Gram Negative Rods Lecture 6 SM PDF

GRAM NEGATIVE RODS ENTEROBACTERIACEAE & VIBRIO SHIGELLA  General Characteristics of Shigella Coliform bacilli (enteric rods) Nonmotile gram-negative facultative anaerobes Four species Shigella sonnei (most common in industrial world) Shigella flexneri (most common in developing cou...

GRAM NEGATIVE RODS ENTEROBACTERIACEAE & VIBRIO SHIGELLA  General Characteristics of Shigella Coliform bacilli (enteric rods) Nonmotile gram-negative facultative anaerobes Four species Shigella sonnei (most common in industrial world) Shigella flexneri (most common in developing countries) Shigella boydii Shigella dysenteriae Non-lactose fermenting Produce no gas from glucose Resistant to bile salts SHIGELLA Shigella dysenteriae - Gram-negative, enteric, facultatively anaerobic, rod prokaryote; causes bacterial dysentery. This species is most often found in water contaminated with human feces. CLASSIFICATION OF SHIGELLA 4 groups according to O antigen:  Group A Shigella dysenteriae  Group B Shigella flexneri  Group C Shigella boydii  Group D Shigella sonnei SHIGELLA Shigella (S. flexneri, S. boydii, S. sonnei, S. dysenteriae) all cause bacillary dysentery or shigellosis, (bloody feces associated with intestinal pain). The organism invades the epithelial lining layer but does not penetrate. Usually within 2-3 days, dysentery results from bacteria damaging the epithelial layers lining the intestine, often with release of mucus and blood (found in the feces) and attraction of leukocytes (also found in the feces as "pus"). However, watery diarrhea is frequently observed with no evidence of dysentery. SHIGELLA S. flexneri, S. boydii, S. sonnei, S. dysenteriae Bacillary dysentery Shigellosis Bloody feces Intestinal pain Pus 7  Epidemiology and Clinical Syndromes of Shigella Shigellosis = Generic term for disease Low infectious dose (102-104 CFU) Humans are only reservoir Transmission by fecal-oral route (4F, fingers, flies, food and feces ) Incubation period = 1-3 days Watery diarrhea with fever; changing to dysentery Major cause of bacillary dysentery (severe 2nd stage) in pediatric age group (1-10 yrs) via fecal-oral route Outbreaks in daycare centers, nurseries, institutions Estimated 15% of pediatric diarrhea in U.S. Leading cause of infant diarrhea and mortality (death) in developing countries  DEFINITIONS Enterotoxin = an exotoxin with enteric activity, i.e., affects the intestinal tract Dysentery = inflammation of intestines (especially the colon (colitis) of the large intestine) with accompanying severe abdominal cramps, tenesmus (straining to defecate), and frequent, low-volume stools containing blood, mucus, and fecal leukocytes (PMN’s) Bacillary dysentery = dysentery caused by bacterial infection with invasion of host cells/tissues and/or production of exotoxins  Pathogenesis of Shigella Two-stage disease: Shiga toxin (powerful enterotoxic and cytotoxic activities) Early stage: Watery diarrhea attributed to the enterotoxic activity of Shiga toxin following ingestion and noninvasive colonization, multiplication, and production of enterotoxin in the small intestine Fever attributed to neurotoxic activity of toxin Second stage: Adherence to and tissue invasion of large intestine with typical symptoms of dysentery Cytotoxic activity of Shiga toxin increases severity SHIGELLOSIS within 2-3 days  epithelial cell damage Gut lumen 11  Pathogenesis and Virulence Factors (cont.) Virulence attributable to: Invasiveness Attachment (adherence) and internalization with complex genetic control Large multi-gene virulence plasmid regulated by multiple chromosomal genes Exotoxin (Shiga toxin) Intracellular survival & multiplication  Characteristics of Shiga Toxin Enterotoxic, neurotoxic and cytotoxic Encoded by chromosomal genes Two domain (A-5B) structure Similar to the Shiga-like toxin of enterohemorrhagic E. coli (EHEC) NOTE: except that Shiga-like toxin is encoded by lysogenic bacteriophage SHIGELLA  Diagnosis:  Methylene blue staining of feces (presence of PMN)  Specimen  Culture  (selenite or tetrathionate broth)  Biochemical tests  Serological tests  TREATMENT OF SHIGELLA DISEASES  Supportive treatment  Managing of dehydration is of primary concern by fluids and electrolytes. Indeed, mild diarrhea is often not recognized as shigellosis.  Patients with severe dysentery are usually treated with antibiotics (e.g. ampicillin, sulfamethoxazole- trimethoprin, quinolone).  Control and prevention:  Sanitation and personal hygiene  All of the followings are true for family Enterobacteriaceae EXCEPT: a. Gram negative non-spore forming bacilli and b. Reduce nitrate to nitrites facultative anaerobes. c. All species are oxidase negative d. All species ferment lactose. e. All species ferment glucose  WHICH OF THE FOLLOWING DISEASES AND BACTERIA ARE MATCHED UP INCORRECTLY? A. Streptococcus pneumoniae- meningitis B. Gastritis - Heliobacter pylori C. Streptococcus pyogenes- Tonsillitis D. Cellulitis - Staphylococcus aureus E. coli- UTI F. Enterobacter- TB SALMONELLA General Characteristics of Salmonella Coliform bacilli (enteric rods) Motile gram-negative facultative anaerobes Non-lactose fermenting Resistant to bile salts H2S producing from thiosulfate Produce acid only (S. typhi) or acid and gas (S. paratyphi) SALMONELLA Salmonella - rod prokaryote (dividing); note the flagella. Causes salmonellosis (food poisoning). CLASSIFICATION AND TAXONOMY OF SALMONELLA  O (group specific),H (type specific), Vi (virulence) antigens  Kauffman-White antigenic schema classified Salmonella to groups (A, B, C, D, …etc) using O and H antigens Classification and Taxonomy of Salmonella Old: Serotyping& biochemical assays used to name individual species within genus (e.g., Salmonella enteritidis, S. choleraesuis, S. typhi) Over 2400 O-serotypes (referred to as species) (Kauffman-White antigenic schema) Bioserotyping (e.g., S. typhimurium) New: DNAhomology shows only two species Salmonella enterica (six subspecies) and S. bongori Most pathogens in S. enterica ssp. enterica  Clinical Syndromes of Salmonella Salmonellosis = Generic term for disease Clinical Syndromes Enteric fever (prototype is typhoid fever and less severe paratyphoid fever) (S. typi, S. typhyimurium) Salmonella food poisoning -Enteritis (acute gastroenteritis) (S. enteritidis, S. typhyimurium) Septicemia (particularly S. choleraesuis, S. typhi, and S. paratyphi) Asymptomatic carriage (gall bladder is the reservoir for Salmonella typhi) DISEASE IN HUMANS  Incubation period:  Gastroenteritis: 12 hrs to 3 days  Enteric fever: 10 to 14 days  Asymptomatic to severe  All serovars can produce all forms  Reptile-associated is the most severe TYPHOID FEVER CAUSED BY SALMONELLA The severest form of salmonella infections, "typhoid" (enteric fever), caused by Salmonella typhi, S. typhyimurium. The organism is transmitted from a human reservoir or in the water supply (if sanitary conditions are poor) or in contaminated food. It invades the intestinal epithelium and, during this acute phase, gastrointestinal symptoms are noted. The organisms penetrates (usually within the first week) and passes into the bloodstream where it is disseminated in macrophages. Typical features of a systemic bacterial infection are noted. The septicemia usually is temporary with the organism finally lodging in the gall bladder (intestinal carriers). Organisms are shed into the intestine for some weeks (intestinal carriers). At this time, the gastroenteritis (including diarrhea) is noted again. The organism if excreted in urine (Urinary carriers) The Vi (capsular) antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one person (e.g. a food handler, files act as vectors) can cause a lot of spread. Antibiotic therapy is essential.  TYPHOID septicemia - occurs 10-14 days – lasts 7 days macrophage gall bladder –shedding, weeks acute phase, gastroenteritis 4 gastroenteritis CLINICAL DIAGNOSIS OF TYPHOID FEVER Isolation of organism From Blood: samples inoculated into N.B and subculture to MacConkey’s or SS or DCA (deoxycholate) media From stool: samples inoculated into selenite F or tetrthionate broth and subculture on MacConkey’s or SS or DCA (deoxycholate) media From urine: on MacConkey’s media Non lactose fermenters----identified (morphology, biochemical and serological) Biochemical Tests: IMVIC, TSI Serological ---Widal test TREATMENT Chloramphenicol Recently, quinolones Prevention and control: Pasteurized milk Pure water supply Proper sanitary Disposal of sewage Detection of carriers Destroy of flies Vacccination Killed vaccine Attenuated vaccine Vibrio cholera, The causative agent of Cholera  John Snow--father of epidemiology Case maps-over 500 cases of cholera in London Bad water vrs:bad air Hypothesis driven research Mapping the water supply Removing the handle from the Broad street pump 1854 Rapid end of the epidemic 1884 Koch’s discovery of Vibrio cholera John Snow was a physician working in London during an outbreak of cholera in the 18th Century. He noted the houses where individual cases of cholera clustered in a small block of streets near Piccadilly in central London. He further noted that the cases had all used a water pump in Broad Street. Previously, cholera was believed to be spread by “bad air”. He fought to have the handle removed from the broad Street water pump and was eventually successful. This stopped the local outbreak dramatically and was perhaps the first use Epidemiology to control a contagious disease John Snow’s Discovery of the “Broad Street Pump” Vibrio species: ✓ Gram negative, curved motile rods. ✓Sensitive to low pH; ✓Tolerant to salt ✓Aquatic habitat V. cholera V. parahaemolyticus V.vulnificus Cholera Vibrios Vibrio cholerae on 5% sheep blood agar (left). Colonies of V. cholerae grow well on most laboratory media. Growth is enhanced by the addition of 1% salt and it tolerates 3%. V. cholerae on thiosulfate-citrate-bile slats sucrose (TCBS) agar (right). Colonies appear yellow due to sucrose fermentation. Most other gram negative bacteria are inhibited in their growth on this medium. Oral fecal route High infective dose Cyclic epidemics CHOLERA CAUSING SEROTYPES  Phage infected strains  Enterotoxigenic V. cholerae had fallen into serotype O1  Eltore serotype  V. cholerae 0139 in Bangladesh The “rice-water” stool of cholera V. cholera produces several toxins including cholera toxin As much as 20 liters of fluid can be lost in a single 24 hrs period Although V. cholera encodes many virulence determinants, CT is sufficient to cause diarrhea Cholera is an acute disease with no gut pathology and no carrier state Moderate Dehydration Up to 10% body weight lost Pallor is striking Initial Irritability gives way to listlessness Sunken eyes from loss of water from retro-orbital fat pad Loss of skin turgor and Elasticity. There is no gross tissue damage in cholera Recovery depends on regeneration of poisoned intestinal cells For cholera toxin to be effective It must reach a critical concentration Near the cell. To do this requires Bacterial colonization --- adhesion to gut cells. V. cholera has several adhesions…most important among These are the TCP pili--- flagella Tcp pili It has been known for some time that not all V. cholera produce cholera toxin; and some strains have more than one copy of the CT gene ---also, new strains of pathogenic V. cholera arise in unexplained ways. Cholera toxin is encoded by a phage!! And TCP pili are the phage receptor... Treatment=rehydration Vaccine…its been difficult Prevention: ,,,,,,,,,,,,, HELICOBACTER PYLORI ✓Helical, gram negative bacterium ✓Microaerophilic ✓Acid-tolerant ✓Urease producer ✓Polar, sheathed flagella ✓Lives in mucus and on epithelial surfaces of human stomach ✓Genome size 1.7 MB HISTORY  The observation of Dr Robin Warren & Dr Marshall 1981  Successfully cultured H. pylori after many unsuccessful trails  Microaerophillic  Nobel prize announced - 2005  gastric adenocarcinoma Helicobacter pylori asymptomatic lymphoma colonization chronic atrophic peptic ulcer gastritis disease The Cancer Connection Bacteria Inflammation Cell damage H. PYLORI VIRULENCE DETERMINANTS  Adhesins  Flagella  Urease  Iron acquisition  Neutrophil activating protein (napA) MODE OF TRANSMISSION  Infection is acquired via ingestion orally  Transmitted during childhood in most cases  Prevalence varies geographically  Risk factors—increased age, lower level of education, developing country  May be asymptomatic (90% of infected) o 20 – 50% prevalence in middle age adults in industrialised countries o >80% prevalence in middle age adults in developing countries o :may reflect poorer living conditions LABORATORY DIAGNOSIS: NON-INVASIVE TESTS Serology : detect an immune response by examining a blood sample for abs to the organism (ELISA) : poor accuracy Urea breath test : a urea solution labelled with C14 isotope is given to patient. The solution subsequently exhaled by the patient contains the C14 isotope and this is measured. A high reading indicates presence of H. pylori  Faecal antigen test : detect H. pylori antigens in faecal specimens Polymerase chain reaction (PCR) : can detect HP within a few hours. Not routine in clinical use.  INVASIVE TESTING 1. Histological examination of biopsy specimens of gastric/duodenal mucosa take at endoscopy 2. Test urease-production by the organism->NH3 production->rise in pH=>change in the colour indicator of the kit High sensitivity and specificity INVASIVE TESTING 3. Culture : no more sensitive than skilled microscopy of histological sections  Used for antibiotic resistance testing  Requires selective agars and incubation periods  Tests for Helicobacter pylori Infection McColl K. N Engl J Med 2010;362:1597-1604 TREATMENT Goal of treatment to eradicate infection Triple therapy regimens consist of one anti- secretory agent and two antimicrobial agents for 7 to 14 days Triple therapy regimens must - have cure rate of approximately 80% - be without major side effects - minimal induction of resistance PSEUDOMONAS AERUGINOSA  Hospital-acquired Pseudomonas aeruginosa infctions Individuals at highest risk for infection: Burn victims Chemotherapy (neutropenia) Catheterized or other (Foley, central venous, endotracheal tubes) Diabetics (impaired circulation, cutaneous lesions, transplant/steroids) Surgery patients (particularly spinal or brain) IV drug abuse Cystic Fibrosis > 3 days Hospitalized Pseudomonas aeruginosa Opportunistic pathogen in people plant disease, insects (lab)  Pseudomonas aeruginosa Primary (and “model”) pathogen of the Pseudomonaceae O2 O2 O2 Polar Flagellum O2 Highly Motile Gram negative rods Organisms found anywhere there is water and oxygen LABORATORY: QUITE EASY TO IDENTIFY No fermentation of glucose, other sugars Grow on almost all media Highly motile (irregular colony shapes) Produce PIGMENTS (esp. pyocyanin) Characteristic FRUITY ODOR Beta hemolysis on Blood Agar Plates OXIDASE POSITIVE Pseudomonas aeruginosa: an amazingly versatile pathogen  Clinical Manifestations Pulmonary: Range from asymptomatic colonization to severe necrotizing bronchopneumonia Diffuse bilateral bronchopneumonia possible abscess and tissue necrosis Cystic Fibrosis, ICU respiratory equipment, hospitalized - neutropenic chemotherapy, chronic lung disorders Skin: Range from self-limiting folliculitis to vascular damage, tissue necrosis and blood stream invasion (life-threatening bacteremia) Severe burns ≥20-25% of surface area, hot tub users  Clinical Manifestations Urinary Tract: Can progress to life-threatening bacteremia Long-term indwelling catheters, Paraplegics, Quadriplegics Ear Infections: Range from mild “Swimmer’s Ear” to Malignant external otitis Frequent swimmers and deep-sea divers, Diabetic / Elderly Eye Infections: Range from corneal ulcers to loss of eye Requires physical abrasions to the cornea Contact Lens Wearers exposed to contaminated water Endocarditis: Tricuspid, aortic, mitral valves Intravenous drug abuse, contaminated IV fluids in hospital Other (Rare): Osteomyelitis, ecthyma, gangrenous Pathogenesis determinants of Pseudomonas aeruginosa  Pseudomonas aeruginosa VIRULENCE FACTORS STRUCTURAL: Alginate: Enormous polysaccharide capsule LPS: Endotoxin Flagellum: Dissemination in host Pili: Adherence and colonization-regulated by N ANTIMICROBIAL RESISTANCE: Chromosomal and/or Plasmid-mediated, Includes multi-drug efflux pumps  Pseudomonas aeruginosa VIRULENCE FACTORS-Secreted Exotoxin A: Exotoxin S: ADP-ribosylation of GTP-binding proteins (exoenzyme S) 2 Elastases: Cleaves elastin, collagen, immunoglobulin, complement components Production is regulated by iron availability Phospholipase: Hydrolyze eukaryotic phospholipids Production is regulated by availability of phosphate Alkaline protease: Cleaves IL-1, interferong and TNFa  PRIMARY VIRULENCE MECHANISMS Severe and Disseminated Use pili to colonize initial site (burns, heart valves) Secrete major virulence factors Exotoxin A and Exotoxin S Phospholipase Elastase and Alkaline Protease Inactivates IL-1b, TNFa, IFNg and complement Cleaves IgG and IgA Degrades laminin, fibrin, collagens Cleaves lymphocyte surface molecules (CD4+ T cells) Use flagella for systemic spread (bacteremia) Endotoxin for septic shock Quorum sensing-biofilm formation Mucoid = copious production of ALGINATE Biofilm formation is complex and essential for Virulence of PA ✓ DNA, polysaccharides are an essential biofilm constitutuent ✓ Treatment with dnase prevents biofilm formation in vitro ) THANK YOU