Bacteriology Finals Lecture Outline


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  • enteric gram-negative rods , enteric bacteria , coliforms natural habitat – intestinal tract of humans, animals, & insects; either opportunistic or true pathogens large, heterogeneous group; well studied approximately 25 of the 97 described enterics – frequently isolated from clinical specimens
  • & may or may not produce gas in the process (aerogenic vs anaerogenic) colicins (bacteriocins)– high-molecular weight bactericidal proteins, controlled by plasmids; produced by E. coli, serratia, pseudomonas, bacteriocin-producing strains are resistant to their own bacteriocin; thus, bacteriocins can be used for “typing” of organisms
  • O Ag = most external part of the LPS;
  • Variation from smooth to rough colonial forms is accompanied by progressive loss of smooth O Antigen.
  • H Ag = denatured or removed by heat or alcohol Phase variation: 1 – designated by lower case letters, 2- arabic numerals
  • external to O antigens on some;
  • LIA – Lysine decarboxylase + (purple slant/purple butt = k/k)
  • movement of water and ions from the tissues to the bowel resulting in watery diarrhea (up to 20 liters a day) (stool looks like rice water like cholera)
  • LT – binds to specific Gm 1 gangliosides on the epithelial cells of the small intestine where ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP. Increased cAMP alters the activity of Na and Cl transporters producing an ion imbalance that results in fluid transport into the bowel. ST – binds to specific receptors to stimulate the production of cGMP with the same results as with LT
  • movement of water and ions from the tissues to the bowel resulting in watery diarrhea
  • to deliver effector molecules directly into the host cells
  • Antibodies formed protect from colonization
  • s/sx: urinary frequency, dysuria, hematuria, pyuria, flank pain (upper tract infxn) O antigen types, eleborate virulence factors that facilitate colonization = uropathogenic E. coli Produce hemolysin, cytotoxic, facilitates tissue invasion Strains that cause pyelonephritis express K antigen and elaborate specific type of pilus, P fimbriae, which binds to the P blood group antigen
  • Neonatal meningitis, septicemia – high mortality rate
  • ETEC – common cause of “traveler’s diarrhea” & diarrhea in infants in developing countries . characterized by a watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. Transmission is via contaminated food or water. EPEC – Bundle forming pili are involved in attachment to the intestinal mucosa. The type III secretion system inserts the tir (translocated intimin receptor) into target cells, and intimate attachment of the non-fimbrial adhesion called intimin to tir occurs. Host cell kinases activated to phosphorylate tir which then causes a reorganization of host cytoskeletal elements resulting in pedestal formation and development of an attaching and effacing lesion The exact mode of pathogenesis is unclear, but it is probably due to the attachment and effacement. 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 care centers. EIEC – 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.
  • EAEC – 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. Symptoms include watery diarrhea, vomiting, dehydration and occasional abdominal pain. EHEC – The organism attaches via pili to the intestinal mucosa and liberates the shiga-like toxin. Start with a watery diarrhea that progresses to bloody diarrhea without pus and crampy abdominal pain with no fever or a low-grade fever. May progress to hemolytic-uremic syndrome that is characterized by low platelet count, hemolytic anemia, and kidney failure. E. coli O1547:H7 = Differentiated from other strains of E. coli by the fact that it does not ferment sorbitol in 48 hours (other strains do).
  • Primarily pneumonia, wound, & UTI Tribe Klebsiellae
  • Permanent lung damage is a frequent occurrence, mostly in immunocompromised hosts. Rank among the top ten cause of nosocomial infections such as septicemia and meningitis Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia)
  • Enterobacter aerogenes – LD+, OD+ E. agglomerans – LD(-), AD(-), OD(-) E. cloacae – LD (-), AD(+), OD(+) depending upon species Nosocomial infections Pneumonia, UTI, wound & devices infections Bacteremia in burn patients
  • Hafnia alvei is only species Has been isolated from many anatomical sites in humans and the environment Occasionally isolated from stools Delayed citrate reaction is major characteristic
  • , grown on KCN, ferment xylose
  • RTIrespiratory tract infections
  • 4 species, differ antigenically, to a lesser extent, biochemically:
  • K antigens may interfere with serotyping, but are heat labile
  • Shiga toxin S. dysenteriae; S. flexneri & S. sonnei ( smaller amounts) inhibit protein synthesisinactivating 60S ribosomal subunit, cleaving a glycosidic bond in the 28S rRNA constituents → role in ulceration of the intestinal mucosa. Outer membrane and secreted proteins expressed at body temperature; upon contact with M cells in the intestinal mucosa - induce phagocytosis of the bacteria into vacuoles M cells (or microfold cells ) are cells found in the follicle-associated epithelium of the Peyer's patch . They transport organisms and particles from the gut lumen to immune cells across the epithelial barrier, and thus are important in stimulating mucosal immunity . Unlike their neighbouring cells, they have the unique ability to take up antigen from the lumen of the small intestine via endocytosis or phagocytosis , and then deliver it via transcytosis to dendritic cells (an antigen presenting cell ) and lymphocytes (namely T cells ) located in a unique pocket-like structure on their basolateral side. expressed at body temperature & upon contact with M cells in the intestinal mucosa induce phagocytosis of the bacteria into vacuoles
  • Destroy the vacuoles to escape into the cytoplasm Spread laterally by polymerization of actin filaments propels them through the cytoplasm) to epithelial cells where they multiply but do not usually disseminate beyond the epithelium.
  • Transmission - via the fecal-oral route. infective dose required to cause infection is very low (10 3 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). The severity of the disease depends upon the species one is infected with. S. dysenteriae is the most pathogenic followed by S. flexneri , S. sonnei and S. boydii . Tenesmus (rectal spasm)
  • IP incubation period Carrier State Organisms shed in feces Gallbladder is the site of organisms (removal of gallbladder may be the only solution to carrier state)
  • Protease = modifies the gastric mucus and reduces the ability of acid to diffuse through the mucus Urease = yields prodxn of ammonia, thus, buffering the acid in the mucosa.
  • (Lyme, Connecticut) Neurologic –meningitis, facial nerve palsy, painful radiculopathy, cardiac disease with conduction defects, myocarditis Deposition of antigen-antibody complex
  • See table 26-1 textbook, p 320
  • Bacteriology Finals Lecture Outline

    1. 1. MLS 302 BACTERIOLOGY Lecture 11Enteric Gram-Negative Rods (Enterobacteriaceae)
    2. 2. Enterobacteriaceae enteric gram-negative rods, entericbacteria, __________ intestinal tract of humans, animals, &insects; either opportunistic or true pathogens large, heterogeneous group; well studied approximately 25 of the 97 enterics –frequently isolated from specimens
    3. 3. Family EnterobacteriaceaeCertain E .coli strainscan be consideredtrue pathogens (nonmotile) (nonmotile) (nonmotile at 37°)
    4. 4. SITES OF INFECTIONS with members of theEnterobacteriaceae Some are enteric pathogens; others are urinary or respiratory tract pathogens; also cause __________ infections
    5. 5. Enterobacteriaceae Morphology and General CharacteristicsShort gram-negative rodsMotile (peritrichous flagella) or nonmotileFacultative anaerobes/aerobesFerment glucose, mostly aerogenicReduce nitrate to nitriteCatalase (+)Produce __________ (bacteriocins)
    6. 6. EnterobacteriaceaeDifferentiation of species:1.Typical colonies (selective & differential) – Recall Mac, EMB, TCBS, HEA, SSA, XLD – smooth, mucoid, swarming, pigment, etc. – large & dull gray on blood agar (BA); may or may not be hemolytic – Selenite-F, APW = enrichment (fecal sample)
    7. 7. Rapid, Presumptive Identification of Gram-Negative Enteric BacteriaLactose fermented rapidly Escherichia coli: metallic sheen on EMB; motile, flat, nonviscous colonies Enterobacter aerogenes: raised colonies, no metallic sheen; often motile, more viscous growth Klebsiella pneumoniae: very viscous, mucoid growth; nonmotileLactose fermented slowlyEdwardsiella, Serratia, Citrobacter, Arizona, Providencia, Erwinia
    8. 8. Rapid, Presumptive Identification of Gram-Negative Enteric Bacteria• Lactose not fermented • Shigella species: nonmotile; no gas from dextrose • Salmonella species: motile; acid and usually gas from dextrose • Proteus species: “swarming” on agar; urea rapidly hydrolyzed • Pseudomonas species: soluble pigments, blue-green & fluorescing; sweetish smell
    9. 9. Enterobacteriaceae2. Biochemical reaction patterns: – Carbohydrate fermentation patterns (TSI) – Activity of amino acid decarboyxlases – Other enzymes (eg, urease) or products – TSI, LIA, urea or phenylalanine agar = 3 useful media for screening stool cultures (see Table 15-1, page 215, Jawetz’s Med Micro book)
    10. 10. Enterobacteriaceae3. Antigenic structures - 3 major classes:
    11. 11. Enterobacteriaceae3. Antigenic structures - 3 major classes:1. O antigens (somatic O - LPS) >150 different heat-stable; heat & alcohol __________; detected by bacterial agglutination; antibodies to O Ag predominantly IgM
    12. 12. Enterobacteriaceae3. Antigenic structures - 3 major classes:2. H antigens >50 H (flagellar) antigens heat & alcohol __________; antibodies to H Ag (flagellin) mainly IgG; some undergo phase variation; may interfere with agglutination by anti-O antibody
    13. 13. Enterobacteriaceae3. Antigenic structures - 3 major classes:3. K antigens (envelope/capsular) >100 heat-labile K polysaccharides or proteins may interfere with agglutination by O antisera; boil for 15 min. to unmask O Ag associated with virulence (K1) Salmonella typhi capsular antigens = __________
    14. 14. Escherichia coliNormal inhabitant of the •Dry, pink (LF)G.I. tract. colony withMost significant species surrounding pinkin the genus area onCommon isolate from MacConkeycolon flora •May be hemolyticIndex organism for fecal on BA – morecontamination of water common in __________ pathogenic strainsEMB: Greenish metallic sheen; iridescent “sheen” (urine isolates)
    15. 15. Escherichia coli – KEY tests for the normal strain: • TSI - A/A gas+ • Lysine decarboxylase +ve • Mannitol fermenter • Motile • Indole +ve • ß-glucuronidase + (>90%) (substrate 4-methylumbelliferyl- ß- glucuronide, MUG) • Urea –ve • __________ – IMViC: ++--
    16. 16. E. coli Virulence FactorsLipid A component of the LPS (endotoxin)= cause of death in sepsis (septic shock)K1 antigen= strong association with neonatal meningitisEnterotoxins (exotoxin)= cause severe watery diarrhea (ETEC strain)= 2 types: LT (heat labile), ST (heat stable)
    17. 17. E. coli Virulence Factors LT vs ST activity Cyclic guanosine monophosphate • Inhibit the reabsorption of Na+ & Cl- • Stimulate the secretion of H2O, Cl- & HCO3- into the lumen Cyclic adenosine monophosphate
    18. 18. E. coli Virulence Factors Shiga-type toxins 1 & 2 (verotoxin) = STEC or EHEC strain = inhibit protein synthesis (60S subunit), cytotoxic = STEC 0157:H7 Enteroaggregative ST-like toxin = EAEC strain
    19. 19. E. coli Virulence Factors Hemolysins__________ secretion system to deliver effector molecules induce uptake of EIEC into host cells effacing lesion in EPEC characterized by microvilli destruction and pedestal formation
    20. 20. E. coli Virulence FactorsAdhesins (colonization factors)= include pili or fimbriae & nonfimbrial factors (eg intimin); different types= Antibodies protect from colonization•Capsule• Iron capturing ability(enterochelin)• Outer membrane proteins
    21. 21. E. coli Infections URINARY TRACT INFECTIONS – (asleading cause) → lead to acute cystitis(bladder infection) and pyelonephritis(kidney infection)
    22. 22. E. coli InfectionsNEONATAL MENINGITIS & SEPTICEMIA– strains with the K1 Ag (usually)GASTROENTERITIS1.enterotoxigenic E. coli (ETEC)2.enteroinvasive E. coli (EIEC)3.enteropathogenic E. coli (EPEC)4._______________________________5.enterohemorrhagic E. coli (EHEC)
    23. 23. E. coli GASTROENTERITIS
    24. 24. E. coli GASTROENTERITISETEC– common cause of “_____________” & diarrhea in infants in developing countriesEPEC– diarrhea with large amount of mucous without blood or pus, with vomiting, malaise & low gradeEIEC– fever, severe abdominal cramps, malaise, watery diarrhea followed by scanty stools with blood,mucous, and pus
    25. 25. E. coli GASTROENTERITISEAEC– watery diarrhea, vomiting, dehydration, occasional abdominal painEHEC– watery diarrhea that progresses to bloody diarrhea no pus; crampy abdominal pain– progression to hemolytic-uremic syndrome* (HUS) *thrombocytopenia, hemolytic anemia, renal failure– E. coli O1547:H7 does not ferment __________ in 48 hours (other strains do)
    26. 26. E. coli gastroenteritis - EHEC• Sorbitol-MacConkey (SMAC) plate – contains sorbitol instead of lactose; selective• Confirm E. coli O1547:H7 isolate by serological testing then confirm production of the shiga- like toxin before reporting out results
    27. 27. EHEC/ E. coli 0157:H7
    28. 28. E coli Gastroenteritis (one more time)
    29. 29. Escherichia coli Treatment Sulfonamides, ampicillin, cephalosporins, fluoroquinolones, and aminoglycosides Multidrug resistant strains - common (transmissible plasmids) Susceptibility testing - essential
    30. 30. Klebsiella-Enterobacter- Serratia-Hafnia speciesWide variety of infectionsGeneral characteristics:– Simmons citrate positive– H2S negative– Phenylalanine deaminase negative– Some weakly urease positive– MR negative; __________
    31. 31. Klebsiella (most common)• K. pneumoniae subsp. pneumoniae – (-) indole, (+) citrate, urease, malonate – TSI A/A + gas – LIA K/K – MR-, VP+ – Nonmotile – Has O and K antigens• Klebsiella oxytoca – __________, citrate, urease, malonate
    32. 32. KlebsiellaClinical significance Pneumonia, permanent lung damage (immunocompromised) UTI, bacteremia, & other nosocomial infections K. pneumoniae subsp. ozaenae – ozena (nasal mucosa) K. pneumoniae subsp. rhinoscleromatis – rhinoscleroma K. granulomatis (formerly __________) – chronic genital ulcerative disease Drug resistance (ESBL)
    33. 33. Enterobacter speciesCharacteristics TSI, LIA, and urea (species) Citrate +, motile, large gas producersClinical significance • Nosocomial infections • Pneumonia, UTI, wound & devices infections • Ampicillin & cephalosporin resistant strains (ESBL)• E. agglomerans; E. cloacae, E. aerogenes, E. sakazakii (the latter 3 now genus Cronobacter)
    34. 34. Serratia• Serratia marcescens – A free-living saprophyte – LIA usually K/K produce __________ (red pigment) – (+) DNAse, lipase, gelatinase – TSI A/A or K/A; +/- gas, NLF – Citrate +, Motile – Has been found in RT and UT infections – Is resistant to many antimicrobials
    35. 35. Hafnia• Hafnia alvei - only species• Disease: – Gastroenteritis • Pathogenesis: adhesions to epithelial cells• biochemically similar to the Enterobacter• makes the scent of human feces: skatole• Delayed citrate reaction is major characteristic.
    36. 36. Proteus, Providencia, Morganella All motile, with Proteus swarming, NLF PEA, CLED Phenylalanine deaminase + Lysine deamination + (LIA R/A) Urease (+) except Providencia alcalifaciens Indole (+) except __________ Only Proteus is H2S(+)
    37. 37. Proteus, Providencia, and Morganella Virulence factor • Urease – the ammonia produced may damage the epithelial cells of the UT (Proteus) Clinical Significance Infections: UTI, bacteremia, pneumonia, focal lesions Highly alkaline urine – prone to stone formation
    38. 38. Proteus species• P. mirabilis and P. vulgaris – recognized human pathogens• Isolated from urine, wounds, and ear and blood infections
    39. 39. Morganella speciesMorganella __________- only speciesDocumented cause of UTIIsolated from other anatomical sitesUrease positivePhenylalanine deaminase positive citrate(-)
    40. 40. Providencia speciesProvidencia rettgeri pathogen of urinary tract nosocomial outbreaksProvidenicia stuartii nosocomial outbreaks in burn units has been isolated from urineBoth phenylalanine deaminase positive
    41. 41. Citrobacter(C. freundii, C. diversus, C. amalonaticus)TSI K/A or A/A both + gas, H2S+LIA K/A + H2SUrea usually + (slowly)Citrate +, motileResemble __________Opportunistic pathogens UTI, RTI, occasionally wound infections, osteomyelitis, endocarditis, and meningitis
    42. 42. Edwardsiella• Edwardsiella tarda – TSI K/A + gas and H2S – LIA K/K +H2S – Urea – – Citrate – – Indole + – Clinical significance – causes GI disease in tropical and subtropical countries
    43. 43. Shigella species4 pathogenic Group Mannitol Ornithine species DecarboxylaseS. dysenteriae AS. flexneri BS. boydii CS. sonnei D
    44. 44. Shigella speciesBiochemistry TSI: K/A with NO gas NON-MOTILE Ferment glucose Non-Lactose fermenters S. sonnei may show delayed lactose fermentation All ferment mannitol except S. dysenteriae Circular, convex, transparent colonies
    45. 45. Shigella speciesAntigenic structures A, B, C, and D grouping based on O antigen serotyping K antigens may interfere w/ serotyping O antigen is similar to E. coli important to identify as Shigella before doing serotyping
    46. 46. Shigella Virulence FactorsEndotoxinShiga exotoxin S. dysenteriae type 1 (Shiga bacillus) S. flexneri & S. sonnei (smaller amounts) inhibit protein synthesis → role in ulceration of the intestinal mucosaOuter membrane and secreted proteins induce phagocytosis of the bacteria into vacuoles (M cells)
    47. 47. Shigella attachment and penetration Destroy the vacuoles to escape into the cytoplasm Spread laterally by actin polymerization Multiply but do not usually disseminate beyond the epithelium
    48. 48. Shigella Clinical Significance Shigellosis or bacillary dysentery ________ infective dose (103 organisms) “food, fingers, feces, and flies” transmission Short incubation (1-2 days) Fever, cramping, abdominal pain, and watery diarrhea Followed by frequent, scant stools with blood, mucous, and pus with straining & tenesmus
    49. 49. Salmonella-ArizonaComplex Classification:• 1 significant species with 5 subspeciesSalmonella enterica subspecies: I – enterica = human infections II – salamae III – arizonae IV – houtenae VI - indica• reported out as serotypes based on the Kauffman-White scheme of classification
    50. 50. Salmonella• Kauffman-White scheme of classification – Based on O and H (flagella) antigens – Salmonella typhi - Vi capsular antigen – Polyvalent antisera is used followed by group specific antisera.• Salmonella enterica subsp. enterica ser. – A = Paratyphi A – B = Paratyphi B – C1 = Choleraesuis – D = Typhi How to write: ____________________
    51. 51. non-lactose fermenting Salmonella colonies with black centersBiochemistry S. typhi: TSI K/A gas (-) & moustache H2S, IMViC -+--, LD (+), AD (-), OD (-) Others: K/A gas(+), H2S (+), IMViC -+-+, LD (+) S. paratyphi A produces no H2S, LD (-). Motile, NLF Selenite F, tetrathionate broth, SSA, XLD Serological typing (rapid slide agglutination)
    52. 52. Other Diagnostic Tests• Fluorescent antibody technique• WIDAL TEST (Tube dilution agglutination) – High or rising titer “O” >1:320 against H antigen of >1:640 = considered positive – High titer “H” suggests past vaccination or past infection – High titer “Vi” occurs in carrier state• ELISA type kit (monoclonal antibodies), DNA probes in kit form
    53. 53. Typhidot• Rapid detection of specific IgM and IgG antibodies to the OMP of S. typhi• IgM (+) only or IgM (+) & IgG (+) = acute typhoid• IgG (+) only = persistent typhoid, relapse, or reinfection, convalescent, carrier state, previous typhoid• IgM(-) IgG(-) = not typhoid fever
    54. 54. Salmonella Virulence factors– Endotoxin – role in intracellular survival– Capsule (S. typhi, S. paratyphi strains)– Adhesions – fimbrial and non-fimbrial– Type III secretion systems and effector molecules (survive inside macrophages)– Outer membrane proteins– Flagella –move through intestinal mucous– Enterotoxin– Iron capturing ability
    55. 55. Salmonella Invasion
    56. 56. SalmonellaClinical diseases Enteric fevers 7-20 days IP Fever gradual then high plateau, “typhoidal” Early constipation, later bloody diarrhea Septicemias Rapid rise of fever then spiking ‘septic’ temperature Enterocolitis Low-grade fever Nausea, vomiting, diarrhea
    57. 57. Diagnosis of Typhoid FeverBlood cultures: positive the 1st week & afterthe 2nd weekStool cultures: positive after the 2nd weekWidal test: look for a __________ rise in titerbetween acute and convalescent stages10% of those infected become short-termcarriers and a smaller % become long-termcarriers due to persistence of the bacteria inthe gallbladder or urinary bladder.
    58. 58. Salmonella– Antimicrobial therapy • Enteric fevers – chloramphenicol usually, ampicillin, trimethoprim-sulfamethoxazole, or third-generation cephalosporins • Resistant strains have emerged. • Gastroenteritis – usually does not require antimicrobial therapy – Replace lost fluids and electrolytes
    59. 59. • Pseudomonad Group 1. Pseudomonas aeruginosa 2. Burkholderia pseudomallei 3. Burkholderia mallei 4. Burkholderia cepacia 5. Stenotrophomonas maltophilia 6. Other pseudomonads
    60. 60. LEARNING OBJECTIVESAt the end of this unit, the student is able to:1. Characterize the significant organism under Pseudomonad group.2. Explain the pathophysiology of diseases caused by significant Pseudomonads;3. Distinguish features of the Pseudomonas aeruginosa for accurate laboratory diagnosis;4. Recognize their medical implications of significant Pseudomonads to public health.
    61. 61. Pseudomonad Group• Gram negative, motile, aerobic rods• Some may produce water-soluble pigments.• Widely distributed in soil, water, plants & animals• Classification is based on rRNA/DNA homology and common cultural cx.
    62. 62. Classification of some medically important Pseudomonads:rRNA Homology Genus and SpeciesGroup & subgroupI. Fluorescent Group P. aeruginosa Nonfluorescent Group P. fluorescens P. putida P. stutzeri P. mendocinaII. B. pseudomallei B. mallei B. Ccpacia Ralstonia pickettiiIII. Comamonas species Acidovorax speciesIV. Brevundimonas speciesV Stenotrophomonas maltophilia
    63. 63. Pseudomonas aeruginosa• Colonizes human – GIT and human skin• Major human pathogen in the group.• Invasive and toxigenic – Causes infxn among people with abnormal host defenses – Nosocomial pathogen = moist environment
    64. 64. Pseudomonas aeruginosa: Morphology and Idenification• Motile Gram (-) bacilli/rod (0.6 x 2 um)• Arrangement: single, pairs or short chains• CULTURE: – Obligate aerobe and grows in ordinary media – Odor: Sweet /grape like or corn taco-like – Colony: smooth, round w/ fluorescent greenish color ; BAP: some hemolyze blood – Pigments: pyocyanin (blue); pyorubin (dark red); pyomelanin (black pigment); pyoverdin (green)
    65. 65. Pseudomonas aeruginosa: Morphology and Idenification• Growth Cx: – Grows at 37-42°C – Ability to grow at 42 °C, differentiate it from other fluorescent Pseudomonas spp. – Oxidase positive – Do not ferment CHO but most strains oxidize glucose
    66. 66. Oxidation-Fermentation(O-F)media:• Conventional Fermentation Medium (CFM) with 1% peptone• Hugh & Leifson’s oxidative-fermentative (0.02% low peptone) medium• Principle: – To determine the oxidative or fermentative metabolism of a carbohydrate. The OF test is used to determine whether a bacterium has the enzymes necessary for the aerobic breakdown of glucose (ie oxidation) and/or for the fermentation of glucose.
    67. 67. Method• Inoculate two tubes of OF medium for each organism being tested.• Overlay one tube only with sterile paraffin oil/petroleum jelly (vaspar) to exclude all oxygen.• Tubes are incubated at 37C for 48 hrs and results read by noting the color of the bromthymol blue indicator.• If glucose is used, acid end-products are generated, the pH is lowered and the bromthymol blue indicator turns yellow.
    68. 68. Oxidative-Fermentation Reaction• Fermentative Organisms – acid in both tubes – fermentation occurs in closed environment• Oxidative organisms – organisms can also oxidize glucose – Oxidation occurs in an open environment• Nonoxidizer – do not utilize glucose in either tube – produce alkaline reaction
    69. 69. OPEN CLOSED RESULTYellow GreenYellow YellowGreen Green
    70. 70. Pseudomonas aeruginosa: Antigenic Structures and Toxins • Adhesins – fimbriae (N-methyl-phenylalanine pili) – polysaccharide capsule (glycocalyx) – alginate slime (biofilm) • Motility/chemotaxis – flagella • Bacteriocin (pyocin) • Enzymes: – Elastase, proteases, 2 hemolysins (heat labile phospholipase C and heat stable glycolipid) • Exotoxin A – tissue necrosis (blocks CHON synthesis) • Endotoxin – fever, shock….DIC and RDS
    71. 71. Pseudomonas aeruginosa: Clinical Findings• Wound and burn infection = blue green pus• Meningitis = lumbar puncture• UTI = catheters and or other instruments• Necrotizing pneumonia = contaminated respirators• Mild otitis externa = swimmers & diabetic pts.• Eye infection• Pseudomonal sepsis – Ecthyma gangrenosum
    72. 72. Pseudomonas aeruginosa: Diagnostic Laboratory Test• Specimens: – Skin lesions, pus, urine, blood, spinal fluid, sputum, and others• Smears: – Gram (-) rods• Culture: – BAP and differential media(Mac) – Mac Conkey: NLF• Significant test: – Oxidase test – Oxidative Fermentation Reaction – Pigment production
    73. 73. Pseudomonas aeruginosa: TREATMENT• Should not be treated with single drug therapy because... – Success rate is slow – Bacteria rapidly develops resistance• Penicillin based drug (ticarcillin or piperacillin) in combination with aminoglycoside (tobramycin)• Others: aztreonam, imipenem, newer quinolones (Ciprofloxacin), Cephalosporins (Ceftazidime• Susceptibility test should be done
    74. 74. ASSIGNMENT:• Make a table of comparison of the following organisms: 1. Burkholderia pseudomallei 2. Burkholderia mallei 3. Burkholderia cepacia 4. Stenotrophomonas maltophiliaBase on: Gram Staining, Cultural Characteristics, Motility, growth at 42, oxidase test, CHO utilization, specific tests for identification, disease caused
    75. 75. VibriosCampylobacters Helicobacter
    76. 76. At the end of this unit, the student is ableto:1. Characterize the significant organism under LEARNING OBJECTIVES Vibrio, Campylobacter, and Helicobacter.2. Explain the pathophysiology of diseases caused by Vibrio, Campylobacter, and Helicobacter.3. Distinguish features of the Vibrio, Campylobacter, and Helicobacter for accurate laboratory diagnosis;4. Recognize their medical implications of significant Vibrio, Campylobacter, and Helicobacter to public health.
    77. 77. The Vibrio• Found in marine and surface waters• Comma-shaped, curved aerobic rods (2-4 um long) – Prolonged culture: straight rods – Motile w/ polar flagellum
    78. 78. Medically Important VibriosORGANISM HUMAN DISEASEV. cholerae serogroups o1 and Epidemic & pandemico139 choleraV. cholerae serogroups non- Cholera-like diarrhea; mildo1/non-o139 diarrhea; rarely extraintestinal infxnV. parahaemolyticus Gastroeteritis, possible extraintestinal infxnOthers: V. mimicus, V. hollisae,V. Diarrheafluvialis Wound infxn, bacteremia, V. vulnificus gastroenteritis Wound infxn, bacteremia, V. damsela, V. Ear, wound, soft tissue, andanginolyticus, other extraintestinal infxn V. metschnikovii
    79. 79. Vibrio cholerae Morphology and Identification• Microscopy: Grm (-) motile comma shape rods• Culture: – convex, smooth, round colonies that are opaque and granular.• Oxidase positive• Grow best at 37°C on TCBS (Thiosulfate-citrate- bile-sucrose) agar = yellow colonies• Grow at very high pH (8.5-9.5) = killed by acid• Endemic areas: requires direct culture of stool – TCBS – selective medium – APW – enrichment medium
    80. 80. Vibrio cholerae Morphology and Identification• Growth Characteristics: – Ferments sucrose and mannose but not arabinose – Positive oxidase test is the key step – Vibrios are susceptible to compound O/129 (2,4- diamino-6,7-diisopropylpteridine phosphate) = differentiates them to aeromonas (resistant) – Most are Halotolerant (NaCl stimulates their growth) • Growth on 6% Nacl = diff vibrio from aeromonas
    81. 81. Vibrio cholerae Antigenic Structure & Biologic Classification• Many vibrios has a single heat-labile flagellar (H) antigen = poorly immunogenic• Contains O LPS = serologic specificity – At least 139 O antigens known – Strains that contain O1 and O139 = classic cholera – Non-O1/non-O139 = cholera like dse• V. Cholerae serogroup O1 has determinants for further typing: – Ogawa, Inaba, Hikojima
    82. 82. Vibrio cholerae Antigenic Structure & Biologic Classification• 2 Biotypes of Epidemic V. cholerae : – El Tor – (agglutinates chicken RBC, hemolytic, VP +, Polymyxin resistant) – Classical - (opposite of El Tor)• V. cholerae O139 is very similar to V. cholerae O1 El Tor biotype
    83. 83. Vibrio choleraeAntigenic Structure & Biologic Classification• V. Cholerae & related Vibrios – Produces heat labile enterotoxin (A & B subunits) – A subunit causes Î level of cAMP = hypersecretion of water and electrolytes – Diarrhea (20-30 L) = dehydration, shock, acidosis, and death.
    84. 84. Vibrio cholerae Pathogenesis & Pathology• V. Cholerae is pathogenic only for humans• Normal gastric acidity – need > 1010 or more V. Cholerae in water to become infected = Organism is susceptible to acid – < 102 – 1o4 organism in food = buffering capacity of food – Medication or condition that decreases stomach acidity = predisposes a person to cholera
    85. 85. Vibrio cholerae Pathogenesis & Pathology• Cholera – non-invasive infxn (- bloodstream) – s/s: nausea, vomiting, profuse diarrhea & abdominal cramps – Rice water stool: mucus, epithelial cells, and î no of vibrios – El Tor causes milder disease than classic biotype.
    86. 86. Vibrio cholerae Diagnosis:• SPECIMEN: mucus from stool• SMEAR: Grm (-) comma shape rods – Dark field or phase contrast microscopy = motility• CULTURE: – Rapid growth in peptone agar, BAP w/ pH 9, TCBS – TCBS – see typical colonies
    87. 87. Vibrio cholerae Treatment:• Water and electrolyte replacement• Oral tetracycline – Note: some V. Cholerae acquired resistance already (transmissible plasmids)
    88. 88. Vibrio parahaemolyticus, Vibrio vulnificus V. V. vulnificus V. cholerae Parahaemo lyticusHalophilic Yes (10%) Yes (5%) No (halotolerant only)Disease gastroenteritis Severe wound infection Cholera Bacteremia Possible gastroenteritisCause Ingestion of Infected oyster Contaminated contaminated food & water seafoodsTCBS Green colonies Blue green colonies YellowOxidase positive positive positiveKanagawa + (detect - -test pathogenic(haemolysin) strain)
    89. 89. CAMPYLOBACTER• Campylobacter jejuni HUMAN PATHOGENS• Campylobacter coli =DIARRHEA• Gram (-) rods with comma, S, or “gull- wing” shapes• Motile w/ single polar flagellum
    90. 90. Campylobacter jejuni & C. Coli Laboratory Diagnosis:• SPECIMEN: STOOL• SMEAR: “gull-wing” shaped rods• CULTURE: – Selecteive media is used • Skirrow’s medium (w/ Vancomycin, polymyxin B, trimethoprim) = Colorless or gray, maybe watery or spreading – Incubated at reduced atmospheric Oxygen (5% O2) w/ 10% CO2 = anaerobic incubation jar – Temperature for incubation: 42°C • Prevent growth of enteric bacteria
    91. 91. Campylobacter jejuni & C. coli• Clinically Indistinguishable• Catalase & oxidase positive• Do not oxidize & ferment CHO
    92. 92. Campylobacter jejuni & C. Coli Pathogenesis• Virulence Factor: – LPS w/ endotoxic activity – cytopathic toxin and enterotoxin• MOT: oral route from contaminated food & water• Organism multiplies in SI invade epithelium inflammation rbc , wbc in stool – Sometimes bloodstream is invaded• s/s: crampy abdominal pain, profuse diarrhea w/ blood, headache, malaise & fever• Self-limiting (5-8 days)• Susceptible to erythromycin
    93. 93. HELICOBACTER• Helicobacter pylori – Agent of antral gastritis, duodenal peptic ulcer, gastric ulcer & gastric carcinoma• Characteristics: – Spiral shaped microaerophilic gram (-) rod – Mutiple flagella at one pole – can’t grow at 42C – Oxidase and catalase positive; nitrate (-) – Strong producer of urease
    94. 94. Helicobacter pylori Pathogenesis• H. pylori grows at pH 6.0- 7.0• Dies in pH within the gastric lumen• H.pylori thrives deep in the epithelial surface (suitable pH)• H.pylori produces protease & urease• Bacteria invade the epithelial surface releases toxins and LPS damage the mucosal cells
    95. 95. Laboratory Diagnosis• MICROSCOPY: – Special silver stain or Modified Giemsa = spiraled organism• CULTURE: – on selective media at 37ºC, in a microaerophilic atmosphere• BIOCHEM: – show strong & very rapid urea production
    96. 96. DIAGNOSIS• Blood = determination of serum antibodies• Stool antigen tests• Carbon urea breath test• Endoscopy (to provide a biopsy sample for testing for:) • “rapid urease test" • histology • microbial culture
    97. 97. CHEMOTHERAPY• Triple therapy – Metronidazole + bismuth subsalicylate or bismuth subcitrate + amoxycillin or tetracycline – 14 days• Acid suppressing agent given 4-6 wks – ulcer healing• Proton pump inhibitor – directly inhibits H. pylori and inhibits urease production
    98. 98. Haemophilus, Bordatella, Brucella, & Francisella
    99. 99. I. Haemophilus Species:• Pleomorphic Gram negative coccobacilliCulture• Requires enriched media (blood/blood products)• IR: 35°C or 37°C w/ 5-10% CO2• Species: – H. influenzae type b – H. ducreyi
    100. 100. Haemophilus Species:• Blood loving• Growth Requirement: – Hemin / hematin • Heat stable • Released during hemoglobin degradation – Nicotinamide-adenine dinucleotide (NAD) • Heat labile • Produced by certain bacteria & yeast or exogenous sources (potato or yeast extracts)
    101. 101. ISOLATION of Haemophilus:• SBA – not recommended for the isolation H. influenzae – Due to its requirement for NAD – Sheep RBC releases NADase – Except: w/ colonies of Staphylococci (grow)• Best: Horse or Rabbit blood – Preferably: Chocolate agar – Heating RBC to at least 80°C – To release hematin
    102. 102. Ways of Isolating Haemophilus:1. Staphylococcus Streak – PRINCIPLE: • SBA  SAU (synthesize NAD)  Haemophilus spp requiring V factor  forms small “dewdrop” colonies • When grown on SBA, beta hemolytic strain of S. aureus release X factor (hemin) as the red cells are hemolyzed.
    103. 103. 2. Horse blood-bacitracin agar – Bacitracin: inhibits the NF – Observe for hemolyitc reactions (H. haemolyticus & parahaemolyticus)3. Levinthal agar – Clear medium containing X & V factors – Encapsulated H. influenzae appears iridescent – Non-encapsulated strain appear as transparent, bluish & noniridescent.
    104. 104. 4. use of X and V Factors – Purpose: • Speciation of Haemophilus (basis: X and V) – Principle: • Use MH agar • Test for X & V requirements • Check for growth
    105. 105. • RESULTS & INTERPRETATION: Haemophilus spp XV X V Hemolysis (BAP)Influenze (H. aegyptius) + - - -parainfluenzae + - + -haemolyticus + - - +parahaemolyticus + - + +ducreyi + + - -
    106. 106. 5. Delta-aminolevulinic acid test (ALA porphyrin test) – Determine the requirement for X factor – H. parainfluenzae are ALA positive (does not require exogenous X factor) • possess the nz to convert ALA to porphobilinogen & porphyrin to hemin (X factor) – Species that require exogenous X factor such as H. influenzae are ALA negative
    107. 107. Pathogenecity of Haemophilus species (Diseases) Species DiseaseH. influenzae capsulated Meningitis, arthritis, osteomyelitis, (mainly type a, b) cellulitis, epiglottitis, bacteremiaH. influenzae non- Exacerbations of chronic bronchitis, capsulated sinusitis, otitis media, others.H. aegyptius ConjunctivitisH. ducreyi Chancroid (Sexually Transmitted)H. parainfluenzae Commensals of the upperH. Haemolyticus Respiratory tract; rarelyH. Parahaemolyticus Cause disease in immunocompromised patients
    108. 108. Haemophilus influenzae• Habitat: – URT of humans (nonencapsulated strains)• Most important pathogenic species: – meningitis (children) & RTI in children & adults• Characteristics: 1. Gram – negative coccobacilli 2. Grows on chocolate agar (smooth drop like colonies) 3. Needs X and V factors for growth on nutrient agar 4. Shows satelletism near Staphyloccocus aureus colonies 5. Shows pleomorphism from clinical specimens 6. Divided into Capsulated and Non-capsulated strains.
    109. 109. Haemophilus influenzae Encapsulated Strainsa) Capsule is made of Polysaccharidesb) It is antigenicc) It increases the pathogenecity of strainsd) Prevents phagocytosise) 6 Serotypes a b c d e ff) Identified through TYPING
    110. 110. Antigenic Structure:• Caplsular Polysaccharides• Somatic antigen – Outer membrane proteins – Lipooligosaccharides (endotoxin)
    111. 111. H.influenzae invasive InfectionsInclude: a) Meningitis b) Epilottitis c) Cellulitis d) Arthritis e) Osteomyelitis• Usually accompanied with bacteriaemia• Usually caused by type b• Major virulence factor is polysaccharide capsules• Affects children between 5 months to 5 years.• Infants under age 3 months (maternal IgG
    112. 112. Specimens depends on the type of diseases a. CSF b. Blood c. Sputum d. Swab e. Synovial fluid• Direct smear shows pus cell + pleomorphic GN coccobacilli• Culture on chocolate agar• detection for presence of capsular antigens by agglutination
    113. 113. Immunization• Early preparations consists of purified type b polysaccharide vaccines. But found not to be effective in children ↓ 18 months old.• Haemophilus b conjugate (HbOC) vaccine which consist of polysaccharide combined with one or two carrier molecule: – HbOC w/ protein carrier CRM197 mutant C. diphtheriae toxin protein; or N. meningitidis outer membrane complex – This vaccine is protective for children younger than 2 years.
    114. 114. Treatmenta) Ampicillin (amoxycillin) if not β lactamase Producer * β lactamase producer it destroy ampicillin and amoxycillinb) Cephalosporinsc) Cefotaxime (intravenous for excellent treatment)d) Chemoprophylaxis: Contacts of patient with H. influenzae type b infection can be given Rifampicin for 4 days as prophylaxis.
    115. 115. Other Haemophilus spp.• Haemophilus aegyptius • Haemohilus – Conjunctivitis, Brazilian parainfluenzae purpuric fever – Requires V factor only• Haemophilus aphrophilus – Infective endocarditis & – Infective endocardidtis & urethritis pneumonia • Haemophilus suis• Haemophilus ducreyi – Diseases in hogs – Causes soft chancre • Haemophilus – Requires X factor only haemoglobinophilus• Haemophilus haemolyticus – Requires X factor – Requires X & V – Found in dogs but not – Markedly hemolytic humans organism
    116. 116. The BordatellaeB. pertussis – causes WHOOPING COUGHB. parapertussis – causes a milder form of whooping cough.B. Bronchiseptica (B. bronchicanis) – causes kennel cough in dogs, snuffles in rabbits; pertussis like illness in humansB. avium – causes turkey coryza w/ no human infection.
    117. 117. Bordatella pertussisMicroscopy:• Small GN cocobacilli• Has a bipolar metachromatic granules seen in toluidine blue stain• EncapsulatedCulture:• Bordet-Gengou medium w/ Penicillin G• Incubated at 35-37C for 3-7 days in moist environment• non-motile• Strict aerobes; forms acid but not gas from glucose & lactose
    118. 118. Bordatella pertussis• Possess: – heat stable endotoxin LPS – Exotoxins:• Filamentous hemagglutinin• Pertussis toxin / Adenylate cyclase toxin:• Tracheal cytotoxin• Dermonecrotic toxin
    119. 119. Pathogenesis• Non invasive disease (do not cause bacteremia)• Human are the only natural host• Incubation period 1-2 weeks• Initial stage: colonization of ciliated epithelium of the bronchi and trachea.• Production of cytotoxin which paralyses cilia• Production of pertussis toxin• Can also predispose to secondary bronchopneumonia.
    120. 120. PERTUSSIS• 3 Stages Catarrhal - mild coughing and sneezing * Patient highly infectious but not very ill Paroxysmal – explosive coughing w/ inspissatory whoop – Copious amt of mucus  narrowed epiglottis  whoop – cough interferes with swallowing – Swallowing mucus may induce vomiting  dehydration – Hypoxia during prolonged attacks may lead to seizure, hypoxic encephalopathy or coma.
    121. 121. • Convalescent - cough episodes slowly decrease and gradual recovery over 3-16 weeks• Secondary complications: – Pneumonia – otitis media – rectal prolapse – meningo-encephalitis
    122. 122. LABORATORY DIAGNOSIS:• SPECIMEN: saline nasal wash (best) – nasophayngeal swabs and “cough plate”• Direct Fluorescent Antibody (FA) Test – Useful in identifying B. pertussis after culture on solid media• CULTURE: – Bordet-Gengou medium• IDENTIFICATION: FAT/DFA or slide agglutination w/ specific antiserum• PCR
    123. 123. Culture Media: Minute colonies grow over 3-4 days • Bordet-Gengou Agar – pearl like clusters surrounded by a zone of hemolysis • Jones-Kendrich Charcoal Agar – For transportation and cultivation • Regan-Lowe Agar – Transport & selective medium – Half strength charcoal agar medium with horse blood & cefalexin. • Steiner Scholte Agar – Selective medium
    124. 124. TREATMENT & CONTROL:Sanitation: This very contagious disease requires quarantine for a period of 4-6 weeks.• Immunological: Pertussis vaccine "DPT"• Chemotherapeutic: – ERYTHROMYCIN: reduce symptoms when given early - catarrhal stage (first week of illness). – hospitalization for supportive care and management of coughing paroxysms, apnea, cyanosis, feeding difficulties, and other complications such as pneumonia.
    125. 125. Vaccination1. 3 injections of pertussis vaccine (1st yr of life)  booster2. Give Intramuscularly as part of the TripleVaccine DPT D = DIPTHERIA P = PERTUSSIS T = TETANUS
    126. 126. THE BRUCELLAE• Obligate parasite of animals and humans (intracellularly) Species: –Brucella melitensis –Brucella suis –Brucella abortus –Brucella canis• DNA studies proved that there is only 1 species in the Genus, Brucella melitensis, w/ multiple biovars
    127. 127. Morphology & Identification• Typical Organisms: – Short GN coccobacillary forms – Aerobic, non-motile & non-spreforming• CULTURE: – Enriched media: small, convex, smooth colonies• Growth Cx: – Complex nutritional requirements – Amino acids, vitamins, salts & glucose – Grows in ambient air except B. abortus – Catalase & oxidase (+); Some are H2S producers – Reduce nitrates to nitrites
    128. 128. Antigenic Structures• Hazardous organsism  performed only in reference public health lab w/ biologic safety cabinets.• Facultative intracellular org• LPS endotoxin in the cell wall• Cell wall anitgens – A & M
    129. 129. DISEASES:• Malta fever, Undulant fever, Bang Disease, Brucellosis – 1 - 3 week incubation – interferes with phagocytosis by inhibiting fusion – allows for intracellular replication – LPS endotoxin - disseminated in blood
    130. 130. Pathogenesis & Pathology• Routes of infection: – intestinal tract – Mucous membrane – Skin Organism  enters GIT lymphatic channels & regional LN thoracic duct  bloodstream  distributed to the parenchymatous organs * Granuloma formation in lymphatic tissue, liver, spleen, bone marrow & other RES
    131. 131. Severity of disease:• B. abortus – mild disease w/o suppurative complications & non- caseating granulomas of RES• B. canis – mild disease• B. suis – chronic & suppurative lesions w/ caseating granulomas• B. melitensis – more acute & severe
    132. 132. Laboratory Diagnosis:• Specimens: Blood culture, biopsy, serum• Culture: Brucella agar – Biphasic blood culture media (5-10% CO2) for 30 days or in Castaneda medium – Bone marrow & blood = Brucella are most isolated – tiny GN coccobacilli ; catalase, oxidase, CUA (+)  Reference lab (H2S, dye inhibition, agglutination by sera)• Serology: Agglutination test & Blocking Antibodies
    133. 133. Dye Sensitivity Test:• Thionine & Fuchsin to differentiate the 4 species. – Brucella abortus – thionine (S); Fuchsin (R) – Brucella suis – thionine (R) ; fuchsin (S) – B. canis – thionine (R) ; fuchsin (S) – B. melitensis – thionine (R) ; fuchsin (R)
    134. 134. Treatment and prevention• Treatment: – Tetracycline or Ampicillin – RECOMMENDED: Prolonged & combination therapy • Streptomycin and a tetracycline • Intracellular location of the organism• Prevention: – Pasteurization – Quarantine testing and vaccination of animals – Careful handling of animals – use of biohazard hood
    135. 135. Francisella tularensis• Found in animal reservoir• Transmissible to humans thru: – Biting arthropods – Direct contact to infected animal tissue – Inhalation of aerosols – Ingestion of contaminated food & water• Disease caused: TULAREMIA
    136. 136. Francisella tularensis• No spore, & non-motile• Encapsulated GN pleomorphic coccobacilli• Aerobic to f. anaerobic• Biochemically inactive• Biological weapon: extremely invasive & infectious (aerosol transmission)• Biohazard 3 – safety measures
    137. 137. • Clinical Disease – Ulceroglandular (70-80%) - contaminated bites • TULAREMIA or RABBIT FEVER – Glandular (2-12%) - direct contact – Oculoglandular (1-2%) - direct contact – Oropharyngeal (2-4%) - contaminated food – Typhoidal (7-14%) - contaminated food – Peribronchial inflammation & Pneumonia (8- 13%) -aerosols
    138. 138. Laboratory Diagnosis• SPECIMEN: – ulcer scraping, lymph node biopsy, sputum, & serum• MICROSCOPY: – tiny, gram (-) coccobacillus –• CULTURE: – slow grower unless grown on special media • Glucose Cystine Blood Agar (metallic gray, mucoid) • Supplemented Charcoal-Yeast Agar • Buffered Charcoal Yeast Agar
    139. 139. • SEROLOGY: – Rise in Ab titer – Fluorescent antibody identification – ELISA
    140. 140. • TREATMENT: – Streptomycin or Gentamicin – Tetracycline• PREVENTION: – Observe safety measures when handling animals – Cook meat thoroughly – Immunization w/ live attenuated strains among lab personnel.
    141. 141. Yersinia & PasteurellaShort, Pleomorphic Gram negative RodsExhibit bipolar stainingCatalase (+); Oxidase (-)Microaerophilic or facultative anaerobesZoonotic; causes serious disease in humans
    142. 142. Yersinia pestis• Agent of plague• Plague – infection of wild rodents – Rodents to humans by bites of fleas• Characteristics: – GNB that exhibits striking bipolar staining – Non-motile – Facultative anaerobes – Grow rapidly in media containing blood or tissue fluids (30°C)
    143. 143. Yersinia pestis• Antigenic Structure – Possess LPS w/ endotoxic activity – Antiphagocytic property: envelope containing CHON (fraction I) – V-W antigens – 72 kb plasmid• ENZYMES: – Produce coagulase at 28° C
    144. 144. Yersinia• Yersinia enterocolitica – Rodents & farm animals as reservoir – MOT: Ingestion of contaminated water supplies, unpasteurized milk, contaminated food – Spread from person-person is possible• Yersinia pestis – Rodents & their flea – reservoir – MOT: Flea bite or handling of infected animal • Pneumonia – transmitted from person to person
    145. 145. Pasteurella• GENERAL CHARACTERISTICS:• Gram negative pleomorphic coccobacilli, bipolar staining• Non-motile, facultative anaerobe• Grows on CAP and BAP• Oxidase and catalase positive
    146. 146. CLINICAL SYNDROME• Pasteurella multocida – May lead to more serious systemic disease in infants and elderly or other immunocompromised host • Pneumonia • Empyema • Lung abscess • Tonsillitis • Meningitis
    147. 147. MISCELLANEOUS GRAM NEGATIVE BACILLILegionellae, Bartonella, Gardnerella & Streptobacillus
    148. 148. Legionella pneumophila• Major cause of disease in humans• Widely publicized outbreak of pneumonia in Philadelphia (American Legion convention)• Legionnaires disease• Pontiac Fever• Virulence: intracellular survival within macrophages• Natural habitats: bodies of water and soil
    149. 149. Legionella in BCYE & DFARequires cysteine forgrowth Small, GN rods that stain poorly byBuffered charcoal yeastextract w/α-ketoglutarate Gram’s Motile, Catalase +Pinpoint, ground-glassappearance, round or flat, Direct fluorescentcolorless or varying color, antibody testingtranslucent or speckled
    150. 150. Bartonella species• B bacilliformis • B henselae & B – Oroya fever quintana – Verruga peruana – Cat-Scratch disease • Fever, – Transmitted by lymphadenopathy sandflies – By contact with a cat (Lutzomyia) (scratch, lick, bite, or flea bite)• Limited to Peru, Colombia, Ecuador GN RODS, PLEOMORPHIC SLOW GROWING SEEN IN INFECTED TISSUES W/ WARTHIN-STARRY SILVER IMPREGNATION STAIN
    151. 151. Gardnerella vaginalis• Cause of Bacterial vaginosis• Vaginal discharge• No inflammatory cells – Many anaerobes – pH is over 4.5• Direct wet mount: many characteristic desquamated epithelial cells with attached organisms
    152. 152. Streptobacillus moniliformis• Causes rat-bite fever (rat bites) – Septic fever, blotcy & petechial rashes – Painful polyarthritis• Causes Haverhill fever• Pleomorphic GN rods, irregular chains• Culture: L forms – best at 37C; ceases to grow at 22C – media w/ serum protein, egg yolk, starch• Serum agglutination tests
    153. 153. ASSIGNMENT1. Enumerate the medically significant anaerobic bacteria according to the following classifications: a) Gram positive cocci b) Gram negative cocci c) Gram positive bacilli d) Gram negative bacilli2. What enzymes are lacking among obligate anaerobes that make them susceptible to the lethal effects of oxygen?3. Describe the performance of an anaerobic cultivation method.
    154. 154. Spirochetesand other unusual pathogens Mycoplasma Chlamydia Rickettsia
    155. 155. Order Spirochaetales• 2 families: 1. Spirochaetaceae  Free-living, large spiral organisms 1. Treponemataceae  Human pathogens »Treponema »Borrelia »Leptospira
    156. 156. Treponema species• Treponema pallidum subspecies pallidum• T pallidum subspecies pertenue• T pallidum subspecies endemicum• T pallidum subspecies carateum
    157. 157. Treponema pallidum morphology• Slender spirals (0.2 µm in width & 5-15 µm in length)• Rotate steadily around their endoflagella even after attaching to cells by their tapered ends• Not readily seen with ordinary dyes• Immunofluorescent stain, dark-field illumination, or iron staining• Seen in tissues with silver impregnation method
    158. 158. Treponema pallidum Culture• Has never been cultured on artificial media• Experimentally, infected skin, testes, & eye of rabbits;• Grown in tissue culture, short periods of time• Microaerophile; best in 1-4% oxygen• May remain motile in 3-6 days at 25°C• Remain viable for at least 24 hrs in whole
    159. 159. Treponema pallidum ControlWhat kills the spirochete?• Drying• Elevation of the temperature to 42°C• Trivalent arsenical, mercury, bismuth• Penicillin
    160. 160. Treponema pallidum Antigenic Structures• Outer sheath (glycosaminoglycan coating)• Outer membrane surrounds the periplasmic space and the peptidoglycan- cytoplasmic membrane complex• Endoflagella (axial filaments)• Hyaluronidase• > 100 protein antigens, i.e. cardiolipin• Develop reagin (antibody-like substance)
    161. 161. Treponema pallidum = SYPHILISA. ACQUIRED SYPHILIS  Sexual contact  Infectious lesions o On skin or mucous membranes of genitalia o Intrarectal, perianal, or oral o Multiply locally, spread to nearby lymph nodes, then reach the blood stream  4 stages
    162. 162. SYPHILIS1. PRIMARY STAGE  Hard chancre (heals after 4-6 weeks)  Regional lymphadenopathy2. SECONDARY STAGE  Hair loss, fever, weight loss  Generalized lymphadenopathy  Red rash (palms & soles)  Condyloma latum  May also be: syphilitic meningitis, chorioretinitis, hepatitis, nephritis, or periostitis
    163. 163. 3. LATENT STAGE  No symptoms but found specific antitreponemal Abs  2/3 (66%) resolution  1/3 (33%), 6-40 years progress to tertiary syphilis  Untreated infection remains latent4. TERTIARY STAGE  GUMMAS (skin, CNS, bones, eyes, heart)  CNS, Eyes, Heart→ blindness  → aortic damage or aneurisms
    164. 164. Treponema pallidumCongenital syphilis treponemes cross the placenta, infecting the fetus usually latent stage, 10th to 15th week of gestation fetal death, miscarriages, stillborn at term live but with congenital syphilis in childhood:  Interstitial keratitis, Hutchinson’s teeth, Saddlenose, Periostitis, damage to mental development or other neurological symptoms
    165. 165. T. Pallidum Diagnostic Lab Testsa. Observation of organisms in lesions 1. Darkfield examination 2. Immunofluorescence • Fluorescein-labeled antitreponeme seruma. Serologic Tests 1. Nontreponemal tests 2. Treponemal antibody tests
    166. 166. Diagnostic Lab Tests1.NONSPECIFIC, NONTREPONEMAL TESTSScreening purposes; efficacy of therapyLipid-antigen particles dispersed in serum and flocculate with reaginAntigens: cardiolipin, cholesterol, & purified lecithinDetect reagin or Wasserman antibodiesVenereal Disease Research Laboratory (VDRL)Unheated serum reagin (USR) testRapid plasma reagin (RPR) card testToluidine red unheated serum test (TRUST)
    167. 167. Diagnostic Lab Tests2. SPECIFIC, TREPONEMAL TESTS  Confirm positive nontreponemal results  T pallidum-particle agglutination (TP-PA)  T pallidum hemagglutination* (TPHA)  Microhemagglutination T pallidum* (MHA-TP)  Enzyme immunoassay (EIA) for T pallidum  Fluorescent treponemal antibody absorbed (FTA-ABS)
    168. 168. Borrelia recurrentis Borrelia recurrentis – epidemic relapsing fever Fever, chills, headaches, malaise Fatal cases: involve spleen, liver, other organs, hemorrhagic lesions (kidneys and GIT) Ultimate recovery: after 3-10 relapses
    169. 169. Borrelia recurrentis: Epidemic relapsing fever Direct observation from blood specimens of febrile patients Darkfield (wet prep), move rapidly RBCs Thick & thin smear, Wright or Giemsa stains Serology not reliable; ANTIGENIC SHIFTSTx: tetracyclines, erythromycin, penicillin
    170. 170. Borrelia burgdorferi• Lyme disease• Stage 1: – Erythema migrans (characteristic skin lesion) – Flulike symptoms• Stage 2: — arthralgia & arthritis — Neurologic manifestions• Stage 3: — chronic skin, CNS, or joint involvement
    171. 171. Leptospira interrogans Tightly coiled, 5-15 µm long, form a hook Darkfield examination or thick smears with Giemsa stain Grown on semisolid media Many serovars, see Table 24-1, p. 309 Jawetz’ book
    172. 172. Leptospira interrogans ser. icterohemorrhagiae : Weil disease• Rat urine, water Dx: Serology –• Enter cuts/abrasions Agglutination tests of skin or mucous membranes Tx: doxycycline, ampicillin, amoxicillin• IP: 1-2 weeks Fever Jaundice Hemorrhages Aseptic meningitis
    173. 173. Spirillum minor(Spirillum morsus muris) – Rat-bite fever (sodoku) – Small, rigid spiral – Worldwide – Bite of rat – Local lesion, regional gland swelling, skin rashes, fever
    174. 174. Mycoplasmas:• Order Mycoplasmatales, Family Mycoplasmataceae• 2 medically important genera: (1) Mycoplasma (2) Ureaplasma• 4 species of primary importance: – M. pneumoniae – U. urealyticum – M. hominis – M. genitalium
    175. 175. Mycoplasmao Plasma membrane - outermost part oUnique, high sterol content, prevent osmotic lysiso Very small (125-250 nm)o Highly pleomorphico Resistant to penicillino Ureaplasma: metabolize ureao Virulence: Protein P1
    176. 176. M. pneumoniae U. urealyticum• Tracheobronchitis • Nongonococcal• Walking pneumonia urethritis• fever with a dry, • burning on urination, nonproductive hacking with yellow mucoid cough discharge from the – Patchy infiltrates urethra – Usually in children, adolescents, and • Lung disease in young adults premature low-birth- – tetracyclines, weight infants erythromycin
    177. 177. Chlamydiae• Order Chlamydiales• 3 medically important species – Chlamydia trachomatis – Chlamydia (Chlamydophila) pneumoniae – Chlamydia (Chalmydophila) psittaci• Obligate intracellular parasites• Cell wall similar to GNB, but lack peptidoglycan• Complex developmental cycle
    178. 178. Chlamydial Diseases SPECIES DISEASESChlamydia trachomatis • Trachoma Serotypes A, B, & C (leading cause of blindness in the world) Serotypes D thru K • Inclusion conjunctivitis (newborn, contracted in the birth canal) • Infant pneumonia • Cervicitis, PID • Nongonococcal urethritis in men Serotypes L1, L2, L3 • Lymphogranuloma venereum (LGV)Chlamydia psittaci • Atypical pneumonia, psittacosis, FUOChlamydia pneumoniae • Atypical pneumonia, bronchitis, pharyngitis, serogroup TWAR sinusitis
    179. 179. Chlamydia Diagnostics• Grown on cell lines• C trachomatis contain Glycogen ininclusions and stain with iodine; aresusceptible to sulfonamides.• C pneumoniae & C psittaci = resistant to sulfonamides• Serology: CF & IF tests, elevated titers
    180. 180. Rickettsia & Ehrlichia• Family Rickettsiaceae• 4 medically important genera – Rickettsia – Coxiella – Orientia – Rochlimaea• Obligate intracellular parasites• Infections – fever, rashes, vasculitis• Transmitted to humans by arthropods
    181. 181. ORGANISM DISEASE VECTOR RESERVOIRRickettsia prowazekii Epidemic typhus, Louse Humans Brill-Zinsser diseaseRickettsia typhi Endemic typhus, Flea Rodents murine typhusOrientia Scrub typhus Mite RodentstsutsugamushiRickettsia rickettsii Rocky Mountain Tick Rodents, dogs spotted feverRickettsia akari Rickettsial pox Mite MiceCoxiella burnetii Q fever (no rash) None, Sheep, cattle, airborne goatsRochalimaea quintana Trench fever Louse HumansEhrlichia chaffeensis Human monocyte Tick Deer, dogs, ehrlichiosis (no humans rash)
    182. 182. Rickettsial Diagnosis & Serology• Direct detection in tissues (Giemsa stain or IF test)• Culture: chick yolk sac, tissue culture (Exception: Rochlimaea on artificial media with blood)• Serology: CF, ELISA and Weil-Felix reaction – antibodies formed will agglutinate OX strains of Proteus vulgaris.
    183. 183. Weil-Felix Reaction DISEASE OX-19 OX-2 OX-KRMSF + + ̶Rickettsial pox ̶ ̶ ̶Epidemic + ̶ ̶typhusEndemic typhus + ̶ ̶Scrub typhus ̶ ̶ +Trench fever ̶ ̶ ̶Q fever ̶ ̶ ̶
    184. 184. Rickettsia Virulence Factors– Damage endothelial cells lining blood vessels– Induced phagocytosis– Recruitment of actin for intracellular spread– resistant to lysosomal enzymes, has endospore form (only Coxiella burnetti)– Cell-to-cell spread →
    185. 185. Rickettsiae• Treatment – Doxycycline, chloramphenicol• Eradicate lice• Wear protective clothing• Use insect repellents