Microbial Diseases of GIT Part 1 PDF

MICROBIAL DISEASES OF THE GIT Prepared by: FRITZ VON T. GELLA, RMT, MD College of Medicine Davao Medical School Foundation, Inc.  NORMAL MICROBIAL FLORA OF THE GIT v Mouth (1 mL saliva) = millions of bacteria v Stomach and small intestine – few or...

MICROBIAL DISEASES OF THE GIT Prepared by: FRITZ VON T. GELLA, RMT, MD College of Medicine Davao Medical School Foundation, Inc.  NORMAL MICROBIAL FLORA OF THE GIT v Mouth (1 mL saliva) = millions of bacteria v Stomach and small intestine – few organisms due to HCl and rapid movement of food v Large intestine – 100 billion bacteria per gram of feces, 40% of fecal mass Microbial cell material (Lactobacillus, Bacteroides, Enterobacter, E. coli, Proteus spp.) S. aureus may colonize the gluteal fold and perinatal area 2 NORMAL MICROBIAL FLORA OF THE GIT 3  NORMAL MICROBIAL FLORA OF THE GIT Advantages of Normal Residential Flora v Antibacterial bacteriocins and colicin competition for space and nutrients with potentially pathogenic bacteria v Benefits to the host Release organic acids to help digestion Produce vitamin K and some B vitamins Stimulates immune system 4  NORMAL MICROBIAL FLORA OF THE GIT Normal Flora become pathogenic when: Antibacterial v Host is immunocompromised v Chronic antibiotic use weakens “good” intestinal bacteria 5  GASTROINTESTINAL DEFENSES v Mechanical barriers – tight junctions between epithelial cells prevent entry into tissues v Mechanical actions – peristalsis v GI secretions and intestinal fluids, electrolytes and mucus– flush away potential pathogens or macromolecules, acid gastric pH (especially important in inhibition of gram (–) bacilli) v Gut flora v Immune cells – e.g. Gut associated lymphoid tissue (GALT), Peyer’s patches, leukocytes, IgA 6  PREDISPOSING FACTORS FOR DIARRHEAL DISEASES v Extremes of Age (young and old) v Recent travel v Unsafe water v Unclean food v Malnutrition v Poor personal hygiene/sanitation v Antibiotic prolonged usage v Underlying illness v Institutionalization 7  GENERAL MECHANISMS OF GI PATHOGENS v Toxigenic Effects are due to toxins released by the gut bacteria v Enteroinvasive Effects due to multiplication and invasion of organisms into gut tissues v Interference of secretion and absorption v Spread to contiguous and distant sites v Large numbers obstruct the lumen (due to intestinal worms) 8 9  DEFINITION OF TERMS v Enterotoxin an exotoxin with enteric activity (i.e. affects the intestinal tract) v Dysentery inflammation of the intestines (esp. colitis) with accompanying severe abdominal cramps, tenesmus (straining to defecate), and frequent, low-volume stools containing blood, mucus, and fecal leukocytes (PMNs) v Bacillary dysentery caused by bacterial infection with invasion of host cells/tissues and/or production of exotoxins v Diarrhea loose, watery stools bloody or mucus – DYSENTERY 10  DEFINITION OF TERMS v Abdominal cramps, nausea and vomiting defense mechanism to rid body of harmful material v Gastroenteritis inflammation of stomach or intestinal mucosa 11  BACTERIAL DISEASES OF THE LOWER GI Infections Toxins Pathogens enter GI tract Ingestion of a preformed and multiplies toxin Bacteria may penetrate the intestinal mucosa or may pass to other systemic organs Delay in appearance of Sudden onset of symptoms while symptoms (few hours) pathogen increases in number or invades tissues Usually with fever Fever not always 12 present GRAM NEGATIVE BACILLI 13  ENTEROBACTERIACEAE v Large family of small, non-spore forming gram-negative rods (bacilli) v Many members inhabit the soil, water, and decaying matter; and are common occupants of large bowel of animals including humans v Most frequent cause of diarrhea through enterotoxins v Enterics, along with Pseudomonas sp., account for almost 50% of nosocomial infections v Facultative anaerobes, grow best in air v All ferment glucose, reduce nitrates to nitrites, oxidase (-), and catalase (+) v Divide into lactose fermenters and nonlactose fermenters v Enrichment, selective and differential media utilized for screening samples for pathogens 14  ANTIGENIC STRUCTURE v Endotoxin; some also produce exotoxins v Antigens O: cell envelope or O antigen H: flagellar (motile cells only) antigen K: capsular polysaccharide antigen Vi (virulence): Salmonella capsular antigen 15  ANTIGENIC STRUCTURE v O antigens most external part of the cell wall made of lipopolysaccharide some O-specific polysaccharides contain unique sugars resistant to heat and alcohol detected by bacterial agglutination agglutinate with IgM antibodies single organism may carryseveral O antigens (responsible for cross-reactions) E.g. Shigella share one or more O antigens with E coli. E coli may cross-react with some Providencia, Klebsiella, and Salmonella species. 16  ANTIGENIC STRUCTURE v K antigens can be external to O antigens, be polysaccharides (e.g. K antigens of E coli) or proteins interfere with agglutination by O antisera associated with virulence: K1 antigen of E. coli in neonatal meningitis K antigens of E. coli cause attachment of the bacteria to epithelial cells before GI or UT invasion 17  ANTIGENIC STRUCTURE v H antigens located at the flagella Denatured or removed by heat or alcohol preserved by treating motile bacterial variants with formalin agglutinate with IgG antibodies The determinantsin H antigens are a function of the amino acid sequence in flagellar protein (flagellin) 18  ANTIGENIC STRUCTURE v Colicins (Bacteriocins) produced by gram-negative organisms bactericidal produced by certain strains of bacteria active against some other strains of the same or closely related species (protective) production controlled by plasmids (can jump from species to species) bacteriocin-producing strains resistant to their own bacteriocin Example: Colicin of E. coli is called colicin Colicin of Serratia is called marcescens Colicin of Pseudomonas is called pyocins 19  MORPHOLOGY AND IDENTIFICATION v Typical Organisms short rods capsulated or non-capsulated v Klebsiella species are large and regular capsule v Enterobacter species less regular and less prominent 20  CULTURE v E coli colonies (considered a prototype organism because it is encountered the most) and most of the other enteric bacteria circular, convex, smooth colonies with distinct edges some E coli strains produce hemolysis on blood agar v Enterobacter colonies similar but somewhat more mucoid 21  CULTURE v Klebsiella large and very mucoid (represents the capsule)and tend to coalesce with prolonged incubation v Salmonellae and Shigellae colonies similar to E coli but do not ferment lactose 22  CULTURE v Blood agar v Eosin methylene blue or MacConkey agar (differentiate lactose fermentation) Lactose fermenters (colored colonies) Non–lactose fermenters (colorless colonies) 23 CULTURE 24  CULTURE v Triple sugar iron (TSI) agar - complex media composed of: 0.1% glucose 1% sucrose 1% lactose ferrous sulfate (detection of H2S) tissue extracts (protein growth substrate) pH indicator (phenol red) Differentiate salmonellae and shigellae from other enteric gram-negative rods in stool cultures 25  CULTURE v Triple sugar iron (TSI) agar Procedure: Poured agar into a tilted test tube to producea slant with a deep butt and inoculated by stabbing bacterial growth into the butt. If glucose is fermented: Slant and butt initially turn yellow from the acid production Slant turns red due to the alkalinity from oxidation of fermentation products (CO2 and H2O) and formation of amines (oxidative decarboxylation of proteins) If lactose or sucrose is fermented: Slant and butt remain yellow (acid) from too much acid production 26  CULTURE v Salmonellae and shigellae Alkaline (red/orange) slant with an acid butt v Proteus, Providencia, and Morganella species Alkaline slant with acid butt Rapid formation of red color in Christensen’s urea medium v Other enteric bacteria Acid on the slant Acid and gas (bubbles) in the butt 27  CULTURE v Triple sugar iron (TSI) agar 28  GROWTH CHARACTERISTICS v Identified further by Biochemical differentiation which involves: Carbohydrate fermentation patterns Activity of amino acid decarboxylases Other enzymes 29  GROWTH CHARACTERISTICS v Glucose and Acid production – all Enterobacteriaceae v H2S – Proteus mirabilis & Salmonella 30  GROWTH CHARACTERISTICS v Indole test (indole production from tryptophan) commonly used in rapid identification systems (+) for E. coli, Klebsiella oxytoca, Morganella morganii 31  GROWTH CHARACTERISTICS v Methyl Red 32  GROWTH CHARACTERISTICS v Vogues-Proskauer Test 33  GROWTH CHARACTERISTICS v Citrate Utilization Test 34  GROWTH CHARACTERISTICS v IMViC Test 35  GROWTH CHARACTERISTICS v IMViC Test 36  GROWTH CHARACTERISTICS v Motility Test 37  GROWTH CHARACTERISTICS v Urease Test 38 PRESUMTIVE IDENTIFICATION 39  ESCHERICHIA COLI v Distinguishing Features Gram-negative rod Facultative anaerobic, oxidase negative E. coli is a lactose fermenter: colonies with iridescent green sheen on EMB Triple sugar iron (TSI) agar: A/A, gas (+), H2S (-) 40  ESCHERICHIA COLI v Reservoir: human colon; may colonize vagina or urethra; contaminated crops where human fecal fertilizer is used; enterohemorrhagic strains: bovine feces v Transmission Endogenous Fecal-oral Maternal fecal flora Enterohemorrhagic strains: bovine fecal contamination (raw or undercooked beef, milk, apple juice from fallen apples) 41  ESCHERICHIA COLI: DISEASE SYNDROMES - GASTROENTERITIS Enteropathogenic E coli (EPEC) v Diarrhea in infants v Adhere to mucosal cells of small bowel v Pathogenicity factors: EAF- EPEC adherence factor = encodes bundle forming pilus LEE- locus of enterocyte effacement = promotes tight adherence v After attachment, there is loss of microvilli (effacement), formation of filamentous actin pedestals or cuplike structures, and entry of EPEC cells into mucosal cells (occasionally) 42  ESCHERICHIA COLI: DISEASE SYNDROMES - GASTROENTERITIS Enterotoxigenic E coli (ETEC) v Traveler’s diarrhea v Diarrhea in children < 5 yrs v Colonization factor antigens (CFA) v Causes severe diarrhea due to heat-labile toxin and heat-stable toxin which stimulate secretion and fluid loss; also has fimbriae v Cholera-like diarrhea, only milder v Heat labile toxin: adenylcyclase activated, cyclic AMP ↑, secretion water/ions ↑ v Heat stable toxin: guanylate cyclase activated, cyclic GMP ↑, uptake water/ions↓ 43  ESCHERICHIA COLI: DISEASE SYNDROMES - GASTROENTERITIS Enteroinvasive E. coli (EIEC) v causes inflammatory disease of the large intestine v organism attaches to the intestinal mucosa via pili v outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa v there is lateral movement of the organism from one cell to adjacent cells 44  ESCHERICHIA COLI: DISEASE SYNDROMES - GASTROENTERITIS Enterohemorrhagic E. coli (EHEC) v O157:H7 strain v causes hemorrhagic syndrome and kidney damage v Vero toxin - “shiga-like” v Hemolysins v Historically, a cause of outbreaks v Inhibits 60S Ribosome resulting in intestinal epithelial cell death v Hemorrhagic colitis (severe form of diarrhea) v Hemolytic uremic syndrome (acute renal failure, microangiopathic hemolytic anemia, thrombocytopenia) 45  ESCHERICHIA COLI: DISEASE SYNDROMES - GASTROENTERITIS Enteroaggregative E. coli (EAEC) v A cause of persistent, watery diarrhea with vomiting and dehydration in infants v The bacteria adheres to the intestinal mucosa and elaborate enterotoxins (enteroaggregative heat-stable toxin, EAST) v The result is mucosal damage, secretion of large amounts of mucus, and secretory diarrhea 46  ESCHERICHIA COLI: DISEASE SYNDROMES - GASTROENTERITIS EPEC P pediatric EIEC I inflammatory ETEC T traveler EHEC H hamburger A. ETEC- Traveler’s Diarrhea, ST/LT toxins, choleralike watery diarrhea; children in developing countries B. EIEC- invades intestinal mucosal cells dysentery; resembles Shigellosis, older children and adults C. EPEC- infant diarrhea; non-bloody stools; bundle-forming pili and intimin D. EHEC- cytotoxin; bloody diarrhea; hemolytic anemia, thrombocytopenia, acute renal failure (Hemolytic-Uremic Syndrome); STEC O157:H7 E. EAEC- enterotoxins, ST-like toxin, hemolysin; acute and chronic diarrhea; persistent diarrhea in HIV 47 ESCHERICHIA COLI: DISEASE SYNDROMES - GASTROENTERITIS 48  KLEBSIELLA PNEUMONIAE v Distinguishing Features Gram-negative rods with large polysaccharide capsule Mucoid, lactose-fermenting colonies on MacConkey agar Oxidase negative 49  KLEBSIELLA PNEUMONIAE v Reservoir: human colon and upper respiratory tract v Pathogenesis : capsule (impedes phagocytosis); endotoxin (causes fever, inflammation, and shock [septicemia]) v Diseases: Pneumonia, Urinary tract infection, Septicemia v Diagnosis: culture of sputum or clean catch urine sample; lactose fermenter v Treatment: third-generation cephalosporin with or without an aminoglycoside; carbapenem for ESBL- producing strains v Prevention: good catheter care, limit use 50  SERRATIA MARCESCENS v Distinguishing Features Produces a characteristic red pigment called prodigiosin v Diseases Meningitis, Pneumonia, UTI v Treatment Cephalosporins (3rd Generation) 51  PROTEUS MIRABILIS/PROTEUS VULGARIS v Distinguishing Features Gram-negative rods, non-lactose fermenting Highly motile; “swarming” motility on surface of blood agar Urease produced Facultative anaerobe (Enterobacteriaceae), oxidase negative TSI test results: Alkaline/Acid, (Non-lactose fermenters), Gas is produced and H2S (+) Capable of producing urease 52  PROTEUS MIRABILIS/PROTEUS VULGARIS v Reservoir: human colon and environment (water and soil) v Pathogenesis Urease raises urine pH to cause kidney stones (staghorn renal calculi) Motility may aid entry into bladder Endotoxin causes fever and shock when septicemia v Disease(s): urinary tract infection; septicemia v Diagnosis: culture of blood or urine for lactose- negative organisms with swarming motility v Treatment: fluoroquinolone, TMP-SMX, or third- generation cephalosporin for uncomplicated UTI; remove stones if present 53  YERSINIA PESTIS v Distinguishing Features Small gram-negative rods with bipolar staining Facultative intracellular parasite Coagulase positive 54  YERSINIA PESTIS v Transmission: wild rodents flea bite lead to sylvatic plague; human-to-human transmission by respiratory droplets v Pathogenesis Coagulase-contaminated mouth parts of flea Endotoxin and exotoxin Envelope antigen (F-1) inhibits phagocytosis. Type III secretion system suppresses cytokine production and resists phagocytic killing 55  YERSINIA PESTIS v Disease(s) Bubonic plague o Flea bites infected animal and then later uninfected human o Symptoms: rapidly increasing fever, regional buboes, conjunctivitis; if untreated, leads to septicemia and death Pneumonic plague o Arises from septic pulmonary emboli in bubonic plague or inhalation of organisms from infected individual o Highly contagious o Bioterrorism o Hemoptysis, chest pain, dyspnea 56  YERSINIA PESTIS v Diagnosis Clinical specimens and cultures are hazardous Serodiagnosis or direct immunofluorescence “Safety pin” staining on blood stain (Wright or Wayson) v Treatment: aminoglycosides 57  YERSINIA ENTEROCOLITICA v Distinguishing Features motile at 25.0 C (77.0 F), nonmotile at 37.0 C (98.6 F); cold growth v Reservoir: zoonotic v Transmission: unpasteurized milk, pork; prominent in northern climates (Michigan, Scandinavia) v Pathogenesis Enterotoxin, endotoxin Multiplies in the cold 58  YERSINIA ENTEROCOLITICA v Disease(s) Enterocolitis: presentations may vary with age o Very young: febrile diarrhea (blood and pus) o Older kids/young adults: pseudoappendicitis (also caused by Yersinia pseudotuberculosis) o Adults: enterocolitis with postinfective sequelae like reactive arthritis Blood transfusion–associated infections 59  YERSINIA ENTEROCOLITICA v Diagnosis: stool culture, 25.0 C (77.0 F), cold enrichment v Treatment: supportive care; fluoroquinolone or third-generation cephalosporin for immunocompromised 60  SHIGELLA SPECIES v Distinguishing Features Gram-negative rods, nonmotile, non-lactose fermenters v Reservoir: human colon only (no animal carriers) v Transmission: fecal-oral spread, person to person 61 SHIGELLA SPECIES 62  SHIGELLA SPECIES Pathogenesis v Endotoxin triggers inflammation v No H antigens v Shigellae invade M cells (membrane ruffling and macropinocytosis), get into the cytoplasm, replicate, and then polymerize actin jet trails to go laterally without going back out into the extracellular milieu. This produces very shallow ulcers and rarely causes invasion of blood vessels. v Shiga toxin: Produced by S. dysenteriae, type 1 Three activities: neurotoxic, cytotoxic, enterotoxic AB component toxin is internalized in human cells; inhibits protein synthesis by clipping 60S ribosomal subunit 63  SHIGELLA SPECIES v Disease(s): enterocolitis/shigellosis (most severe form is dysentery) 1–4 day incubation Organisms invade, producing bloody diarrhea Fever (generally >38.3 C [101.0 F]); lower abdominal cramps; tenesmus; diarrhea first watery, then bloody; invasive but rarely causes septicemia; shallow ulcers 64  SHIGELLA SPECIES v Diagnosis: isolation from stool during illness and culture on selective media v Treatment: fluid and electrolyte replacement (mild cases); antibiotics (severe cases); resistance is mediated by plasmid-encoded enzymes; many strains are ampicillin- resistant 65  SALMONELLA ENTERICA TYPHI v Distinguishing Features Gram-negative rods, highly motile with the Vi capsule Facultative anaerobe, non–lactose fermenting Produces H2S Species identification with biochemical reactions Sensitive to acid v Reservoir: humans only; no animal reservoirs v Transmission: fecal-oral route from human carriers (gall bladder); decreased stomach acid or impairment of mononuclear cells as in sickle cell disease predisposes to Salmonella infection 66  SALMONELLA ENTERICA TYPHI v Pathogenesis and Disease: typhoid fever (enteric fever), S. typhi (milder form: paratyphoid fever; S. paratyphi) *PRELAB DISCUSSION v Diagnosis: organisms can be isolated from blood, bone marrow, urine, and tissue biopsy from the rose spots if present; antibodies to O, Vi, and H antigens in patient’s serum can be detected by agglutination (Widal test) v Treatment: fluoroquinolones or third-generation cephalosporins 67  Salmonella Subspecies other than typhi (S. enteritidis, S. typhimurium) v Distinguishing Features Facultative gram-negative rods, non–lactose- fermenting on EMB, MacConkey medium Produces H2S, motile (unlike Shigella) Speciated with biochemical reactions and serotyped with O, H, and Vi antigens v Reservoir: enteric tracts of humans and domestic animals, e.g., chickens and turtles v Transmission: raw chicken and eggs in kitchen; food-borne outbreaks (peanut butter, produce, eggs); reptile pets (snakes, turtles) 68  Salmonella Subspecies other than typhi (S. enteritidis, S. typhimurium) v Pathogenesis Invades mucosa in ileocecal region, invasive to lamina propria -> inflammation -> increased PG -> increased cAMP -> loose diarrhea; shallow ulceration v Disease(s) Enterocolitis/gastroenteritis (second most common bacterial cause after Campylobacter): 6–48 hour incubation; nausea; vomiting; only occasionally bloody, loose stools; fever; abdominal pain; myalgia; headache 69  Salmonella Subspecies other than typhi (S. enteritidis, S. typhimurium) v Diagnosis: culture on Hektoen agar, H2S production v Treatment: antibiotics are contraindicated for self-limiting gastroenteritis; ampicillin, third- generation cephalosporin, fluoroquinolone, or TMP-SMX for invasive disease 70  PSEUDOMONAS AERUGINOSA v Distinguishing Features Oxidase-positive, Gram-negative rods, nonfermenting Pigments: pyocyanin (blue-green) and fluorescein Grape-like odor Slime layer Non–lactose fermenting colonies on EMB or MacConkey Biofilm 71  PSEUDOMONAS AERUGINOSA v Pathogenesis Endotoxin causes inflammation in tissues and gram-negative shock in septicemia Pseudomonas exotoxin A ADP ribosylates eEF- 2, inhibiting protein synthesis (like diphtheria toxin) Liver is primary target Capsule/slime layer allows formation of pulmonary microcolonies; difficult to remove by phagocytosis 72  PSEUDOMONAS AERUGINOSA v Disease(s) Healthy people: transient GI tract colonization (loose stool 10% population); hot tub folliculitis; eye ulcer (trauma, coma, prolonged contact wear) Burn patients: GI tract colonization - skin - colonization of eschar cellulitis (blue-green pus) -septicemia Neutropenic patients: pneumonia and septicemia (often superinfection, i.e., infection while on antibiotics) 73  PSEUDOMONAS AERUGINOSA v Disease(s) Chronic granulomatous disease: pneumonias, septicemias (Pseudomonas is catalase- positive); [diabetic] osteomyelitis (diabetic foot) Otitis externa: swimmers, diabetics, those with pierced ears Septicemias: fever, shock +/- skin lesions (black, necrotic center, erythematous margin, ecthyma gangrenosum) Catheterized patients: urinary tract infection Cystic fibrosis: early pulmonary colonization, recurrent pneumonia; always high slime- producing strain 74  PSEUDOMONAS AERUGINOSA v Diagnosis: Gram stain and culture v Treatment: antipseudomonal penicillin and an aminoglycoside or fluoroquinolone v Prevention: pasteurize or disinfect water- related equipment, hand washing; prompt removal of catheters; avoid flowers and raw vegetables in burn units 75  CAMPYLOBACTER JEJUNI v Distinguishing Features Gram-negative curved rods with polar flagella (“gulls’ wings”) oxidase-positive microaerophilic grows at 42°C on Campy or Skirrow agar 76  CAMPYLOBACTER JEJUNI v Reservoir: intestinal tracts of humans, cattle, sheep, dogs, cats, poultry v Transmission: fecal-oral, Skirrow agar primarily from poultry v Pathogenesis: low infectious dose (as few as 500); invades mucosa of the colon, destroying mucosal surfaces; blood and pus in stools (inflammatory diarrhea); rarely penetrates to cause septicemia 77  CAMPYLOBACTER JEJUNI v Disease: Gastroenteritis Common cause of infectious diarrhea worldwide >/= 10 stools/day (may be frankly bloody) Abdominal pain, fever, malaise, nausea, and vomiting Generally self-limiting in 3–5 days but may last longer Complications: o Guillain-Barré syndrome (GBS) - 30% of GBS in U.S. Serotype O:19, antigenic cross-reactivity between Campylobacter oligosaccharides and glycosphingolipids on neural tissues o Reactive arthritis (HLA-B27) 78  CAMPYLOBACTER JEJUNI v Diagnosis: culture on Campylobacter or Skirrow agar at 42.0° C (107.6 F); microaerophilic v Treatment: mostly supportive, i.e., fluid and electrolyte replacement; erythromycin, fluoroquinolones; resistant to penicillins 79  HELICOBACTER PYLORI v Distinguishing Features: Gram-negative spiral gastric bacteria with flagella oxidase-positive, urease-positive microaerophilic grows at 37°C on Campy or Skirrow agar 80  HELICOBACTER PYLORI v Reservoir: humans v Transmission: fecal-oral; oral-oral v Pathogenesis Motile Urease-positive: ammonium cloud neutralizes stomach acid, allowing survival in stomach acid during transit to border Mucinase aids in penetration of mucous layer (rapid shift down to neutral as it penetrates) Invasive into stomach lining where pH is neutral Inflammation is prominent Two biotypes (I and II); type I produces vacuolating cytotoxin 81 HELICOBACTER PYLORI 82  HELICOBACTER PYLORI v Diseases: chronic gastritis and duodenal ulcers Associated with several forms of stomach cancer (gastric adenocarcinoma, gastric mucosa- associated lymphoid tissue lymphoma [MALToma], B-cell lymphomas) Now classed by WHO as type I carcinogen 83  HELICOBACTER PYLORI v Diagnosis Biopsy with culture; histology with Giemsa or silver stain Urea breath test: 13C-urea swallowed; ammonia+13C-CO2 exhaled Serology v Treatment: myriad of regimens Omeprazole + amoxicillin + clarithromycin is one example of triple therapy Treat for 10–14 days Quadruple therapy is used in areas where clarithromycin resistance is >/=15%, e.g., PPI + bismuth + 2 antibiotics (metronidazole + tetracycline) 84  VIBRIO SPECIES v Genus Features Gram-negative curved rod with polar flagella Oxidase positive Vibrionaceae Growth on alkaline, but not acidic, media (TCBS, thiosulfate citratebile salt sucrose medium) v Species of Medical Importance Vibrio cholerae Vibrio parahaemolyticus Vibrio vulnificus 85  VIBRIO CHOLERAE v Distinguishing Features: rice-water diarrhea; growth on TCBS v Reservoir Human colon; no vertebrate animal carriers (copepods or shellfish may be contaminated by water contamination) Human carriage may persist after untreated infection for months after infection; permanent carrier state is rare. v Transmission Fecal-oral spread; sensitive to stomach acid Requires high dose (>107 organisms), if stomach acid is normal 86  VIBRIO CHOLERAE v Pathogenesis Motility, mucinase, and toxin coregulated pili (TCP) aid in attachment to the intestinal mucosa. Cholera enterotoxin (choleragen) similar to E. coli LT; ADP ribosylates (Gs alpha) activating adenylate cyclase -> increased cAMP -> efflux of Cl- and H2O (persistent activation of adenylate cyclase) v Disease: cholera Rice water stools, tremendous fluid loss Hypovolemic shock if not treated 87  VIBRIO CHOLERAE v Diagnosis: culture stool on TCBS producing yellow colonies; oxidase positive v Treatment: fluid and electrolyte replacement; doxycycline or ciprofloxacin to shorten disease and reduce carriage; resistance to tetracycline reported v Prevention: proper sanitation; new vaccine 88 OTHER VIBRIO SPECIES 89  VIBRIO CHOLERAE v Distinguishing Features: rice-water diarrhea; growth on TCBS v Reservoir Human colon; no vertebrate animal carriers (copepods or shellfish may be contaminated by water contamination) Human carriage may persist after untreated infection for months after infection; permanent carrier state is rare. v Transmission Fecal-oral spread; sensitive to stomach acid Requires high dose (>107 organisms), if stomach acid is normal 90  CLOSTRIDIUM DIFFICILE v Reservoir: human colon/gastrointestinal tract v Pathogenesis Toxin A: enterotoxin damaging mucosa leading to fluid increase; granulocyte attractant Toxin B: cytotoxin: cytopathic v Disease(s): antibiotic-associated (clindamycin, cephalosporins, amoxicillin, ampicillin) diarrhea, colitis, or pseudomembranous colitis (yellow plaques on colon) v Diagnosis: culture is not diagnostic because organism is part of normal flora; stool exam for toxin production 91  CLOSTRIDIUM DIFFICILE v Treatment Metronidazole for severe disease; vancomycin only when no other drug is available to avoid selecting for vancomycin-resistant normal flora Fecal transplant for chronic infections Discontinuation of other antibiotic therapy for mild disease v Prevention: use caution in overprescribing broad- spectrum antibiotics 92  CLOSTRIDIUM PERFRINGENS v Distinguishing Features Large gram-positive, spore-forming rods (spores rare in tissue), nonmotile Anaerobic: “stormy fermentation” in milk media Double zone of hemolysis v Reservoir: soil and human colon v Transmission: foodborne and traumatic implantation 93  CLOSTRIDIUM PERFRINGENS v Pathogenesis Spores germinate under anaerobic conditions in tissue Vegetative cells produce alpha toxin (phospholipase C), a lecithinase, which disrupts membranes Enterotoxin produced in intestines in food poisoning: disrupts ion transport -> watery diarrhea, cramps (similar to E. coli); resolution watery diarrhea v Diseases Gastroenteritis: nonbloody, vomiting Eye infection (rare) 101  BACILLUS CEREUS v Diagnosis Clinical grounds Culture and Gram stain of implicated food v Treatment: self-limiting; vancomycin for eye infection 102  STAPHYLOCOCCUS AUREUS v Distinguishing Features Small, yellow Staphylococcus aureuscolonies on blood agar B-hemolytic Coagulase positive (all other Staphylococcus species are negative) Ferments mannitol on mannitol salt agar 103  STAPHYLOCOCCUS AUREUS v Pathogenesis Enterotoxins: fast acting, heat stable v Treatment Gastroenteritis is self-limiting. 104 STAPHYLOCOCCUS AUREUS 105  TYPHOID ASSAY (PRELAB DISCUSSION) Prepared by: FRITZ VON T. GELLA, RMT, MD College of Medicine Davao Medical School Foundation, Inc.  SALMONELLA ENTERICA TYPHI v Distinguishing Features Gram-negative rods, highly motile with the Vi capsule Facultative anaerobe, non–lactose fermenting Produces H2S Species identification with biochemical reactions Sensitive to acid v Reservoir: humans only; no animal reservoirs v Transmission: fecal-oral route from human carriers (gall bladder); decreased stomach acid or impairment of mononuclear cells as in sickle cell disease predisposes to Salmonella infection 107  SALMONELLA ENTERICA TYPHI v Pathogenesis and Disease: typhoid fever (enteric fever), S. typhi (milder form: paratyphoid fever; S. paratyphi) v Diagnosis: organisms can be isolated from blood, bone marrow, urine, and tissue biopsy from the rose spots if present; antibodies to O, Vi, and H antigens in patient’s serum can be detected by agglutination (Widal test) v Treatment: fluoroquinolones or third-generation cephalosporins 108  TYPHOID FEVER v also known as enteric fever, is a potentially fatal multisystemic illness usually through ingestion of contaminated food or water v characterized by prolonged fever, headache, nausea, loss of appetite, and constipation or sometimes diarrhea v Etiology: Salmonella enterica Subsp. enterica Serotype Typhi 109  TYPHOID FEVER v Taxonomy of Salmonella Family Enterobacteriaceae Genus Salmonella Species: S. enterica, S. bongori Salmonellaenterica subspecies: o I – enterica (the most common pathogen) o II - salamae o IIIa – arizonae(frequently found in cold-blooded o IIIb–diarizonaeanimals (reptiles); rare in humans) o IV - boutenae o VI - indica 110  TYPHOID FEVER v Microbiology of Salmonella Facultative anaerobe Non-spore forming, motile Differentiated from other Salmonella sp. being non-gas forming Differential media: EMB, McConkey, Deoxycholate, Bismuth sulfate (black colonies) Selective media: Salmonella-Shigella (SS), Hektoen Enteric agar, XLD (xylose-lysine decarboxylase), Deoxycholate citrate 111 TYPHOID FEVER Yellow or colorless due to absence of lactose fermentation 112 TYPHOID FEVER Black colonies with a metallic sheen formed due to production of H2S. 113 TYPHOID FEVER produce colonies with black centers 114 TYPHOID FEVER 115  TYPHOID FEVER 3 kinds of Antigens: H, O, Vi v H – Flagellar denatured by heat or alcohol preserved by treating motile bacterial variants with formalin aggregate with anti-H antibodies, mainly IgG the determinants of H antigens are a function of the amino acid sequence in flagellar protein (flagellin) 116  TYPHOID FEVER 3 kinds of Antigens: H, O, Vi v O– Somatic most external part of the cell wall lipopolysaccharide consist of repeating units of polysaccharide resistant to heat and alcohol usually detected by bacterial agglutination antibodies to O antigens are predominantly IgM 117  TYPHOID FEVER 3 kinds of Antigens: H, O, Vi Vi – capsular polysaccharide (limited to S. typhi & paratyphi) Heat-labile may interfere with agglutination by O antisera may be associated with virulence *H & O – common antigens in Gram negative bacilli *Vi – specific in Salmonella typhi and paratyphi 118  TYPHOID FEVER v Mode of Transmission Fecal – Oral o Foodborne o Water-borne o Person to person Role of carriers Risk Factors in Endemic Areas: o Eating food prepared outside the home (e.g. street vendors, ‘carinderia’) o Drinking contaminated water o Having close contact with recent typhoid fever patient o Poor housing facilities and personal hygiene o Recent use of antimicrobial drugs 119  TYPHOID FEVER v Pathogenesis Adherence to mucosal cells Incubation period: ↓ o 7 to 14 days (average) Invades mucosa and internalized into M o 3 to 60 days (range) cells overlying Peyer’s patches ↓ Lymphoid follicles and mesenteric nodes ↓ Released into the bloodstream (primary bacteremia) *concentrate in the lymph areas *1st symptom experienced: abdominal ↓ discomfort/ bloatedness RES of liver and spleen *organ system of choice: 120 Reticuloendothelial system  TYPHOID FEVER Adherence to mucosal cells ↓ Invades mucosa and internalized into M cells overlying Peyer’s patches ↓ Lymphoid follicles and mesenteric nodes ↓ Released into the bloodstream (primary bacteremia) ↓ RES of liver and spleen 121  TYPHOID FEVER v Pathogenesis Secondary Infection o liver, spleen, bone marrow, gall bladder, Peyer’s patches and terminal ileum o secondary bacteremia: any other organ system (e.g. Pneumonia) 122 TYPHOID FEVER 123  TYPHOID FEVER v Clinical Feature Fever - initially low grade, rises progressively; by the 2nd week it is often high and sustained (39 – 40 C) “Step ladder pattern of fever” Flu-like symptoms Chills, frontal headache Malaise, anorexia, nausea Poorly localized abdominal discomfort Dry cough, myalgia Bowel changes – constipation or diarrhea 124  TYPHOID FEVER v Physical Sign Tender abdomen Hepatomegaly Splenomegaly Rose spots o Salmon-colored, blanching, maculopapular rash located primarily on the trunk and chest o not common o seen in fair-skinned o evanescent 125  TYPHOID FEVER v Complications 3 most important complications: 1. GI bleeding – most common 2. Intestinal perforation - most serious 3. Encephalopathy - often accompanied by shock, is associated with a high mortality 126  TYPHOID FEVER v Laboratory organisms can be isolated from blood, bone marrow, urine, and tissue biopsy from the rose spots if present antibodies to O, Vi, and H antigens in patient’s serum can be detected by agglutination (Widal test) 127  TYPHOID FEVER v Laboratory Blood Culture - the gold standard Bone marrow culture - more sensitive; (+) yield even if the patient has been on antibiotic Stool culture - 2nd to 3rd week Urine, rose spots, blood-mononuclear platelet fraction Gastric/intestinal secretions More specific tests but impractical for developing countries: DNA probes Polymerase Chain Reaction 128  TYPHOID FEVER v Laboratory Widal’s Test o detects presence of antibodies to O and H antigens o antibodies that are produced in serum when exposed to bacterial suspensions containing homologous antigens, cause the formation of agglutination. The agglutination process is visible as clustering. o cross reactions with other Salmonella serotypes and other Enterobacteriaceae; ideally done by testing paired acute and convalescent sera at the same time, noting for a 4-fold rise in Ab titer o Previous infection or immunization may produce detectable antibodies o single serum specimen requires a higher antibody titer cut-off: O Ag - 1:320 H Ag - 1:640 129  TYPHOID FEVER v Laboratory Widal’s Test Two methods: o Rapid slide test: Rapid slide test may be qualitative or quantitative and is conducted on slides. o Tube test: It’s conducted in tubes, is quantifiable and requires an incubation period. It’s believed to be more precise than slide tests. o Positive: Agglutination occurs within one minute o Negative: No Agglutination 130  TYPHOID FEVER v Laboratory Widal’s Test Advantages: o It’s a fast test to determine the presence of enteric fever in regions with the highest incidence. o If culture facilities aren’t readily accessible Tests using Widal are helpful. o It is able to identify diseases due to Salmonella Typhi and Salmonella Paratyph. 131  TYPHOID FEVER v Laboratory Widal’s Test Disdvantages: o The Widal test can be lengthy (to detect the level of antibody) and, most of the time once the the diagnosis has been confirmed, it’s for too late to initiate an antimicrobial treatment. o Cross-reactivity can occur in other Salmonella species, it is not possible to differentiate between an ongoing illness and an infection that was present before or an earlier vaccine against Typhoid. 132  TYPHOID FEVER v Laboratory Typhidot Test o Principle: Enzyme Immunoassay o Immunochromatographic assay. A kit that makes use of antigen to detect IgM and IgG antibodies against the outer membrane protein (OMP) of the S. typhi. o The Typhidot test becomes positive within 2-3 days of infection and separately identifies IgM and IgG antibodies. o IgM recent infection; IgG past infection 133 TYPHOID FEVER 134  TYPHOID FEVER v Laboratory Tubex TF o Principle: detects the presence of anti-O9 antibodies in the patient's serum by assessing their ability to inhibit the reaction between the antigen-coated brown and antibody-coated blue reagents. The level of inhibition is proportional to the concentration of anti-O9 antibodies in the sample. Separation is enabled by magnetic force. Result is read visually against a color scale. 135 TYPHOID FEVER 136  TYPHOID FEVER v Laboratory Tubex TF o Principle: detects the presence of anti-O9 antibodies in the patient's serum by assessing their ability to inhibit the reaction between the antigen-coated brown and antibody-coated blue reagents. The level of inhibition is proportional to the concentration of anti-O9 antibodies in the sample. Separation is enabled by magnetic force. Result is read visually against a color scale. 137 TYPHOID FEVER 138 INTERPRETION INTERPRET INTERPRETION INTERPRET INTERPRETION  VACCINATION TYPHOID FEVER