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Bacteriology Update 2020

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Bacteriology Update 2020 by Dr. Morgan

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Bacteriology Update 2020

  1. 1. Bacteriology Update 2020 Margie Morgan, PhD, D(ABMM)
  2. 2. Definitions • Obligate Aerobe – require high level of oxygen (20%) to grow • Obligate Anaerobe – >30 min of oxygen exposure can be deadly • Facultative anaerobes – grow in both aerobic and anaerobic conditions, most so-named “aerobic” bacteria are actually facultative (ie. E. coli) • Microaerophilic – grow better with reduced oxygen and elevated carbon dioxide % • Aerotolerant anaerobes– anaerobe is not killed by prolonged exposure to oxygen, but grow best anaerobically, example: Clostridium tertium • Lag Phase - >24 hrs old on agar plates, growth is slowing, not appropriate for biochemical or susceptibility testing • Log Phase – <=24 hr growth that is appropriate for all testing • Stationary phase – Organisms alive but not replicating, appropriate for transporting specimens
  3. 3. Specimen Collection for Aerobic Bacteriology Throat / Wound collection 1. Collection swabs should be of polyester fiber or flocked (prickly sponge) 2. Cotton swab fibers should not be used for they trap bacteria and are potentially toxic 3. Specimen is collected on swab then placed in Stuart’s or Amie’s transport media (buffered solution with peptones) for transport and storage up to 72 hrs. 4. Transport media preserves viability of the bacteria but does not promote growth of bacteria, provides stasis of numbers prior to plating onto solid media Urine collection – two methods most often used 1. Container with boric acid used to induce organisms into stationary phase 2. Refrigerate urine within one hour after collect Both methods maintain original colony count and viability of organisms Must plate urine within 24 hours of collection Tissues/Sterile body fluid collection – collect in sterile container
  4. 4. Blood Cultures • Two most important blood culture collection issues • Preventing contamination: Cleanse collection site using Chlorhexidine • National Benchmark: Blood culture contamination rate should be <=3% • Adequate volume of blood drawn per blood culture bottle • Adult blood culture should approach 8-10 ml of blood per bottle • One Blood culture consists of two bottles: • One aerobe and one anaerobe bottle • Incubated at 35*C for 5 days • Automated detection instruments for positivity is a the standard of practice • Growth is detected by increasing number of bacteria causing an increase in the amount of C02 in the bottle air space. This triggers a fluorescent indicator to produce a positive result.
  5. 5. Gram stain Procedure 1 minute Rinse Primary stain Mordant 1 minute Rinse 5-10 seconds RinseDecolorization Counter stain 1 minute Rinse Peptidoglycan in high amount in Gram positive cell wall traps the Crystal violet, hence blue color for Gram positive organisms. Document the color (red/blue) and shape of the stained organism. Prepare thin film of specimen on glass slide Heat or methanol
  6. 6. Gram Stain to Assess Quality of Sputum for Performing Bacterial Culture • Expectorated sputum is examined for presence of epithelial cells and neutrophils (WBCs) • If <10 epithelial cells/Low Power Field (LPF) is observed – acceptable for bacterial culture Should also have >25 WBCs/LPF (except in leukopenia) • If >10 epithelial cells /LPF • Sputum is judged to be spit • Bacterial culture is not performed • Request made to nursing staff for a “deep cough” specimen Bad Sputum Good Sputum 10X objective
  7. 7. Most commonly used agar plated media Blood agar- 5% sheep’s blood agar • Used to gauge hemolytic reaction of bacteria (alpha, beta, gamma) • Culture Gram positive & Gram negative bacteria, and Yeast Chocolate agar • “Caramelized” blood agar – more enriched than blood agar medium • Supports the growth of the same organisms as 5% Sheep’s blood agar plus fastidious bacteria – such as Haemophilus influenzae and Neisseria gonorrhoeae
  8. 8. Commonly used agar media • MacConkey agar – Selective and differential medium • Selective - Supports growth of Gram negative rods but crystal violet inhibits growth of Gram positive organisms • Differential - Lactose fermentation can be determined by pink color produced by colony, neutral red indicator turns pink from acid production • Lactose fermentation positive = pink • Non-lactose fermentation negative = no color • Major branchpoint in enteric Gram negative rod identification
  9. 9. MALDI-TOF Mass Spectrometry Matrix-Assisted Laser Desorption/Ionization – Time of flight – Identification by analyzing protein fingerprints of bacteria – Replaced many/most biochemical tests for identification of bacteria
  10. 10. MALDI-TOF Theory • Laser is fired at target containing matrix and sample • Laser energy is absorbed by the matrix and converted to heat energy and ionizes the sample. • Positive ions (proteins) are accelerated through a vacuum by an applied electrical field • The time taken for the proteins to reach the detector depends on their mass/charge ratio (m/z) and creates spectrograph. • Each organism species has a different protein composition, thus giving rise to a specific mass spectrograph. • The mass spectrograph produced by a sample is then compared with many thousands stored in a spectrograph database to see which one it most closely matches. Thus an identification is achieved.
  11. 11. Gram Positive Cocci Staphylococcus Streptococcus Enterococcus
  12. 12. Staphylococcus Gram positive cocci in clusters – clusters formed due to bound coagulase or “clumping factor” Catalase enzyme test = Positive Staphylococcus aureus Coagulase Negative Staph Coagulase Enzyme Positive Coagulase Enzyme Negative +/- Yellow colony Beta hemolysis on Sheep’s blood agar plate White colony Most not hemolytic on Sheep’s blood agar plate
  13. 13. Catalase Enzyme Reaction Negative Positive Bacteria placed in Hydrogen Peroxide/ bubbles=positive reaction Slide Coagulase reaction Staphylococcus organism emulsified in rabbit plasma/ mix well/ agglutination is positive rxn. Positive Coagulase rxn = Staph aureus Tube Coagulase Reaction Rabbit plasma inoculated with organism / Incubate at 35˚C / observe for clot at 4 hours and if negative read again at 24 hours Negative tube coagulase No clot formed/liquid Coagulase negative Staph Positive Tube Coagulase Clot formed Staph aureus Slide Coagulase Reaction
  14. 14. Staphylococcus aureus • Virulence mechanisms: • Protein A – Primary virulence factor, surface protein, ability to bind immunoglobulin and combat the immune response • Toxins - act as super antigens, recruit host defense cells that liberate cytokines with systemic effects • Disease associated toxins: • Toxic shock syndrome (TSST-1 toxin) • Scalded skin syndrome (Exfoliatin (SSS) toxin) • Soft tissue infection (Panton valentine leukocidin toxin – PVL) • Food poisoning / Enterotoxins – Toxins stable to heating at 100*C for 30 minutes • Bacteremia • Endocarditis • Septic arthritis – Primary cause of adult septic arthritis Exfoliatin toxin Onion skin peeling PVL – soft tissue abscess
  15. 15. Methicillin Resistant Staphylococcus aureus – Due to altered Penicillin binding proteins (PBP2a) produced by the mecA gene that codes for resistance to oxacillin/methicillin /nafcillin resistance (semisynthetic penicillin antibitotic) – Ways to detect MRSA 1. Can use molecular PBP2a, produced by the mecA gene to detect resistance 2. Cefoxitin susceptibility testing is considered a more sensitive indicator of resistance to these antibiotics and provides more sensitive detection of MRSA – All MRSAs are reported resistant to oxacillin, nafcillin and methicillin and all cephalosporin antibiotics. None of these antibiotics should be used for therapy – Vancomycin becomes antibiotic of choice – Of note: Recently there has been an emergence of mecC producing MRSA, these are not detected using a mecA test. They are best detected by testing for resistance to cefoxitin.
  16. 16. The “D” Test for Inducible Clindamycin Resistance • Is Staph aureus really susceptible to Clindamycin? • During therapy, S aureus isolates resistant to Erythromycin possess enzymes capable of inducing Clindamycin resistance • This cannot be detected by regular susceptibility testing • With the D test – the zone around Clindamycin KB disk will be blunted to form a “D” shape, meaning Clindamycin was induced by Erythromycin to be resistant – so called “INDUCIBLE RESISTANCE” • Clindamycin should be reported as resistant by clindamycin induction testing and clindamycin should not used for therapy. D test positive Inducible resistance to clindamycin Do not use for therapy D test negative Susceptible to Clindamycin Clindamycin used for therapy
  17. 17. Nares is primary colonization site and primary site used for surveillance cultures Culture methods used for nares surveillance cultures: Chromogenic media is commonly used – It is selective for MRSA due to the addition of cefoxitin. It is differential due to chromogenic substrates that turn a specific color to identify Staph aureus Molecular assays (MA) can also be used to screen nares for MRSA colonization. MA increase the sensitivity of detection over culture methods by 5-10% but greatly increase laboratory costs. Mupirocin therapy can be placed in nares to eradicate carriage Chlorhexidine bathing is used to decolonize skin Methicillin Resistant Staphylococcus aureus (MRSA) Surveillance cultures to assist with Hospital Epidemiology
  18. 18. Coagulase negative Staph (CNS) @ 15 species infect humans • Staph epidermidis – most common in humans – Major aerobic component of normal skin flora – Common cause of subacute bacterial endocarditis – Pathogenicity from cell adhesion factors form biofilm on biologics and plastics such as heart valves, Catheters and replacement joints, making treatment difficult • Staph saprophyticus – – Urinary tract infection in the child bearing age female, – This CNS adheres in greater #’s to epithelial cells – Only CNS resistant to Novobiocin (KB disk test) White non-hemolytic colony Resistant to Novobiocin
  19. 19. • Staphylococcus hemolyticus • Cause catheter line related sepsis • Beta hemolytic on 5% sheep’s blood agar • Do not confuse with Staph aureus! • Staphylococcus lugdunensis • ? greater pathogenicity than other CNS • Infections mostly abscess and bacteremia • Biochemical test of note: PYR test positive = pink • Closely related organism: • Micrococcus species • Gram positive cocci in tetrads • Mustard yellow colony – environmental contaminate • Catalase = positive • Coagulase enzyme = negative • Does not ferment glucose • Staphylococcus ferment glucose Neg Pos PYR Test Coagulase Negative Staph (CNS)
  20. 20. Streptococcus Gram positive cocci in chains and pairs Catalase enzyme = negative Three groups based on hemolytic reaction produced with growth on 5% sheep’s blood agar • Alpha – greening of agar, partial hemolysis of RBCs • Viridans Streptococcus, Streptococcus pneumoniae, Granulicatella and Abiotrophia • Beta – clearing of agar, complete hemolysis of RBCs • Beta hemolytic Streptococcus such as Streptococcus pyogenes and Streptococcus agalactiae • Gamma – no clearing of agar, intact RBCs • Streptococcus bovis (gallolyticus)
  21. 21. Beta Hemolytic Streptococcus typing • Beta hemolytic Streptococcus are grouped by the “C” carbohydrate (CHO) present in the bacteria cell wall. This typing system is known as the Lancefield typing system. • Classifies the Beta Strep groups – A, B, C, F, and G, the groups most commonly isolated with human infections • The “C” CHO in the cell wall is the antigen used in a slide agglutination test and it bonds with specific monoclonal antibody of each individual Streptococcus group. Shown in picture is Strep group A typed using monoclonal antibody coated latex beads to group A - A
  22. 22. Streptococcus pyogenes • Group A Streptococcus [GAS] – very beta hemolytic on blood agar • Biochemical tests used for identification: • Bacitracin KB sensitivity test – GAS are inhibited by antibiotic Bacitracin • This test is not specific for GAS, inhibition also occurs with Beta hemolytic Streptococcus group C • PYR (pyrrolidonyl arylmidase) reaction • Organism spotted onto moist PYR disk • 2 min – RT incubation • Add Cinnamaldehyde reagent • Pink = positive = Streptococcus pyogenes • This test is not exclusive for Strep pyogenes – Enterococcus and Staph lugdunensis also pink (+) • Therapy : Penicillin, Amoxicillin or Cephalosporin antibiotics No resistance reported to these agents PYR
  23. 23. • Streptolysin O and Streptolysin S toxins • Comprise the ASO titer assay that assists in the diagnosis of GAS sequelae • Cell toxins lead to evasion from the immune system • Toxin activity can be demonstrated on 5% Sheep’s blood agar media • O toxin is oxygen labile S toxin is oxygen stable • When both toxins are present, the stabbed area of the media will demonstrate increased beta hemolysis. Primary virulence factors: M Protein – prevents phagocytosis Capsule – hyaluronic capsule prevents phagocytosis
  24. 24. Streptococcus pyogenes Infections • Diseases – • Pharyngitis • Impetigo (1) • Erysipelas (2) • Cellulitis (3) • Necrotizing fascitis (4) • Puerperal sepsis • Toxic Shock • Scarlet fever (5) 1 2 3 4 5
  25. 25. Sequelae of Strep pyogenes Infection Can occur 10 -30 days following impetigo or pharyngitis infection Rheumatic fever • Due to inadequate treatment of GAS skin or pharyngitis infection • Family history (genes), strain of GAS and multiple exposures to GAS can more likely evolve into sequelae • Usually occurs in children 5 – 15 years • Pathogenicity due to molecular mimicry: there is similarity between the proteins of Strep A and human muscle tissue that cause an autoimmune mechanism that lead to confusion. The immune system is armed to attack heart (heart valves, muscle), joint, and bones • ASO titers will be elevated • Usually leads to need for valve replacement surgery Glomerulonephritis • Post infection with Nephritogenic strain of Strep pyogenes • Leads to immune mediated destruction of the renal glomeruli • ASO titers will be elevated • Usually resolves without therapy
  26. 26. Streptococcus agalactiae (GBS) • Biochemical tests: – Camp test – performed using a Staph aureus strain that contains Camp factor streaked perpendicular to group B Strep, incubate 24 hr. / intensifies toxin production that produces arrow shaped hemolysis (see pix below) – Rapid hippurate hydrolysis – – Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyze hippurate into glycine and benzoic acid by action of hippuricase enzyme present in GBS – 4 hour test. Positive test = purple pos Staph aureus Strep group B Camp TestHippurate Hydrolysis Increased area of hemolysis
  27. 27. Strep agalactiae [GBS] • Pathogen of the elderly – Causes bacteremia and urinary tract infection, acquisition most likely from the intestine • Pathogen of neonate bacteremia or CNS – In utero or perinatal organism acquisition during birthing process, infection in @ 1/2000 births • Early onset infection – within 7 days of birth • Late onset infection - within 8 – 28 days of birth • Treatment: Penicillin or Cephalosporin (3rd generation) • @ 25% of pregnant women colonized in the cervix and/or rectal area with GBS • All pregnant must be screened at 35 – 37 weeks of pregnancy for GBS (Regulation) • Enrichment methods for GBS screening are standard of practice • Cervix and Rectal swab incubated in an enrichment broth for 18 hours at 35 ˚C then cultured onto Blood agar. Enrichment broth can also be used to increase sensitivity in molecular testing methods • Ampicillin drug of choice to treat GBS positive pregnant woman during delivery • Susceptibility testing for alternative therapies must be performed in PCN allergy • Clindamycin testing with Inducible resistance testing confirmation must be done prior to using Clindamycin
  28. 28. • Two most common species • E. faecium and E. faecalis • No defined virulence factors • Group “D” CHO in the cell wall • Biochemical tests: • Bile esculin agar = growth/black • 6.5% NaCl tolerance (turbidity/growth) • PYR = positive • E. faecium = arabinose fermentation positive • E. faecalis = arabinose fermentation negative Enterococcus + - PYR Neg Pos
  29. 29. Enterococcus • Pathogen of opportunity • Usual intestinal normal flora • Infections include UTI, bacteremia, and abdominal abscess • Antimicrobial therapy: • Natural resistance to cephalosporin antibiotics • Ampicillin plus Aminoglycoside can be synergistic for therapy in cases of endocarditis • Vancomycin is an antibiotic of choice • Unique susceptibility issues • Acquired resistance to vancomycin known as vancomycin resistant enterococcus or VRE. Resistance due to acquisition of genetic material: • Van A (E. faecium) resistance gene • Van B (E. faecalis) resistance gene
  30. 30. Streptococcus bovis (gallolyticus) (Taxonomy is once again in the process of change) • Streptococcus gallolyticus ssp. gallolyticus (S. bovis biotype 1) Isolation is associated with colon cancer (73%) when isolated from a patients blood culture • Streptococcus gallolyticus ssp. pasteurianus formerly S. bovis biotype II- associated with neonatal meningitis • Biochemical reactions: Bile esculin slant positive 6.5% NaCl no growth PYR reaction negative Susceptible to Penicillin Bile Esculin Positive 6.5% No Growth 6.5% Growth PYR Negative PYR Positive Strep gallolyticus Enterococcus
  31. 31. Streptococcus pneumoniae Alpha hemolytic Gram positive bullet (lancet) shaped cocci in pairs Polysaccharide capsule = virulence factor and antiphagocytic Identification: Bile soluble – colonies dissolve Inhibited by Optochin – ethyl in sodium deoxycholate (bile) hydrocupreine hydrochloride Zone of inhibition must be >=14 mm Autolytic Changes
  32. 32. Streptococcus pneumoniae • Normal inhabitant of the upper respiratory tract • Infections: Upper and Lower respiratory tract infection (Lobar pneumonia), Sepsis, Meningitis, middle ear, ocular, sinus • Asplenic and immune suppressed patients particularly at risk • 13 valent pneumococcal conjugate vaccine aids in preventing invasive infections – those at risk need vaccination • Susceptibility issues: • Acquired Resistance to Penicillin due to Penicillin binding proteins (PBP) • If test susceptible,1st line therapies include Penicillin or 3rd generation Cephalosporin (Ceftriaxone)
  33. 33. Viridans Streptococcus Several species of viridans group Streptococcus are NF in mouth and upper respiratory tract. Most commons species include: S. mutans S. salivarius S. sanguis S. mitis • Bile esculin = negative • Bile solubility = negative • Optochin resistant with a zone size <=13 mm • Cause 30 – 40% cases of sub acute endocarditis on native valve usually due to bad dentition • Can cause abscess and various infections throughout the body in the immune suppressed host • Variable susceptibility patterns, some isolates with elevated MICs to Penicillin. Usual therapy is 3rd generation cephalosporin. Viridans Streptococcus Streptococcus pneumoniae
  34. 34. Viridans Streptococcus unique species • Streptococcus anginosis group: includes • S. anginosus S. constellatus S. intermedius • Normal flora in human mouth • More virulent than “normal” viridans Streptococcus, due to capsule • Grows best when incubated in 5 – 10% CO² incubation = Microaerophilic • Butterscotch odor to colony • Cause deep tissue abscess, bacteremia, endocarditis, intra abdominal infections… • Variable susceptibilities – so best to do susceptibility testing, always susceptible to vancomycin
  35. 35. Nutritionally Variant Streptococcus • Vitamin B6 (pyridoxal) deficient – • Will not grow on agar medium without B6 supplementation • Will grow in blood culture bottle due to vitamin B6 in patient’s blood • Will not grow on 5% Sheep’s blood agar plate • Will grow with Staph aureus streak that supplies vitamin B6 • Nutritionally variant Streptococcus will satellite @ S. aureus streak • Two species: • Abiotrophia defectiva • Granulicatella adiacens • Endocarditis – • More destructive to valve than “regular” viridans Streptococci • Higher MIC’s to Penicillin, susceptible to 3rd generation Cephalosporins. • Combination therapy: PCN and Gentamicin Satelliting streptococcus next to S. aureus streak
  36. 36. Rare Opportunistic Gram pos cocci • Aerococcus ureae – Gram positive cocci in pairs and clusters • Alpha hemolytic on blood agar , difficult to identify, often confused with viridans Streptococcus • Urinary tract pathogen • Rothia mucilaginosa – Gram positive cocco-baccilli • Neutropenia and gut problems predispose to infection • Normal flora in the oral cavity and upper respiratory tract • Pathogen in dental caries and periodontal disease • Bad teeth can lead to bacteremia with endocarditis • Gemella morbillorum– • Easily over decolorized, Gram positive in pairs – requires CO2 to grow • Normal flora in oral cavity • Bacteremia with endocarditis • Leuconostoc mesenteroides– Gram positive cocci in chains • Intrinsic resistance to vancomycin • Bile esculin = negative • Bacteremia in immune suppressed • Watch out! Do not confuse with vancomycin resistant enterococcus (VRE)
  37. 37. Gram Negative Cocci Neisseria spp Moraxella catarrhalis
  38. 38. Gram Negative Cocci • Neisseria species and Moraxella catarrhalis • Small kidney bean shaped cocci in pairs within WBC • Oxidase enzyme = positive • CTA (Cysteine Trypticase Agar) carbohydrate fermentations for identification – glucose, maltose, lactose, sucrose • N. gonorrhoeae Gluc + Mal - Lac - Suc - • N. meningitidis Gluc + Mal + Lac - Suc - • N. lactamica Gluc + Mal + Lac+ Suc- • M. catarrhalis All negative DNA’ase enzyme positive • N. gonorrhoeae will NOT grow on 5% Sheep’s blood agar • N. meningitidis will grow on 5% Sheep’s blood agar
  39. 39. CHO Fermentation Reactions Compare (+) yellow reactions to negative (red) control well + + + Oxidase enzyme spot test: Detects production of enzyme cytochrome oxidase Add reagent N,N trimethyl-p- phenylenediamine dihydrochloride to filter paper with organism smear positive = blue to purple color control Full size CHO fermentation tubes Growth on Chocolate Agar Oxidase Enzyme Positive Glucose + Glucose+/Maltose + N. gonorrhoeae N. meningitidis No growth BAP Grows on BAP
  40. 40. Neisseria meningitidis • Colonization occurs in nasopharynx (10-20%) • African meningitis belt – highest prevalence in world • Meningitis, usually occurs in children and young adults • Hallmark - petechiae (organisms crowd into capillaries) leads to tissue necrosis and DIC (endotoxin) • Infection can be rapidly fatal (<24hrs) • Capsular polysaccharide is primary virulence factor • N. meningitidis serotypes A,B,C Y and W, most common • Complement deficiencies in factors 7,8,and 9, Eculizumab, asplenia, and HIV predispose to infection • Adrenal necrosis in this infection referred to as Waterhouse Friderichsen syndrome • Immunization at ages 2m, 12 yr, 16 yr, and in HIV
  41. 41. Neisseria gonorrhoeae • STD – sites of infection include urethrae, endocervix, ocular, rectal, oropharynx, septic arthritis • 10-20 % female ascend to PID but only 0.5% disseminate throughout body • Gram stain of urethral discharge useful for male diagnosis only • Gram stain of cervix can be problematic due to NF look alike organisms, such as Acinetobacter species in the female • Transport for culture – charcoal swabs at room temperature • Media: Selective Thayer Martin or Martin Lewis agar, chocolate type agar with increased nutrition and antibiotic trimethoprim • Amplification methods [PCR] have become standard of practice, improved sensitivity comparted to culture methods • Has both beta lactamase enzyme and Chromosomal resistance mechanisms • Therapy: Ceftriaxone + Azithromycin or Doxycycline, combination therapy to prevent development of resistance
  42. 42. Amplification Testing for Neisseria gonorrhoeae and Chlamydia trachomatis • Amplification methods (Polymerase Chain Reaction (PCR) have become the standard of practice, combo testing due to possible co-infection • Urine, cervix/vaginal, throat and rectal – sites most often tested • More sensitive than culture • Sensitivity/Specificity @ 96%/99% • Female: most sensitive specimen is cervix • Urine @ 10 – 15% less sensitive • Males: Equal sensitivity with urine and urethral, urine specimen of choice • The ancient ways: C. trachomatis culture - Iodine staining of inclusions in McCoy cell culture – previous method of choice Fluorescent antibody stain of C. trachomatis infected cell – positive cell contains green staining Elementary bodies
  43. 43. Moraxella catarrhalis • Infections: Pneumonia, ocular, sinusitis, otitis media • Gram stain of sputum can be helpful in diagnosis of pneumonia (Polys with gram negative diplo-cocci) • Hockey puck colony – able to push colony across the agar surface • Biochemical Tests: • Oxidase enzyme = positive • DNA’ase enzyme = positive • Produces a beta lactamase enzyme • Therapy: Augmentin or 2nd or 3rd generation Cephalosporin
  44. 44. Gram Positive Rods Corynebacteria Bacillus Listeria Erysipelothrix
  45. 45. Corynebacterium • Over 20 species, most are saprophytic • Human normal flora, especially skin and nares • Most gamma hemolytic gray colonies on 5% Sheep’s blood agar • Catalase = positive • “Diphtheroid” morphology on Gram stain – Gram positive rods in Chinese letter forms • No spores produced
  46. 46. Corynebacterium diphtheriae • Agent of Diphtheria • Diphtheritic adherent pseudo membrane produced in throat • Non-toxin producing strains can cause wound infection • Phage mediated exotoxin is distributed from the membrane causing respiratory paralysis • Exotoxin detected by Elek immuno-diffusion test • Grows well on 5% Sheep’s BAP • (1) Selective medium Cysteine Tellurite agar • Produces black colonies with brown halos • (2) Metachromatic granules produced from growth on egg containing Loeffler medium -stain with methylene blue to observe colorful storage granules Elek plate Tellurite agar Metachromatic granules
  47. 47. Other Corynebacterium • Corynebacterium jeikeium – • Normal skin flora bacteria / thrives on lipid • Infects patients with indwelling plastic catheters and devices leading to bacteremia • Biofilms are formed on the plastic surface, layers are formed protecting the organism from antibiotic therapy • Resistant to most antibiotics • Susceptible to vancomycin and tetracycline • Corynebacterium urealyticum – • Urease = positive • Cause of urinary tract infection in post renal transplants • Resistant to many antibiotics but vancomycin susceptible Red is (+) for Urease reaction +
  48. 48. Bacillus species • Large Gram positive rods with square ends – boxcar shape • Can over-decolorize easily and appear reddish on Gram • Spores produced – clearing in bacillus on Gram • Catalase enzyme = positive spores
  49. 49. Bacillus anthracis Anthrax • Category A agent – highest threat to public health and bioterrorism – spores are viable for decades in soil • Natural infection of herbivores – infected animals can have fatal infection and contaminate the environment for years • Virulence factors: anthrax toxin and capsular polypeptide • Infections: • Wool sorter’s disease – skin infection acquired from handling infected imported hides produces a unique black eschar skin lesions • Pneumonia, sepsis, and meningitis
  50. 50. • Bacillus anthracis • Irregular shape to colony border Medusa head colonies on BAP No hemolysis on 5% Sheep’s blood agar Non-motile Susceptible to penicillin Alert public health department – possible B. anthracis • Bacillus cereus – • Two diseases most common: • (1) Food poisoning • Rapid onset of vomiting within 1 – 6 hours • Preformed emetic toxin produced in food and ingested • Fried rice is one of the most common food sources • (2) Traumatic wound infection from contaminated soil • Beta hemolytic colony on 5% Sheep’s blood agar • Motile • Resistant to Penicillin
  51. 51. Listeria monocytogenes • Small Gram positive rod/ no spores • Catalase = positive • Subtle beta hemolysis on 5% blood agar • Similar to Streptococcus agalactiae • More motile at 25˚C than 35˚C • Tumbling motility on wet mount • Umbrella motility in tubed media • Cold loving – Grows well at 4˚C • Infection from refrigerated foods • Cheese – non pasteurized • Deli case foods • Milk products approaching expiration date • Ampicillin drug of choice – • Intrinsic resistance to Cephalosporins
  52. 52. Erysipelothrix rhusiopathiae • Small Gram positive rod • Catalase enzyme = negative • Alpha hemolysis produced on blood agar plate • Only G+R that produces hydrogen sulfide (H2S) - can detect on TSI agar slant – Human infections acquired from swine with Erysipelas infection – Isolated rarely from infections post cat bite – Bacteremia in humans uncommon • Occurs in IV drug addict • High % endocarditis /extensive valve damage – Intrinsic resistance to Vancomycin H2S production on Triple sugar iron agar (TSI) Erysipelas – lacy skin lesions of swine
  53. 53. Gram negative bacilli
  54. 54. • Escherichia coli • Normal flora in human intestine • #1 cause of UTI [@80% of cases] • Also, bacteremia, neonatal meningitis, and abdominal infections • Ferments lactose when grown on MacConkey agar • Spot indole reaction = positive / robin’s egg blue color • Detects breakdown of tryptophan from growth on blood agar • Green sheen produced when grown on Eosin methylene blue agar (EMB) • Pathogen of diarrhea: • Enterotoxigenic (ETEC) E. coli is the cause of traveler’s diarrhea • Enterohemorrhagic E. coli (EHEC) (such as 0157:H7) • Bloody diarrhea acquired from eating undercooked meat from an infected cow – pathogenicity from Shiga toxin production • Hemolytic uremic syndrome (HUS) can result [hemolytic anemia, thrombocytopenia, and renal failure] particularly in young children • When grown on MAC agar with sorbitol / does NOT ferment sorbitol/ most all E. coli except EHEC ferment sorbitol Green sheen on EMB agar Indole positive Lactose fermentor
  55. 55. • Enterobacter species • Enterobacter cloacae complex, most common species • Environmental GNRs with low pathogenicity • Usually infects a compromised host • Enterobacter (Cronobacter) sakazakii associated with neonatal meningitis • Klebsiella species • K. pneumoniae most common species • Mucoid colony due to capsule • Currant jelly sputum in alcoholics due to blood mixed with capsular polysaccharide in sputum
  56. 56. Enterics that do NOT ferment lactose! • Proteus species Colonies swarm in layers on agar surface • Proteus vulgaris – spot indole positive • Proteus mirabilis – spot indole negative • Normal flora in intestine - Common in UTI, abdominal infections • Serratia marcescens • Produces red pigmentation - intensifies at room temp • Environmental contaminate • Causes infection in • Immune suppressed • Ventilator associated pneumonia • Bacteremia
  57. 57. 1. Glu/lac/suc fermented with gas 2. Glucose fermented 3. Glucose fermented with H2S 4. No CHO fermentation Non fermenter Triple Sugar Iron Agar (TSI)– Used to detect fermentation of glucose, lactose and/or sucrose and production of hydrogen sulfide [H2S] in GNRs CHO Fermentation= yellow medium Gas production= Disruption of the agar H2S No CHO fermentation = Red medium 1 2 3 4
  58. 58. • Salmonella species • Diarrhea with +/- fever – polys in the stool • Infection from eating contaminated food (such as raw eggs) or direct contact with a sick animal – must ingest large #’s of organisms to make you ill (1,00,000 bacteria), normal levels of stomach acid is protective • Does not ferment lactose • Produces hydrogen sulfide on TSI slant and selective agars • Motile • Identification based on biochemical reactions and serologic typing • Kaufman White serologic typing for speciation of Salmonella • O Somatic (cell wall) antigen – Salmonella group “B” • H flagellar antigens – 2 phases [h1 & h2] • Vi capsular antigen – Salmonella typhi only
  59. 59. Salmonella typhi • Typhoid fever – fever, sepsis, treatable with antibiotics • Human pathogen - most cases in US (75%) from international travel • Post typhi infection there can be carriage in gallbladder (passed in feces), spread by bad hygiene and food handled by person shedding S. typhi • Ingested organisms enter the bowel, then move into the blood stream and eventually the bone marrow • Diagnosis best made with blood cultures and in late stages (>1 month) bone marrow culture • Vi capsular antigen can aid in serologic identification • Moustache of H2S produced in TSI medium slant
  60. 60. Shigella • Diarrhea, +/-vomiting, fluid loss, polys and blood in stool • Infection : Human to human transmission /control with good hand hygiene • Ingestion of low #’s of organisms make you ill [10 – 100 bacteria] • Does not ferment lactose • Non motile • No H2S produced • 4 species based on somatic antigen • S. boydii Group C • S. dysenteriae Group A • S. flexneri Group B • S. sonnei Group D
  61. 61. Salmonella Shigella Agar (SS agar) Salmonella and Shigella are colorless due to lactose not being fermented – H2S produced by Salmonella turning colony black Hektoen agar – Salmonella produces H2S [Hydrogen sulfide] producing black colonies Shigella – green colonies Normal flora – orange colored due to fermentation of lactose (E. coli) Non-Lactose fermenter Shigella Salmonella Normal Flora Lactose fermented H2S
  62. 62. Yersinia enterocolitica • Major reservoir – swine • Humans infected by eating raw or undercooked pork • Infections: • Diarrhea • Septicemia in patients with iron overload syndromes • Mesenteric adenitis – Infection symptomatic for Right Lower Abdominal pain which mimics appendicitis • Infected blood products from transfusion have been reported • Oxidase = neg, Indole = neg, Urease = pos, Grows well at 4 °C • CIN agar (Cefsulodin-irgasan-novobiocin) selective agar for Y. enterocolitica
  63. 63. Yersinia pestis - Plague • Category A agent – call public health! • Obligate flea/ rodent/ flea cycle in nature • Human infection usually from a rat flea bite - leads to Bubonic plague that is the infection of the lymphatic system, forms painful buboes (lymph node swelling) at site of the bite • Hemorrhagic lymph nodes spread Y. pestis into blood stream • Pneumonia develops from blood stream infection • Fatality >=50% • Endemic in SW USA • Grows well on blood agar • catalase +, oxidase - • Bipolar staining “safety pin”
  64. 64. • Vibrio cholera • Natural environment saltwater • Halophilic (salt loving) – salt enhances growth • Rice water diarrheal stool from mucus flecks (classic) • Virulence due to enterotoxin production – – Receptor on epithelial cell in small bowel – – Activates adenyl cyclase – – Increases cAMP with hyper secretion of NaCl and H20 – Death from dehydration and metabolic acidosis • Curved “C” shape Gram negative rod • Selective medium – thio citrate bile sucrose agar, (TCBS) yellow color from sucrose fermentation • Oxidase = positive , grows in 1% salt solution TCBS Agar
  65. 65. • Vibrio parahaemolyticus • Diarrhea from ingestion of raw oysters • Usually self limited but serious in immune suppressed • TCBS medium = green = sucrose fermentation negative • Vibrio vulnificus • Infection by ingestion of raw shellfish • Infections: • Diarrhea • Skin infection from injury in water • Bacteremia - leads to formation of painful skin lesions on lower extremities with muscle necrosis / This form seen in patients with liver disease (cirrhosis) – patients have increased serum iron - 50% fatality rate
  66. 66. Classic Gram Negative Rod Gram Stains Campylobacter Vibrio species Enteric gram negative rod Sea gull wings C shaped Plumb and rectangular
  67. 67. Acinetobacter species – • Environmental saprophyte and normal flora on human skin • Gram negative coccoid-bacilli • Lactose non fermenter • Oxidase enzyme negative • Ac. baumannii – nosocomial/common opportunistic pathogen particularly endo-trachs • Glucose oxidizer • Can acquire resistance to many antibiotics from antibiotic exposure • Ac. lwoffi – less commonly isolated • Glucose non oxidizer • Stenotrophomonas maltophilia • Rapid maltose oxidizer • Gram negative bacillus • Gun metal gray pigment • Intrinsically resistant to many antibiotics • Nosocomial pathogen – super-colonizer after long term carbapenem therapy due to intrinsic resistance to the carbapenems (Imipenem and Meropenem)
  68. 68. Pseudomonas aeruginosa • Fluorescent & blue-green pigment (pyocyanin) • Oxidase enzyme positive • Grape-like odor • Grows at 42˚C • Ps fluorescens/putida group– no growth at 42°C • Major pathogen of cystic fibrosis • Mucoid strains produced due to polysaccharide capsule • In combination with Burkholderia cepacia can cause major lung damage • Nosocomial pathogen associated with exposure to water and moist environments • Intrinsically resistant to many antibiotics
  69. 69. • Burkholderia cepacia • Gram negative tod • Low virulence – Environmental saprophyte • Problem organism in cystic fibrosis, particularly in co-infection of lung with Pseudomonas aeruginosa • Dry , yellow colony • Oxidase positive • Esculin hydrolysis positive • Chryseobacterium (Elizabethkingia) meningosepticum • Infections: • Newborns: fatal meningitis and septicemia in the newborn • Elderly/immune suppressed: bacteremia • Low virulence • Environmental source – water • Yellow colony, • Oxidase and Indole positive
  70. 70. Haemophilus influenzae • Transmission – close contact/secretions • Virulence factor – capsular polysaccharide • Small pleomorphic Gram negative rod • Requires 2 nutritional factors for growth: • X factor = hemin • V factor = NAD (nicotinamide adenine dinucleotide) • Grows on chocolate agar (has X and V factor) • Will not grow on 5% sheep’s blood agar • Requires C0₂ [5 – 8%] for growth • Effective vaccine targets invasive infections with H. influenzae type B (HIB) – effectively eliminating most childhood invasive infections • Resistance to Ampicillin due to beta lactamase enzyme productions [25 %], 3rd generation Cephalosporin becomes the antibiotic of choice (Ceftriaxone) for invasive infections
  71. 71. Haemophilus influenza Growth only between X and V strips Staph aureus supplies the X and V factors required Factor X and V test: Demonstrates the need for both X and V factor Demonstration of the satellite phenomenon
  72. 72. Other Haemophilus species • H. parainfluenza – • Requires V (NAD) factor only • Normal flora in the upper respiratory tract • Member of HACEK organism of endocarditis • H. (Aggregatibacter) aphrophilus • No X and V factor requirements for growth • Infections: abscess (liver, lung, brain) & endocarditis • H. ducreyi – • Requires X factor • Cause of Chancroid - venereal disease • Painful necrotizing genital ulcers/inguinal lymphadenopathy • “School of fish” appearance on stains
  73. 73. HACEK group • Oral flora organisms - Due to poor detention or invasive dental procedures organisms introduced into bloodstream and infect the heart valves • Fastidious Gram negative coccobacilli / need 2-4 days to grow in culture • 5 -10% of community acquired native valve endocarditis (not IV drug use) • Haemophilus species oxidase (-) catalase (-) • Aggregatibacter (Actinobacillus) oxidase (-) catalase (+) • Cardiobacterium hominis oxidase (+) • Eikinella corrodens oxidase (+), pits the agar, smells like bleach • Kingella kingii oxidase (+), hemolytic on blood agar • Major cause of septic joint infection in small children
  74. 74. Bordetella pertussis • Whooping cough – 3 stages of disease (1) Prodromal – flu like disease – most contagious stage (2) Catarrhal - cough - with classic whoop in small children toxin adheres to bronchial epithelial cells and cough continues until toxin wears off – can be months (3) Paroxysmal - recovery phase • Human pathogen - inhabits Nasopharynx • With disease, peripheral blood - Lymphocytosis with atypical large, irregular and deeply basophilic lymphocytes • Tiny Gram negative coccobacillus • Regan Lowe Charcoal agar / growth in 3-5 days • PCR is now standard of practice / more sensitive • Reservoir for infection – young adults due to waning immunity. Reason behind initiative for booster shots in young adults
  75. 75. Pasteurella multocida/canis • Normal flora in many animals (zoonotic) • Bite wound infections from cats and dogs • Human pneumonia from close cat and dog contact • Small Gram negative coccobacilli • Growth on 5% Sheep’s blood agar • Non hemolytic grey colony • No growth on MacConkey agar • Oxidase positive • One of very few GNRs that is sensitive to penicillin
  76. 76. Capnocytophaga species • Fusiform shaped Gram negative rods • Fingerlike projections from colonies “Gliding” • Dependence incubation in C02 for growth • Oxidase negative • Catalase negative • Normal mouth flora in humans and animals • C. canimorsus – Associated with dog bites – high % of bite infections lead to bacteremia and endocarditis • Capnocytophaga species (many) NF in humans • Infect mouth ulcers induced by chemotherapy • Can lead to bacteremia
  77. 77. Brucella species • Brucellosis, FUO, significant joint pain, intracellular pathogen of the Reticuloendothelial system • Specimens: Blood and Bone Marrow • Old: Castaneda biphasic blood culture held for 21 days • Current: Automated Blood culture systems with growth @ 5 -6 days • Laboratory acquired Brucellosis is common* Careful • Serology can assist with chronic disease • Culture: Small Gram negative coccobacilli, non hemolytic gray colony, requires C02 to grow • Oxidase positive, urease enzyme positive • Zoonosis – Infection from ingestion of raw milk, animal exposure, inhalation • B. abortus – raw cow milk • B. melitensis – raw goat milk, feta cheese • B. suis – pigs • B. canis - dogs Granuloma in bone marrow
  78. 78. Campylobacter spp. • C. jejuni – Diarrhea, common cause in US, bacteremia in HIV and immune suppressed • Ingestion of undercooked poultry / juice contaminating raw food • Sea gull shaped, poorly staining Gram negative rod • Culture requires selective blood agar with antibiotics / Campy-BAP, Skirrow’s BAP • Incubate at 42˚C in microaerophilic atmosphere (high CO₂, low O₂) • Sequelae - Significant % Guillain-Barre syndrome • C fetus – Bacteremia in the immune suppressed host. • Source cattle and sheep. • Temperature tolerance aids in identification C. jejuni – grows at 37˚C and 42˚ C, hippurate hydrolysis positive C. fetus - grows at 37˚C and 25˚C hippurate hydrolysis negative
  79. 79. • Reservoir – rabbits, rodents, ticks and flies. • Humans infected by insect bites or from exposure to animal blood (such as skinning rabbits with bare hands) • Bacteria can penetrate small breaks in skin: • cause painful skin lesions • enlarged lymph nodes • leading to bacteremia (ulceroglandular tularemia) • Pneumonia • Great hazard to lab workers by aerosolization • Small Gram negative rod/ oxidase negative • Requires cysteine in culture medium for growth • Found in chocolate agar but not blood agar Francisella tularensis
  80. 80. Helicobacter pylori • Acute gastritis – % progress to gastric adenocarcinoma • Human to human transmission/ fecal - oral route, poor hygiene • Rapid and strong urease enzyme – • Can be used for detection directly from gastric antrum biopsy tissue • Small curved Gram negative bacilli • Difficult to grow • Stool antigen/diagnosis and test of cure • Serum antibody detection obsolete/ not clinically useful • Organism stained by silver stains in GI • Treatment – Antibiotics and acidsuppression Immunohistochemical stain of gastric biopsy
  81. 81. Legionella pneumophila • Pulmonary disease – association with water • Requires cysteine in culture medium for growth • Buffered Charcoal Yeast Extract agar/ growth 3-5 days • Will not show on Gram stains using safranin • Carbol fuchsin counterstain will stain Legionella • Use silver impregnation stains in tissue • Urinary antigen test detects L. pneumophila type I infection – the most common cause of infection • Serology • Treatment: Erythromycin (macrolide) BCYEBlood agar No growth
  82. 82. Bacteria without cell walls • Mycoplasma spp and Ureaplasma spp – cell membranes only! Media contain sterols to protect the membrane • Lack of peptidoglycan cell wall – means unable to Gram stain, no colonies produced on agar, cannot be treated by antibiotics that act by inhibiting cell wall formation • M. pneumoniae – • Community acquired pneumonia • PCR for diagnosis • Presence of cold agglutinins • Genital mycoplasmas • M. hominis – fried egg on agar surface, vaginitis, cervicitis, postpartum sepsis, neonatal infections , pre rupture of membranes • Ureaplasma urealyticum – Dark metal-type appearance, rapid urea hydrolysis in broth, NGU & upper genital tract infection, spontaneous abortion, neonatal infections • PCR current method for a more sensitive diagnosis UreaplasmaMycoplasma hominis
  83. 83. Unusual and difficult to grow • Bartonella henselae • Cat scratch disease – exposure to cat and cat excrement • Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV • Bartonella quintana – cause of trench fever/ vector is the body louse • Diagnose Bartonella by Serology/PCR • Chlamydia trachomatis Serovars L1,L2,& L3 Lymphogranuloma venereum – STD that involves lymphatics and lymph nodes • Chlamydia pneumoniae (TWAR agent)- Pneumonia • Chlamydia psittaci- psittacosis, pneumonia, exotic parrot exposure • Diagnosis: Serology and PCR assays
  84. 84. Unusual and difficult to grow • Ehrlichiosis – Rickettsia bacteria • Zoonotic intracellular pathogen • Vector – Ixodes tick *(hard tick) • 2 Genera cause Ehrlichiosis • Anaplasma spp, inclusion (morula) in the PMN • Ehrlichia spp inclusion in the Monocytes • Fever, leukopenia, thrombocytopenia, • Elevated serum aminotransferases, • No rash (differs from Rocky Mountain Spotted Fever caused by Rickettsia rickettsii) • Found in south central, southeast , midwest US • PCR, serology, and exam of blood smear for diagnosis
  85. 85. Spirochetes • Borrelia burgdorferi - Lyme’s disease • Primarily found in NE part of US • Vector = Ixodes tick • Acute disease: Fatigue, headache, fever, and rash • Can have progression to chronic disease • Diagnosis: Serology and PCR • Borrelia species – Tick borne relapsing fever • Western United States • High fever (relapsing) with thrombocytopenia • Muscle and joint aches • Vector: tick (Ornithorodos hermsi) • Diagnosis: Blood smear observe spirochete
  86. 86. Spirochetes • Treponema pallidum • Syphilis • RPR and VDRL for antibody detection • Molecular methods for diagnosis • Brachyspira – • intestinal spirochete found on the brush border of the intestine, • ?? Role in disease • Leptospira interrogans – Leptospirosis • Fever with rash and renal involvement • Urine from rats and other animals contaminate water supplies Darkfield from chancre lesion (1) Leptospirosis – Shepherd’s crook (2) Presence of spirochete in a renal tubule (1) (2)
  87. 87. Unusual and difficult to grow • Granuloma inguinale • Klebsiella (Calymmatobacterium) granulomatis • Rare STD – causes ulcerative genital lesions • Streptobacillus moniliformis • Rat bite fever or Haverhill fever • Infection from rat bite • L form - cell wall deficient bacteria • Inhibited by SPS in blood culture media • Needs serum supplementation to grow • Tropheryma whipplei - Whipple’s disease • Gram positive rod (Actinomycete) distant relative Mycobacterium avium and M. paratuberculosis • Found in soil and farm animals • Diarrhea – can lead to malabsorption Foamy macrophages in the lamina propria
  88. 88. Bacterial vaginosis • A mixed anaerobic/aerobic bacterial infection • The normal balance of vaginal flora is disrupted • Rather benign infection except in pregnancy • Discharge: Fishy like odor of discharge, alkaline pH >=4.5 • Usual NF organism of Lactobacillus are overgrown by: • Gardnerella vaginalis (aerobic Gram variable rod) • Mobiluncus (anaerobic curved Gram negative rod) • Increased #s Gardnerella vaginalis is the bacterial marker for infection – Human blood agar media of choice, Beta hemolytic, Gram variable staining • Clue Cells are diagnostic and more specific than growth in culture • Molecular probe assays and amplification assays available as part of “women’s health” screening panels Clue Cell
  89. 89. Anaerobic Bacteria • Anaerobic infections can occur in virtually every any organ or region of the body • Polymicrobial –with both aerobic and anaerobic species • Endogenous organisms / usually normal flora • Increase in numbers and cause infection due to trauma, vascular or tissue necrosis cutting off the oxygen supply to the involved tissue • Treatment: Usually surgery to restore oxygen and remove necrotic tissue plus antibiotics • Anaerobic culture collection • Gel containing swabs • ESwab • Evacuated vials (port o cult)/ oxygen free • Do not refrigerate specimens – absorb oxygen in cold temperatures and can more rapidly kill anaerobes
  90. 90. Anaerobic culture • PRAS media – pre reduced anaerobically sterile • Media packaged in oxygen free environment • Most common media used • CDC anaerobic enriched blood agar • Kanamycin-vancomycin blood agar • Bile esculin agar • Thioglycollate broth • Chopped meat glucose broth • Anaerobic chambers – perform all culture work in an oxygen free closed cabinet • Anaerobic gas pack jars – for anaerobic incubation of agar plates • Wet pack – add 10 ml water to hydrogen and CO2 generating envelope/ requires palladium coated catalysts – generates heat • Dry pack – (Anaeropack) Absorbs O2 and generates CO2 PRAS
  91. 91. Anaerobic Gram Negative Rods Unusual shapes and sometimes poorly staining by Gram
  92. 92. Bacteroides fragilis group Pleomorphic irregular staining Gram negative rod • Grows in the presence of bile • Growth on Bile esculin media is black • Resistant to Penicillin and Kanamycin • Normal flora in GI tract • Infections: Related to the bowel/ GI abscess • foul smelling/gas produced • B. fragilis group • B. fragilis – most common species • B. ovatus • B. thetaiotamicron ( indole reaction positive) • B. uniformis • B. vulgatus • Resistant to Penicillin by beta lactamase enzyme / Metronidazole is antibiotic of choice, however recent reports of resistance is suggesting a need to do susceptibility testing. Growth on bile/esculin media Black pigment from Esculin production
  93. 93. Prevotella and Prophyromonas –Pleomorphic Gram negative rod –Normal flora in the upper respiratory tract –Infections: respiratory tract abscesses –Will not grow in the presence of bile –Will not turn black on esculin media –Unique** Brick red fluorescence and black pigment formed on Blood agar after one week of incubation
  94. 94. Fusobacterium spp. • F. nucleatum Long thin gram negative bacilli – spindle shaped, pointed ends • Normal flora upper respiratory tract • Infections: mouth and respiratory tract abscess formation and liver abscess • Vincent’s angina – necrotizing oral infection caused by Fusobacterium species and spirochetes • F. necrophorum – pleomorphic gram negative bacilli that filaments or chains of rods • Lemierre’s syndrome - oropharyngeal infection, leads to thrombosis in jugular vein, septicemia, with high fatality rate
  95. 95. Anaerobic Gram Positive Rods
  96. 96. Clostridium species • Gram positive bacilli (boxcar shaped ) form spores • Bacilli may over-decolorize and appear red • Clostridium perfringens • Infections: Food poisoining, necrotic tissue abscesses (Clostridial myonecrosis), bacteremia, cholecystitis • Most common anaerobic G+R in intestine • Double zone of beta hemolysis on BAP • Lecithinase produced on egg yolk agar • Reverse camp test positive Lecithinase Reverse Camp Test
  97. 97. • Clostridium botulinum – Botulism – Adult disease – Preformed heat labile neurotoxin ingested is mass produced or in home-canned foods – Infant disease - spore ingested from nature or product produced in nature such as honey or household dust, neurotoxin produced in gut • Begins with constipation and difficult sucking bottle – Both forms are ife threatening neuroparalytic disease • Clostridium tetani - Tetanus • Gram stained cells appear like Tennis racket • Tetanospasmin toxin enters by penetrating skin injury • Spastic contractions of voluntary muscles, hyper-reflexia, lock jaw (trismus) • Immunization to prevent
  98. 98. • C. septicum – • Bacteremia or Gas Gangrene in patient with underlying malignancy • Hematogenous spread from GI tract – no trauma necessary • Clostridioides (Clostridium) difficile – • Disease: antibiotic associated colitis, pseudomembranous colitis from toxin production • Toxin A – enterotoxin causing fluid accumulation • Toxin B – potent cell cytotoxin – primary virulence • Binary toxin – so called Nap1 strain is produces larger amount of toxin • Diagnosis of infection: • EIA methods [toxin A/B] are insensitive but detect active toxin • PCR methods [toxin A/B] are more sensitive, detect toxin genes • Culture – Cycloserine, Cefoxitin, Fructose Agar [CCFA] • Infants have @ 70% colonization rate – do not test
  99. 99. Actinomyces • Branching gram positive bacilli – • do not form spores • Aerotolerant, but grow best anaerobic • Normal flora oral, GI, vagina, skin • Infections: usu. oral/facial (lumpy jaw) • Form sulfur granules in tissue • Can be found on normal tonsil • Actinomyces israelii – associated with oral, thoracic, and abdominal infections, IUD infections Bread crumb colonies in broth Molar tooth colony on agar plates Penicillin susceptible.
  100. 100. Branching Gram positive rods of Actinomyces – antler like Molar tooth colony Sulfur granule Clumps of Actionomyces
  101. 101. Propionibacterium acnes renamed: Cutibacterium acnes • Pleomorphic Gram positive rod • Catalase positive, Indole positive • Normal flora - skin, oral, GU and GI • Contaminate in blood cultures from skin NF • Pathogen of acne vulgaris • Opportunistic pathogen: cerebral shunt infections and other endovascular and neurosurgical infections • Firmly established as significant cause of prosthetic joint infection – particularly shoulder joints • Cultures should be held up 7- 14 days • Therapy - Ampicillin

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