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Bacteriology 2018 Update



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Bacteriology 2018 Update

  1. 1. Bacteriology Update 2018 Margie Morgan, PhD
  2. 2. Beginning 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 • Microaerophilic – Grow better with reduced oxygen and elevated carbon dioxide % • Aerotolerant anaerobes– anaerobe is not killed by prolonged exposure to oxygen – example: Clostridium tertium • Lag Phase - >24 hrs old, growth is slowing, not appropriate for biochemical or susceptibility testing • Log Phase – Exponential growth – appropriate for all testing • Stationary phase – appropriate for transporting specimens
  3. 3. Specimen collection for Aerobic Bacteriology Throat / Wound collection 1. Swabs made of polyester fiber, do not use a cotton swab, traps bacteria /potentially toxic 2. Swab placed in Stuart’s or Amie’s transport media (buffered solution with peptones) 3. Transport media preserves bacteria viability but do not promote growth of bacteria (stasis) Urine collection – two methods 1. Boric acid / bacteria in stationary phase 2. Refrigerate within one hour Maintain original colony count and maintain viability of organisms Tissues/Sterile body fluids – Sterile containers
  4. 4. Gram stain Procedure 1 minute Rinse Primary stain Mordant 1 minute Rinse 5-10 seconds RinseDecolorizatio n Counter stain 1 minute Rinse Peptidoglycan in Gram positive cell wall traps the Crystal violet Prepare thin film of specimen on glass slide Heat or methanol
  5. 5. Use Gram Stain to Assess quality of Sputum for Bacterial culture • Sputum specimen is rejected for bacterial culture, if sputum judged to be “spit” not actually a deep cough specimen • If <10 epithelial cells/LPF – acceptable (right pix) >25 WBCs/LPF (except in leukopenia) • If >10 epithelial cells /LPF (left pix) • Sputum is judged to be spit / not a sputum specimen • Bacterial culture is not performed / it is rejected • Request made for a new “deep cough” specimen Bad Sputum Good Sputum 10X objective 10X objective
  6. 6. Commonly used Agar Media Blood agar- 5% sheep’s blood agar • Gauge hemolytic reaction of bacteria (alpha, beta, gamma) • Culture a variety of Gram positive, Gram negative bacteria and Yeast Chocolate agar • “Carmelized” 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
  7. 7. Commonly used agar media •MacConkey agar – Selective and Differential • Selective - Supports growth of only Gram negative rods, crystal violet inhibits Gram positive organisms • Differential - Lactose fermentation can be determined • Lactose fermentation = pink (neutral red indicator) • Non-lactose fermentation = no color • After media is plated, incubate in aerobic incubator at 35˚ C ,24-72 hrs
  8. 8. 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 bacterial ID
  9. 9. MALDI-TOF • Laser is fired at target containing matrix/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 bacterial 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.
  10. 10. Gram Positive Cocci Staphylococcus Streptococcus Enterococcus
  11. 11. Staphylococcus Catalase Enzyme Positive Gram positive cocci in clusters due to bound coagulase or “clumping factor” Staphylococcus aureus Coagulase Negative Staph Coagulase Enzyme Positive Coagulase Enzyme Negative +/- Yellow colony Beta hemolysis White colony Most not hemolytic
  12. 12. Catalase Enzyme Reaction Negative Positive Bacteria placed in Hydrogen Peroxide Tube Coagulase Reaction Rabbit plasma inoculated with organism / Incubate at 35˚C Read at 4 hours and if negative read again at 24 hours Negative tube coagulase =no clot Coagulase negative Staph Positive Tube Coagulase = clot Staph aureus Slide Coagulase Reaction
  13. 13. Staphylococcus aureus • Virulence: • 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 • Diseases (associated toxin): • Toxic shock syndrome (TSST-1 toxin) • Scalded skin syndrome (Exfoliatin (SSS) toxin) • Soft tissue infection (Panton valentine leucocidin toxin – PVL) • Food poisoning / Enterotoxins – stable to heating at 100*C for 30 minutes • Bacteremia and Endocarditis • Septic arthritis – Primary cause Onion skin peeling
  14. 14. MRSA = Methicillin Resistant Staphylococcus aureus –Altered Penicillin binding protein (PBP2) produced by the mecA gene codes for resistance to oxacillin/methicillin /nafcillin resistance –Cefoxitin susceptibility testing is considered a more sensitive indicator of resistance to oxacillin and provides more sensitive detection of MRSA –All MRSA are reported resistant to oxacillin, semisythetic penicillins and cephalosporins and the are not used for therapy –Vancomycin becomes a drug of choice
  15. 15. The “D” Test • Is Staph aureus really susceptible to Clindamycin?Is Staph aureus really susceptible to Clindamycin? • During therapy, S aureus isolates resistant to ErythromycinDuring therapy, S aureus isolates resistant to Erythromycin possess enzymes capable of inducing Clindamycin resistancepossess enzymes capable of inducing Clindamycin resistance • Kirby Bauer zone around Clindamycin will be blunted to form aKirby Bauer zone around Clindamycin will be blunted to form a “D” shape, if Clindamycin can be induced by Erythromycin to“D” shape, if Clindamycin can be induced by Erythromycin to be resistant – so calledbe resistant – so called INDUCIBLE RESISTANCEINDUCIBLE RESISTANCE.. • Clindamycin should be reported as resistant by clindamycinClindamycin should be reported as resistant by clindamycin induction and not used for therapy.induction and not used for therapy. D test positive Inducible Resistance (MSLb) D test negative Clindamycin can be used for therapy
  16. 16. Nares is primary colonization site for MRSA and the most common surveillance site Culture methods for surveillance: ChromAgar is commonly used - Selective and differential medium containing chromogenic substrates that turn a specific color to ID Staph aureus and Cefoxitin is added to the medium to select for only MRSA. Molecular assays (MA) can also be used to screen nares for MRSA. MAs increase the sensitivity of detection (@5-10%) but greatly increases laboratory costs. Mupiricin can be used in nares eradicate carriage Staphylococcus aureus (MRSA) Hospital Epidemiology Issue: Nares surveillance for MRSA
  17. 17. Coagulase negative Staph (CNS) @ 15 species infect humans • Staph epidermidis – most common –Major aerobic component of normal skin flora –Common cause of subacute bacterial endocarditis –Pathogenicity from cell adhesion factors forming biofilm on biologics and plastics like 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
  18. 18. • Staphylococcus hemolyticus • Can cause line related sepsis • Hemolytic on blood agar • Do not confuse with Staph aureus • Staphylococcus lugdunensis • ? greater pathogenicity than other CNS species • Isolated from Abscess and bacteremia • PYR test positive = pink • Related Cocci • Micrococcus species • Mustard yellow colony – environmental contaminate • Catalase positive / Gram positive cocci in tetrads • Tube coagulase negative • Does not ferment glucose • Staphylococcus ferment glucose CNS Neg Pos PYR
  19. 19. Streptococcus Gram positive cocci in chains and pairs Catalase enzyme negative Three groups based on hemolytic reaction on sheep’s blood agar • Alpha – greening of agar, partial hemolysis of RBCs • Viridans Streptococcus, Streptococcus pneumoniae, Granulicatella/Abiotrophia • Beta – clearing of agar, complete hemolysis of RBCs • Beta hemolytic Streptococcus groups A - G • Gamma – no clearing of agar, intact RBCs • Streptococcus bovis (gallolyticus)
  20. 20. Beta Hemolytic Streptococcu s • Beta Streptococcus are grouped using the “C” carbohydrate (CHO) in the bacteria cell wall • Identifies Beta Strep groups – A, B, C, F, and G, ones most commonly seen in human infections • Lancefield grouping system uses the “C” CHO in the cell wall of Streptococcus as the antigen in a slide agglutination test with monoclonal antibody (A<B<C<F<G) attached to latex beads - + -
  21. 21. Streptococcus pyogenes • Group A beta Streptococcus [GAS] • Bacitracin KB sensitivity test – inhibited, no growth @ disk • This test is not specific for Group A , it x-reacts with group C • PYR (pyrrolidonyl arylmidase) reaction • Organism spotted onto moist PYR disk • 2 min – RT incubation • Add Cinnamaldehyde reagent • Pink = positive = Strep pyogenes • This test is not exclusive for Strep pyogenes – Enterococcus and Staph lugdunensis also (+) • Therapy : Penicillin or Cephalosporin antibiotics No resistance reported to these agents PYR
  22. 22. • 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 solid agar • O toxin is oxygen labile S toxin is oxygen stable • When both are present - increased hemolysis is apparent on 5% Sheep’s blood agar Primary virulence factors: M Protein – prevents phagocytosis Capsule – hyaluronic capsule prevents phagocytosis
  23. 23. 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
  24. 24. Sequelae of Strep pyogenes Infections 10 -30 days following impetigo or pharyngitis Rheumatic fever • Cause: inadequately treated GAS skin or pharyngitis infections • Family history (genes), strains of GAS, multiple exposures to GAS • Usually in children 5 – 15 years • Molecular mimicry: Similarity between the proteins of Strep A and muscle tissue cause autoimmune mechanisms to lead to confusion and the immune system to attack heart (heart valves, muscle), joint, and bones • ASO titers will be positive • Usually leads to valve replacement surgery Glomerulonephritis • Nephritogenic strain associated proteins /immune mediated destruction • Renal disease with inflammation of the glomeruli • ASO titers will be positive • Usually resolves with therapy
  25. 25. 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 and produces arrow shaped hemolysis –Rapid hippurate hydrolysis – –Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyse hippurate into glycine and benzoic acid by action of hippuricase enzyme present in bacteria – 4 hour test pos Staph aureus Strep group B Camp TestHippurate Hydrolysis
  26. 26. Strep agalactiae [GBS] • Pathogen of the elderly – Bacteremia and urinary tract infection • Pathogen of neonate – in utero or perinatal organism acquisition during birthing process, infection in @ 1/2000 births • Early onset – within 7 days • Late 7 – 28 days from birth process. • Treatment: Penicillin or Cephalosporin (3rd generation) • Pregnant women carry organism in the cervix and/or rectal area (@25%) • All pregnant must be tested at 35 – 37 weeks of pregnancy. • Enrichment methods for GBS are standard of practice and must be used • Swab placed into enrichment broth (LIM) – incubate for 18 hours at 35 ˚C then subculture onto Blood agar. The broth can also be used as an enrichment method to for molecular testing methods • Ampicillin drug of choice for prevention of infection • Susceptibility testing for alternative therapies • Clindamycin testing with Inducible resistance testing confirmation
  27. 27. • Two most common species • E. faecium and E. faecalis • No well defined virulence factors • Group “D” CHO in the cell wall • Biochemical tests: • Bile esculin positive • 6.5% NaCl tolerance • PYR positive • E. faecium = arabinose fermentation positive • E. faecalis = arabinose fermentation negative Enterococcus + - PYR Neg Pos
  28. 28. Enterococcus • Pathogen of opportunity • Intestinal normal flora • UTI, bacteremia, and abdominal infections • Antimicrobial therapy: • Natural resistance to cephalosporin antibiotics • Ampicillin plus Aminoglycoside can be synergistic for therapy in cases of endocarditis • Unique susceptibility issues • Acquired resistance to vancomycin known as vancomycin resistant enterococcus or VRE • Van A (E. faecium) resistance gene • Van B (E. faecalis) resistance gene
  29. 29. Streptococcus bovis (gallolyticus)• Streptococcus gallolyticus ssp. gallolyticus (S. bovis biotype 1) associated with endocarditis and colon cancer (73%) when isolated from blood cultures • Strep gallolyticus ssp. pasteurianus formerly S. bovis biotype II- associated with neonatal meningitis • Biochemical reactions: Bile Esculin Positive 6.5% No Growth 6.5% Growth PYR Negative PYR Positive Strep gallolyticus Enterococcus
  30. 30. Streptococcus pneumoniae Alpha hemolytic Gram positive bullet (lancet) shaped cocci in pairs Polysaccharide capsule = virulence factor, antiphagocytic Identification: Bile soluble – colonies dissolve Inhibited by Optochin – ethyl in sodium deoxycholate (bile) hydrocupreine hydrochloride Colonies dissolve Inhibition zone must be >=14 mmAutolytic Changes
  31. 31. 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 at risk • 13 valent pneumococcal conjugate vaccine aids in preventing invasive infections • Susceptibility issues: • Acquired Resistance to Penicillin due to Penicillin binding proteins (PBP) • Minimum inhibitory concentration (MIC) testing necessary to detect resistance to penicillin – KB testing cannot be used • If susceptible, 1st line therapies include Penicillin or 3rd generation Cephalosporin (Cefotaxime/Ceftriaxone)
  32. 32. Viridans Streptococcus Several species of viridans 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 zone size <=13 mm • Cause 30 – 40% cases of sub acute endocarditis/native valve • Can cause abscess and various infections throughout the body in the immune suppressed host • Variable susceptibility patterns, some isolates with elevated MICs to Penicillin Viridans Streptococcus
  33. 33. 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 • Grow best when incubated in 5 – 10% CO² inclubation • Butterscotch odor to colony • Cause deep tissue abscess, bacteremia, endocarditis, intra abdominal infections… • Variable susceptibilities – so best to do susceptibility testing
  34. 34. Nutritionally Variant Streptococcus • Vitamin B6 (pyridoxal) deficient – • Will not grow on medium without B6 supplementation • Will grow in blood culture bottle due to vitamin B6 contained in patient’s blood • Will not grow on non-supplemented 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 spp • Granulicatella spp • Endocarditis – • More destructive to valve than than “regular” viridans Streptococci • Higher MIC’s to Penicillin • Combination therapy: PCN and Gentamicin Satelliting streptococcus next to S. aureus streak
  35. 35. Rare Opportunistic Gram pos cocci • Aerococcus ureae – Gram positive cocci in pairs and clusters • Alpha hemolysis , difficult to identify, 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 • Bacteremia in immune suppressed • Watch out! Do not confuse with Vancomycin resistant enterococcus (VRE)
  36. 36. Gram Negative Cocci Neisseria spp Moraxella catarrhalis
  37. 37. Gram Negative Cocci • Neisseria species and Moraxella catarrhalis • Small kidney bean shaped cocci in pairs often 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 + • N. gonorrhoeae will NOT grow on 5% Sheep’s blood agar • N. meningitidis will grow on 5% Sheep’s blood agar
  38. 38. Miniature CHO fermentation Reactions Compare (+) yellow reactions to negative (red) control well + + + Oxidase enzyme spot test: Detects presence of enzyme cytochrome oxidase Add reagent N,N dimethyl-p-phylenediamine oxalate to filter paper with organism positive = purple control Full size tube CHO fermentation test Growth on Chocolate Agar Oxidase Positive/GNDC Glucose + Glucose+/Maltose + N. Gonorrhoeae N. meningitidis
  39. 39. Neisseria meningitidis Infection • Carriage in Nasopharynx (10-20%) • African meningitis belt – highest prevalence in world • Meningitis, usually in children and young adults • Hallmark - petechiae (organisms crowd into capillaries) leads to tissue necrosis and DIC (endotoxin) /infection can be rapidly fatal • Capsular polysaccharide is the primary virulence factor • N. meningitidis serotypes A,B,C Y and W, most common • Complement deficiencies in 7,8,and 9 predispose to infection, also Eculizumab, asplenia, and HIV • Adrenal necrosis is referred to Waterhouse Friderichsen syndrome • Immunization – 2m, 12 yr, 16 yr, HIV
  40. 40. Neisseria gonorrhoeae • STD - acute urethritis, endocervix, ocular, rectal, oropharynx, septic arthritis • 10 – 20 % female ascend to PID but only 0.5% disseminate • Gram stain of urethral discharge useful for male diagnosis • Gram stain of cervix can be problematic due to NF look alike organisms, primarily Acinetobacter species • Transport – Appropriate swabs (charcoal) No refrigeration • Media: Selective Thayer Martin or Martin Lewis agar • Amplification methods [PCR] increase sensitivity of detection and superior to culture – standard of practice • Produces a beta lactamase enzyme and has Chromosomal resistance mechanisms: Therapy: Ceftriaxone + Azithromycin or Doxycycline to prevent development of resistance
  41. 41. Amplification Testing forAmplification Testing for Neisseria gonorrhea andNeisseria gonorrhea and Chlamydia trachomatisChlamydia trachomatis • Amplification methods, including Polymerase Chain Reaction (PCR)Amplification methods, including Polymerase Chain Reaction (PCR) have become the standard of practicehave become the standard of practice • Urine, Cervix, and Urethral – sites most often testedUrine, Cervix, and Urethral – sites most often tested • More sensitive than cultureMore sensitive than culture • Sensitivity/Specificity @ 96%/99%Sensitivity/Specificity @ 96%/99% • Female: most sensitive specimen is cervixFemale: most sensitive specimen is cervix • Urine @ 10 – 15% less sensitiveUrine @ 10 – 15% less sensitive • Males: virtual equal sensitivity with urine and urethralMales: virtual equal sensitivity with urine and urethral • The ancient way:The ancient way: C. trachomatis culture - Iodine staining of inclusions in McCoy cell culture – previous method of choice Fluorescent antibody stain of C. trachomatis infected cell – with green Elementary bodies
  42. 42. 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 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
  43. 43. Gram Positive Rods Corynebacteria Bacillus Listeria Erysipelothrix
  44. 44. Corynebacterium •Over 20 species, most saprophytic • Human normal flora, especially skin and nares •Most gamma hemolytic gray colonies on 5% Sheep’s blood agar •Catalase + •Diphtheroid morphology on Gram stain – Gram positive Chinese letter forms •No spores produced
  45. 45. Corynebacterium diphtheriae• Agent of Diphtheria • Diphtheritic adherent pseudo membrane produced in throat • Phage mediated exotoxin is distributed from the membrane causing respiratory paralysis (virulence factor) • 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 (1) Tellurite agar (2) Metachromatic granules
  46. 46. 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 only • Corynebacterium urealyticum – • Urease positive diphtheroid • Cause of urinary tract infection in post renal transplants • Resistant to many antibiotics – vancomycin susceptible Red is (+) for Urease reaction +
  47. 47. Bacillus species •Large gram positive rods with square ends - boxcar •Can over-decolorize easily and appear reddish on Gram •Spores produced – clearing in bacillus on Gram •Catalase enzyme positive spores
  48. 48. Bacillus anthracis Anthrax • Category A agent – highest threat to public health and bioterrorism – spores are viable for decades • Natural infection of herbivores – infected animals can have fatal infection and contaminate the environment for years • Virulence factors: anthrax toxin and capsular polypeptide • Infections: • Woolsorter’s disease – skin infection acquired from handling infected imported hides produces a unique black eschar skin lesions • Pneumonia, sepsis, and meningitis
  49. 49. Bacillus • Bacillus anthracis • Irregular shape to colony, Medusa head colonies on BAP No hemolysis on 5% Sheep’s blood agar Non-motile Penicillin susceptible Alert public health – possible B. anthracis • Bacillus cereus – • Two diseases most common: • (1) Food poisoning •Rapid onset (1-6 hr) vomiting •Preformed emetic toxin produced in food •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
  50. 50. Listeria monocytogenes• Small gram positive rod/ no spores • Catalase positive • Subtle beta hemolysis on 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 reaching outdate • Ampicillin drug of choice – • Intrinsic resistance to Cephalosporins
  51. 51. Erysipelothrix rhusiopathiae • Small gram positive rod • Catalase enzyme negative • Alpha hemolysis 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 –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
  52. 52. Gram negative bacilli
  53. 53. •Escherichia coli • Normal flora in human intestine • #1 cause of UTI [@80%] • Bacteremia, neonatal meningitis, abdominal infections, diarrhea Biochemicals: • Spot indole reaction = positive • Breakdown of Tryptophan found in blood agar • Green sheen produced on Eosin methylene blue agar • Pathogen of diarrhea • Enterotoxigenic E. coli – Traveler’s diarrhea • Enterohemorrhagic E. coli [0157:H7] - EHEC • Bloody diarrhea acquired from eating undercooked meat from an infected cow • HUS – hemolytic uremic syndrome can result [hemolytic anemia, thrombocytopenia, and renal failure] particularly in children • Does not ferment sorbitol – most other E. coli ferment Sorbitol Green sheen on EMB agar Indole positive Lactose fermentor
  54. 54. •Enterobacter species • Enterobacter cloacae complex, most common • Environmental GNRs with low pathogenicity • Enterobacter (Cronobacter) sakazakii associated with neonatal meningitis •Klebsiella species – • K. pneumoniae most common • Mucoid colony - capsule • Currant jelly sputum in alcoholics due to blood mixed with capsular polysaccharide in sputum • Klebsiella aerogenes (formerly Enterobacter aerogenes)
  55. 55. 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
  56. 56. Glu/lac/suc fermented with gas Glucose fermented Glucose fermented with H2S 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] Fermentation= yellow medium Gas = Disruption of the agar H2S No fermentation = Red medium
  57. 57. •Salmonella species • Diarrhea with +/- fever – polys in the stool • Infection from eating contaminated food (raw eggs)or directly from contact with a sick animal –must ingest large #’s of organisms to make you ill (1,00,000 bacteria), 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
  58. 58. Salmonella typhi • Typhoid fever – fever, sepsis, treatable with antibiotics • Human pathogen - most cases in US (75%) from international travel to • Post infection – carriage in gallbladder (feces), spread in food handled by S. typhi shedder • Ingested organisms enter the bowel, then moves onto the blood stream and bone marrow • Diagnosis best made with blood cultures and in late stages bone marrow culture • Vi capsular antigen can aid in serologic identification • Moustache of H2S produced in TSI medium slant
  59. 59. Shigella • Diarrhea, +/-vomiting, fluid loss, polys and blood in stool • Infection : Human to human transmission /control with good hygiene • 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
  60. 60. Salmonella Shigella Agar (SS agar) Salmonella and Shigella are colorless due to lactose not being fermented – H2S produced by Salmonella 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
  61. 61. Yersinia enterocolitica •Major reservoir – swine •Humans infected by eating raw or undercooked pork •Infections: • Diarrhea • Septicemia in iron overload syndromes • Mesenteric adenitis – RLQ pain which mimics appendicitis • Infected blood products from transfusion have been reported •Grows well at 4 °C (like Listeria) •CIN agar (Cefsulodin-irgasan-novobiocin) selective agar for Y. enterocolitica
  62. 62. 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, infection of the lymphatic system, painful buboes (lymph node swelling) at site of the bite • Hemorrhagic lymph nodes spread Y. pestis into blood • Pneumonic form develops from blood stream infection • Fatality >=50% • Endemic in SW USA • Grows well on blood agar • catalase +, oxidase - • Bipolar staining “safety pin”
  63. 63. •Vibrio cholera • Natural environment saltwater • Halophilic (salt loving) – 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 TCBS Agar
  64. 64. •Vibrio parahaemolyticus • Diarrhea from ingestion of raw /oysters • Usually self limited, worse in immune suppressed • TCBS medium = green = sucrose fermentation negative •Vibrio vulnificus • 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 in patients with pre liver disease – increased serum iron - 50% fatality rate
  65. 65. Classic Gram Negative Rod Gram Stains Campylobacte r Vibrio species Enteric gram negative rod Sea gull wings C shaped Plumb and rectangular
  66. 66. Acinetobacter species – •Environmental saprophyte and NF human skin •Gram negative coccoid-bacilli •Lactose non fermenters •Oxidase enzyme negative • Ac. baumannii - nosocomial pathogen • Glucose oxidizer • Acquired resistant to many antibiotics from exposure • Ac. lwoffi – • glucose non oxidizer •Stenotrophomonas maltophilia • Rapid maltose oxidizer • Gram negative bacillus • Gun metal gray pigment • Intrinsically resistant to many antibiotics • Nosocomial pathogen – super-colonizer when on long term Carbapenem therapy due to natural resistance to Imipenem and Meropenem
  67. 67. Pseudomonas aeruginosa • Fluorescent & blue-green pigment (pyocyanin) • Oxidase enzyme positive • Grape-like odor • Growth 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 water & moisture exposure • Intrinsically resistant to many antibiotics
  68. 68. • Burkholderia cepacia • Low virulence – Environmental saprophyte • Problem organism in cystic fibrosis: causes extensive lung damage • 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 enzyme and Indole positive
  69. 69. Haemophilus influenzae • Transmission – close contact/secretions • Virulence factor – capsular polysaccharide • Small pleomorphic Gram negative rod • Requires 2 nutritional factors for growth: • X = hemin • V= 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 eliminated most childhood invasive infections • Resistance to Ampicillin due to beta lactamase enzyme productions [25 %], 3rd generation Cephalosporin becomes the antibiotic of choice (Cefotaxime or Ceftriaxone)
  70. 70. Satellite phenomena Growth between X and V strips Staph aureus supplies the X and V factors required Demonstrates the need for both X and V factor
  71. 71. Haemophilus species • H. parainfluenza – • requires V (NAD) factor only • Norrmal flora in the upper respiratory tract • Also a HACEK organism of endocarditis • H. (Aggregatibacter) aphrophilus – (new taxonomy) • No X and V factor requirements for growth • Infections: abscesses (liver, lung, brain) & endocarditis • H. ducreyi – • Requires X factor • Cause of Chancroid - venereal disease • “school of fish” appearance on stains
  72. 72. HACEK group • Oral flora organisms - due to poor detention or dental procedures enter bloodstream and become agents of endocarditis • Fastidious Gram negative coccobacilli • 5 -10% of community acquired native valve endocarditis unrelated to IV drug use • Requires 2-4 days to grow in patient blood cultures • Haemophilus species oxidase (-) catalase (-) • Aggregatibacter (Actinobacillus) oxidase (-) catalase (+) • Cardiobacterium hominis oxidase (+) • Eikinella corrodens (pits agar) oxidase (+) • Kingella kingii oxidase (+), • hemolytic on BAP, • Septic joint infection in small children
  73. 73. Bordetella pertussis •Whooping cough –three stages of disease – (1) Prodromal – flu like disease – most contagious stage – (2) Catarrhal - cough - with classic whoop in small children – Pertussis Toxin adheres to bronchial epithelial cells and cough continues until toxin wears off – can be months – (3) Paroxysmal - recovery phase • Human pathogen - Inhabits Nasopharynx • Peripheral blood - Lymphocytosis with atypical large, irregular and deeply basophilic lymphocytes • Tiny Gram negative coccobacillus • Regan Lowe Charcoal agar / growth in 3-5days • PCR more sensitive than culture / standard of practice • Reservoir for infection – young adults due to waning immunity. Reason behind initiative for booster shots in young adults
  74. 74. Pasteurella multocida/canis • Normal flora in many animals (zoonotic) • Infections: • 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
  75. 75. Capnocytophaga species • Fusiform shaped Gram negative rods • Fingerlike projections from colonies “Gliding” • Dependence on CO2 incubation for growth • Oxidase negative • Catalase negative • Normal mouth flora in humans and animals • C. canimorsus – Associated with dog bites – high % of these bite infections lead to bacteremia and endocarditis • Capnocytophaga species (many) in humans • Infect mouth ulcers induced by chemotherapy • Can lead to bacteremia
  76. 76. Brucella species • Disease – Brucellosis, fever of unknown origin, significant joint pain, intracellular pathogen of the RES system • Specimens: Blood and Bone Marrow (chronic disease) • Serology can assist with chronic disease • Small Gram negative coccobacilli – difficult/slow to grow in culture • 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 • Zoonosis – ingestion of raw milk, animal exposure, inhalation • Brucella species dependent upon animal species the patient was exposed: • B. abortus – raw cow milk • B. melitensis – raw goat milk, feta cheese • B. suis – pigs • B. canis - dogs Granuloma in bone marrow
  77. 77. 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
  78. 78. • 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 • Requires cysteine in culture medium for growth Francisella tularensis
  79. 79. 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 for past exposure • Organism stained by silver stains in GI • Treatment – Antibiotics and acid suppression Immunohistochemical stain of gastric biopsy
  80. 80. 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) BCYE Blood agar No growth
  81. 81. 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 more sensitive diagnosis UreaplasmaMycoplasma hominis
  82. 82. 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 lymphatics and lymph nodes involved • Chlamydia pneumoniae (TWAR agent)- Pneumonia • Chlamydia psittaci- psittacosis, pneumonia, exotic parrot exposure • Diagnosis: Serology and PCR assays
  83. 83. 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 Rocky Mountain Spotted Fever) •Found in south central, southeast , midwest US •PCR, serology, and exam of blood smear for diagnosis
  84. 84. 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 recurrentis - Relapsing fever • High fever (relapsing) with thrombocytopenia • Vector: body louse • Diagnosis: Blood smear observe spirochete
  85. 85. Spirochetes • Treponema pallidum • Syphilis • RPR and VDRL for antibody detection • Molecular methods • 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)
  86. 86. • 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 Unusual and difficult to grow Foamy macrophages in the lamina propria
  87. 87. 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
  88. 88. 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
  89. 89. Anaerobic culturing • 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 environment • 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
  90. 90. Anaerobic Gram Negative Rods Unusual shapes and sometimes poorly staining by Gram
  91. 91. 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 Growth on bile/esculin media Black pigment from Esculin production
  92. 92. 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
  93. 93. 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 form filaments or chains of rods • Lemierre’s syndrome - oropharyngeal infection, leads to thrombosis in jugular vein, septicemia, with high fatality rate
  94. 94. Anaerobic Gram Positive Rods
  95. 95. 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
  96. 96. • 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
  97. 97. •C. septicum – • Bacteremia or Gas Gangrene in patient with underlying malignancy • Hematogenous spread from GI tract – no trauma necessary •C. 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
  98. 98. 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.
  99. 99. Branching Gram positive rods of Actinomyces – antler like Molar tooth colony Sulfur granule Clumps of Actionomyces
  100. 100. Propionibacterium acnes renamed: Cutibacterium • 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