Nucleus 2 3 Actin fibers 4 Mucosal abscess 5 Blood vessel Shigella survives in stomach: the infective dose is low, 200 cells Shigella triggers endocytosis. Shigella multiplies in cytosol. Shigella invades neighboring epithelial cells, thus avoiding immune defenses. An abscess forms as epithelial cells are killed by the infection, IL-1b is produced, neutrophils recruited. Phagocyte 6 Shigella that enters the blood is quickly phagocytized and destroyed. No bacteremia. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae: Shigella • Shigellosis – Severe form of dysentery: watery diarrhea (enterotoxin), followed by abdominal cramps, fever, pus-containing, bloody stools. – S. sonnei is predominant cause in industrialized nations – S. flexneri predominates in developing countries – S. dysenteriae is particularly virulent – Associated with poor hygiene and sewage treatment – People ingest bacteria on their own contaminated hands or in contaminated food. – Person-to-person spread is possible • 150 million cases occur annually worldwide. • 60% occur in children. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae: Shigella • Shiga toxin – Secreted by S. dysenteriae – Stops protein synthesis in host cells: damage of intestinal epithelial cells, damage to glomerular endothelial cells and renal failure and Hemolytic Uremic Syndrome (HUS) = acute renal failure, thrombocytopenia, microangiopathic hemolytic anemia. – Produces more severe disease: 20% mortality, 1 million people/year • Shigellosis is typically self-limiting – Treated with fluid and electrolyte replacement – Oral antimicrobials to limit spread to other individuals Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae: Yersinia • Normal pathogen of animals • Three important species – Y. enterocolitica – Y. pseudotuberculosis – Y. pestis – All contain virulence plasmids » Adhesins • Allow attachment to human cells » Type III secretion systems • Injection of proteins that causes apoptosis of macrophage and neutrophils, inhibition of cytokine production » Y. pestis: antiphagocytic protein capsule; plasminogen activator protease: degrades C3, C5a, fibrin clots Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae: Yersinia • Y. enterocolitica – Acquired by consumption of contaminated food or water – Pigs, livestock, rodents and rabbits: natural reservoir. – Causes inflammation of the intestinal tract, lasting for weeks or months, and mimicking appendicitis. – North Europe, North America • Y. pseudotuberculosis – Rodents, wild animals: reservoir – Causes less severe inflammation of the intestines • Y. pestis – Plague: one of the most devastating disease in history – Highly virulent, non-enteric pathogen – Causes bubonic and pneumonic plague The natural history and transmission of Yersinia pestis Direct contact Airborne transmission Natural endemic reservoir hosts (rodents) Flea bite Amplifying hosts (most mammals) Normal lymph nodes Bubo Bubonic plague Pneumonic plague • Natural reservoir hosts (rodents) do not develop disease. Fleas are the vectors for the spread of Y. pestis. Y. pestis multiplies in fleas and cause alteration of the esophagus: starving fleas jump from host to host seeking for a blood meal and infecting new hosts. • Amplifying hosts develop disease and die. ZOONOSIS • Bubonic plague is not spread from person-to-person. • Pneumonic plague is transmitted via aerosol and sputum. Bubo in an eight-year-old patient Bubo A swollen lymph node, a characteristic feature of bubonic plague, that develops within a week of a flea bite. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Yersinia pestis – Diagnosis and treatment must be rapid » Fast progression and deadliness of the plague: from high fever and painful bubo, to bacteremia, DIC, subcutaneous hemorrhaging, death of tissues, secondary infection by Clostridium and gangrene: “Black death”. Fatal in one week in 50% of cases if untreated, 15% if treated. » If it spreads to lungs, it causes pneumonic plague: fatal in 100% of cases if untreated. » Characteristic symptoms are usually diagnostic » Many antibacterial drugs are effective Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Yersinia pestis – Epidemiology: » major pandemics: in 500s-700s AD Egypt, North Africa, Europe, Asia, killing 40 million people; 14th century: 25 million in Europe in 5 years (40% of the population); 1860s: pandemic in all the world, from China, to Africa, Europe, Americas. Today: epidemics and sporadic cases. Sylvatic plague is impossible to eradicate. Pathogenic, Gram-Negative, Facultatively Anaerobic Bacilli • Truly Pathogenic Enterobacteriaceae – Yersinia pestis – Epidemiology Sites of infection by some common members of the Enterobacteriaceae Strategies to sense pathogens and tissue damage Ficolins Collectins Biological fluids Complement activation Opsonization C1q Short and long Pentraxins (CRP, PTX3) Lipopeptides, LPS, PGN Capturing Receptors MDP Phagocytosis TLR 1,2,4,5,6 CpG DNA, viral ssRNA, dsRNA NOD 1,2 MyD88 TRIF Endosome Phagosome Cytoplasm NALP3 (inflammosome) NOD-like R Virus dsRNA NFkB IRFs MAPKs RIG-I TLR 3,7/8,9 Signalling receptors IFN RIG-like R Infections, Vascular Inflammation and Tissue Damage TLR agonists, Inflammatory Cytokines (IL-1, TNF) PMN Mø Fibroblasts SMC Endothelium PTX3 Diagnostic and Prognostic potential Pathogen recognition Innate Immunity to A. fumigatus, P. aeruginosa, K. pneumoniae, E. coli (UPEC) Modulation of inflammation and Complement activation (C1q, Factor H, Ficolins, apoptotic cells, P-selectin, FcgRs) Tissue remodelling (fibrin, plasminogen, FGFs) Modelli di infezione Infezioni: batteri, funghi, virus Locali Respiratorie Peritoneale: Legatura e puntura del cieco Tratto urinario Sistemiche Come si analizza la risposta immunitaria all’infezione? Carica microbica (polmone, BAL, peritoneo, rene, vescica, …..) Reclutamento leucocitario (conta totale e differenziale) Produzione citochine locale e sistemica Altri mediatori dell’infiammazione, edema Danno tessutale: immunopatologia Fagocitosi in vitro e in vivo Models of respiratory infections Models of sepsis: intraperitoneal or intravenous injection of microbes Microbial count in biological fluid or tissue homogenate Serial dilution and CFU count Role of PTX3 in innate resistance to Aspergillus fumigatus and Pseudomonas aeruginosa Mortality Microbial burden Ptx3+/+ Ptx3-/(Garlanda et al. Nature 2002) Role of PTX3 in recognition of A. fumigatus conidia by neutrophils Phagocytic Index In vitro phagocytosis PTX3+/+ 120 120 80 p<0.01 p<0.01 PTX3-/* * 40 p=0.01 80 40 0 15' 45' 0 PTX3 - + In vivo resistance to fungal growth Strain Adoptive transfer of a leukocyte subset Adoptive transfer 0 PTX3+/+ PTX3+/+ PMN PTX3-/- PMN PTX3-/- PTX3+/+ PMN PTX3-/- PMN CD4+ T cells Chitin content (pg/ml) 15 30 * * (Jaillon et al J Exp Med 2007) The agar beads mouse model for P. aeruginosa chronic lung infection and PTX3 treatment P. aeruginosa 50 4°C C Cash H.A. et al., 1979 Tryptic Soy Agar (TSA)+ Mineral oil INFECTION 1.5X106 CFU intra trachea RECOVERY Lung, BALF, blood Day Day 0 1 2 3 4 5 6 7 8 9 10 11 12 PTX3 intraperitoneal treatment (10μg/mouse/day) 13 14 PTX3 treatment reduces bacterial load, leukocyte infiltration, inflammation and vascular permeability in P. aeruginosa chronic lung infection ** 107 104 103 102 105 104 103 102 101 101 Saline PTX3 105 104 103 102 101 100 100 * 3.0×1006 106 Total lung CFU Total lung CFU 105 4.0×1006 107 106 106 Total lung CFU * 107 PTX3 treatment from day 7 to day 21 BALF cell number PTX3 treatment from day 7 to day 14 CFU Leukocytes ∗∗ ∗∗∗ Saline PTX3 2.0×1006 1.0×1006 100 Saline PTX3 Saline PTX3 0 s s ls lls phi cyte cyte l ce o o o a r t t n h p To Mo Neu Lym Cytokines and inflammatory mediators p n 0.009 0.04 0.01 0.04 0.04 0.04 24 28 18 8 14 20 10 8 10 10 10 10 0.04 0.8 * 0.7 0.6 Ο.D. 450nm (pg/ml) Saline PTX3 (mean ±SEM) IL-1b 6323±1288 2438±529 CCL2 116±10 90±8 (mean TGF-b1 1266±86 1630±114 IL-17 25±6 9±5 CXCL1 978±148 624±86 CXCL2 858±178 441±71 INF-g nd nd IL-13 nd nd IL-6 nd nd IL-4 nd nd IL-10 nd nd Total proteins 1546±71 1354±72 BAL protein levels: vascular permeability 0.5 0.4 0.3 0.2 0.1 0.0 Saline PTX3 Moalli and Paroni et al., JI, 2011 The PTX3 treatment reduces parenchyma inflammation, acute and chronic lesions in P. aeruginosa chronic lung infection PTX3 Saline Infected bronchi Parenchyma /tot involved (%) bronchi (%) Acute Chronic Saline Saline (n=4) PTX3 (n=5) p BALT PTX3 Chronic lesions /tot bronchi (%) Acute lesion /tot bronchi (%) 46±9 54±8 15±3 39±8 27±8 27±8 7±2 20±7 0.06 0.009 ∗∗ 0.004 ∗∗ 0.03 ∗ Histopathological damage Moalli and Paroni et al., JI, 2011 FITC-CONIDIA (or CFSE-bacteria) PHAGOCYTOSIS BY ALVEOLAR NEUTROPHILS (or in fresh whole blood) 8X107 i.t. + FITC A.fumigatus conidia 4h BAL FITC intensity in leukocytes (CD45+) and neutrophils (CD11b+ Ly6G+) FITC-conidia In vitro and in vivo phagocytosis ns CTR PTX3 ns ∗ ∗∗∗ 50000 Fluorescence intensity in neutrophils CFSE MFI 40000 ∗ ns 30000 20000 ns 10000 0 WT C1q KO C3 KO Fcγ KO Moalli and Paroni et al., JII, 2011 PTX3 acts as an opsonin for specific microorganisms, facilitating phagocytosis PMN CLP cecal ligation and puncture Legatura sotto valvola ileo-cecale. Puntura con ago di diametro variabile: severità variabile. Modello di sepsi polimicrobica con danno settico polmonare: danno d’organo a distanza. Parametri da valutare: Sopravvivenza Carica microbica nel sangue e peritoneo Aspetto clinico Leucociti reclutati localmente e nel polmone Citochine UTI model: intravescical injection of E. coli (uropathogenic strain) Kidneys Bladder ns * 109 107 CFU/2kidneys 107 106 105 104 103 106 105 104 103 102 102 101 101 100 100 Ptx3+/+ Ptx3-/- Ptx3+/+ Day 1 Ptx3-/- Ptx3+/+ 10000 ** Ptx3+/+ Ptx3-/- Day 5 Leukocytes in urine * 108 700 107 600 500 100 400 300 10 200 CXCL1 (pg/Bladder) 1000 800 Urine cells (Cells/ml) * Ptx3-/- Day 1 Day 5 Day 1 CCL2 and CXCL2 (pg/Bladder) * * 108 108 CFU per bladder * 109 ** 106 105 104 103 100 1 0 Ptx3 +/+ Ptx3 CCL2 -/- Ptx3 +/+ Ptx3 CXCL2 -/- Ptx3 +/+ Ptx3 CXCL1 -/- 102 Ptx3+/+ Ptx3-/-