Bacteriology, classification and morphology.pptx

Bacteriology classification and morphology

1. Bacteriology Review

2. Obligate Aerobe – require oxygen (20%) to grow Obligate Anaerobe –>30 min of oxygen exposure can be toxic Facultative anaerobes – grow in aerobic and anaerobic conditions, most “aerobic” bacteria are actually facultative Microaerophilic – Grow better with reduced oxygen and elevated Carbon dioxide % Aerotolerant anaerobes– anaerobe not killed by prolonged exposure to oxygen – example: Clostridium tertium Lag Phase - >24 hrs old, growth slowing, not appropriate for biochemical or susceptibility testing Log Phase – Exponential growth – appropriate for all testing Stationary phase – appropriate for transporting specimens

3. Specimen collection for Aerobic Bacteriology Throat / Wound collected using swab Swab placed in Stuart’s or Amies transport media buffer solution with peptones – Preserve viability but not promote growth of bacteria Swabs made of polyester or sponge like material, cotton traps organisms/potentially toxic Urine – two methods suggested 1. Boric acid to place organism in stationary phase 2. Refrigerate within one hour, Specimen stable for 24 hours Purpose to maintain original colony count Maintain viability of organisms

4. Gram stain Procedure 10 seconds- 1 minute Primary stain Mordant 10 seconds- 1 minute Rinse 5-10 seconds Rinse Rinse Decolorization Counter stain 10 seconds- 1 minute Peptidoglycan in positive cell wall traps the Crystal violet, hence blue color

5. Gram positive cocci in pairs and chains Streptococcus Gram negative bacillus Resembling an Enteric Gram positive rod most likely a Bacillus species Gram positive cocci in clusters, Staphylococcus Gram negative rod – Fusiform Shaped – Fusobacterium species Some Gram stains to remember! Visit the Gram stain Tutorial for more organisms!

6. Gram stain is used to assess quality of Sputum specimen for culture Expectorated sputum specimen is rejected for bacterial culture if judged to be “spit”: Perform Gram stain of sputum specimen If >= 25 epithelial cells /field 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

7. Most used Agar Media in Bacteriology Blood agar- 5% sheep’s blood agar Gauge hemolytic reaction of bacteria (alpha, beta, gamma) Grow variety of Gram positive & Gram negative bacteria and Yeast Chocolate agar – “ carmelized ” blood agar – more enriched than blood agar medium. Will support the growth of the same organisms as blood agar Plus the more fastidious bacteria like Haemophilus influenzae and Neisseria gonorrhoeae

8. Most used agar media in Bacteriology MacConkey agar – Supports growth of Gram negative rods (GNR) only (crystal violet inhibits Gram positive organisms) Selective (only GNR grow ) and Differential (Lactose fermentation can be determined) Lactose fermentation = pink (neutral red indicator) (+) non-lactose fermentation = no color (-) After media is plated incubate in aerobic and/or CO2 incubators at 35˚ C for 24 – 72 hours

9. Newest Method for Organism Identification MALDI-TOF mass spectrometry Matrix-assisted laser desorption/ionization Identification of organisms by analyzing proteins

10. Gram Positive Cocci Staphylococcus Streptococcus Enterococcus

11. Staphylococcus Gram positive cocci inclusters due to bound coagulase or “clumping factor” All Staphylococcus are Positive for Catalase Enzyme Staphylococcus aureus Coagulase Negative Staph Coagulase Enzyme Positive Coagulase Enzyme Negative +/- Yellow colony Beta hemolysis White colony Most not hemolytic

12. Catalase Enzyme Reaction Negative Positive Hydrogen Peroxide plus bacteria Avoid blood agar when collecting Staph – blood has innate catalase activity and will give false positive catalase reaction

13. 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 [at 4 or 24 hrs] = Clot = Staph aureus Tube Coagulase Reaction

14. Slide Agglutination Test to identify Staphylococcus aureus Clumping factor (coagulase) and protein A is found on the S. aureus cell wall. Fibrinogen and IgG to protein A is on the surface of the Staphaurex latex beads S. aureus will clump in the Staphaurex latex bead solution and positively identifies Staphylococcus aureus. Coagulase negative Staphylococcus species remain milky and do not clump in the solution. Advantage over tube coagulase – reaction time is 30 seconds vs. 24 hours clumping No clumping

15. Staphylococcus aureus Primary virulence factor: Protein A – surface protein, ability to bind immunoglobulin and combat the immune response Diseases and associated toxin: Toxic shock syndrome (TSST-1 toxin) Scalded skin syndrome ( Exfoliatin (SSS) toxin) Soft tissue infection (Panton valentine leucocidin toxin – PVL) Septic arthritis – primary cause Food poisoning / Enterotoxins – stable to heating at 100*C for 30 minutes Bacteremia and endocarditis Toxins act as superantigens , recruit host defense cells that liberate cytokines with systemic effects

16. Susceptibility issues –(1) MRSA Methicillin Resistance (MRSA =Methicillin resistant S. aureus ) Altered Penicillin binding protein (PBP2) produced by the mecA gene codes for resistance to oxacillin /methicillin/ nafcillin All MRSA are considered resistant to semisythetic penicillins and cephalosporins Vancomycin is drug of choice – although reduced susceptibility reported Cefoxitin susceptibility testing is considered a more sensitive indicator of resistance and provides better detection of MRSA than oxacillin testing. Shown is a Kirby Bauer (KB) disk test that is resistant to cefoxitin .

17. (2) The “D” Test To accurately determine if Staph aureus /MRSA is susceptible to Clindamycin During therapy, S aureus isolates resistant to Erythromycin possess enzymes capable of inducing Clindamycin to become resistant and therefore not useful for therapy. Kirby Bauer zone around Clindamycin will be blunted to form a “D” if Clindamycin can be induced by Erythromycin to be resistant – so called INDUCIBLE RESISTANCE. Clindamycin should be reported as resistant by clindamycin induction and not used for therapy. D test positive Inducible resistance D test negative Clindamycin can be used for therapy

18. Nares is a colonization site for MRSA and the most common surveillance site cultured. ChromAgar is commonly used to detect MRSA. It is a selective and differential media with chromogenic substrates that turn specific colors to ID Staph aureus. Cefoxitin is added to the medium to select for methicillin resistant Staph aureus (MRSA). Molecular assays (MA) can also be used to screen nares for MRSA. MAs increase the sensitivity of detection (@5-10%) but increases laboratory costs. Mupiricin can be used to eliminate carriage Staphylococcus aureus (MRSA) Hospital Epidemiology Issue: Nasal surveillance for MRSA

19. Coagulase negative Staph (CNS) @ 15 species infect humans Staph epidermidis – is the 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 plastic (Catheters and replacement joints) – can be difficult to treat Staph saprophyticus – Urinary tract infections in the child bearing age female, This species of CNS adheres in greater #’s to epithelial cells Novobiocin resistant by KB disk test can be used as test to identify Staph saprophyticus White non-hemolytic colony resistant

20. CNS & Related Cocci Staph hemolyticus – Can cause line related sepsis This CNS is hemolytic on blood agar Do not confuse with Staph aureus Staph lugdunensis Positive PYR test Perhaps greater pathogenicity than other species Micrococcus species Mustard yellow colored colony Catalase positive / Gram positive cocci in tetrads Tube coagulase negative Does not ferment glucose Staphylococcus ferment glucose Environmental contaminate Pos Neg

21. Streptococcus Gram positive cocci in chains and pairs Catalase enzyme negative Grouped by hemolytic pattern on 5% sheep’s blood agar Alpha – greening of agar, partial hemolysis of RBCs Viridans Streptococcus, Strep 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)

22. Beta hemolytic Streptococcus Beta Streptococcus are grouped using the “C” (CHO) carbohydrate in the cell wall of the bacteria Identifies the Beta Strep groups – A, B, C, F, and G that are most common cause of human infections Lancefield grouping system uses the “C” CHO in the cell wall of Streptococcus (antigen) in a slide agglutination reaction with purchased monoclonal antibody attached to latex beads See below slide for a positive and negative reaction. Immunologic typing is more accurate than biochemical testing to group Streptococcus + -

23. Streptococcus pyogenes Genus/species for Group A beta Streptococcus [GAS] Biochemical identification: Bacitracin KB sensitivity test – inhibited, no growth @ disk This test is not specific for Group A , it x-reacts with group C “A” disk is the Bacitracin disk PYR (pyrrolidonyl arylmidase) reaction Organism spotted on moist disk 2 min – RT incubation Add Cinnamaldehyde reagent Look for color reaction Pink = positive = Strep pyogenes This test is not exclusive for Strep pyogenes – Enterococcus and Staph lugdunensis are also positive for PYR Therapy : Penicillin or Cephalosporin antibiotics

24. Streptolysin O and Streptolysin S toxins Comprise the ASO titer dtermination Cell toxins lead to evasion from the immune system O toxin is oxygen labile S toxin is oxygen stable When both are active - most hemolysis occurs in the 5% Sheep’s blood agar Primary virulence factors : M Protein – prevents phagocytosis Capsule – hyaluronic capsule protects from phagocytosis

25. Streptococcus pyogenes Diseases Diseases – Pharyngitis Impetigo (1) Erysipelas (2) Cellulitis (3) Necrotizing fascitis (4) Puerperal sepsis Toxic Shock Scarlet fever (5) 1 2 3 4 5

26. Sequelae of Strep pyogenes infections Rheumatic fever Cause: inadequately treated Strep throat Rise in the 1990s due to EIA tests being used as the only diagnostic test for pharyngitis – EIA tests are @ 60-70% sensitive – and led to children not receiving antimicrobial therapy. All negative EIA specimens on children must be confirmed with culture Similarity between the proteins of the Strep A and muscle tissue cause immune system confusion which leads to immune system to attack heart, joint, and bones Anti- streptolysin O measures the amount of antibodies against Streptococcus group A in the serum Glomerulonephritis 10-14 days following skin infection or pharyngitis Renal disease with inflammation of the glomeruli ASO titers will be positive Usually resolves with therapy

27. Streptococcus agalactiae (Group B) Lancefield agglutination test demonstrates “B” cell wall CHO Biochemical tests for identification: Camp test – performed using a Staph aureus strain with Camp factor streaked perpendicular to group B Strep, incubate 24 hr. / intensifies toxin and produces arrow shaped hemolysis Rapid hippurate hydrolysis - 4 hr identification of GBS - Hippurate disk in Saline inoculated with Group B Strep Ninhydrin reagent added for color formation Positive = Purple pos Arrow-like Staph aureus Strep group B

28. Strep agalactiae [GBS] Pathogen of the elderly – mostly bacteremia and urinary tract infection Pathogen of neonate – in utero or perinatal organism acquisition during birthing process Early onset – within 7 days Late 7 – 28 days from birth process. Treatment: Penicillin or Cephalosporin (3 rd generation) Pregnant women carry organism in the cervix and/or rectal area. All pregnant should be tested at 35 – 37 weeks of pregnancy. Enrichment methods for GBS is standard of practice and must be used Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then subculture onto Blood agar. This broth can also be used as an enrichment method to enrich for molecular testing. Carrot enrichment broth turns orange with growth of group B Strep Carrot Broth

29. It used to be Streptococcus bovis Taxonomy update – S. gallolyticus Streptococcus gallolyticus ssp. gallolyticus (formerly S. bovis biotype 1) associated with colonic cancer and endocarditis Strep gallolyticus ssp. pasteurianus (formerly S. bovis biotype II) associated with neonatal meningitis S. gallolyticus (both ssp ) have same biochemical reactions Bile esculin slant – positive (turns black) 6.5% NaCl – no growth PYR reaction - negative Susceptible to Penicillin

30. Two most common species E. faecium E. faecalis No well defined virulence factors Possesses the Group “D” CHO in the cell wall Biochemical tests: Bile esculin positive – growth and black precipitin Growth in 6.5% salt PYR positive (Group A beta Strep and Staph lugdunensis also positive)* E. faecium = arabinose fermentation positive E. faecalis = arabinose fermentation negative Enterococcus Bile esculin PYR + - Neg Pos Neg Pos 6.5% Salt PYR

31. Enterococcus Variety of infections / Pathogen of opportunity Intestinal normal flora Cause 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 genes Van B (E. faecalis) resistance genes

32. Alpha hemolytic Streptococcus Streptococcus pneumoniae Gram stain = Gram positive bullet (lancet) shaped cocci in pairs Polysaccharide capsule = virulence factor, antiphagocytic Identification: Bile soluble – colonies dissolve in sodium deoxycholate (bile) Optochin sensitive – 14mm or greater zone of inhibition around the 6mm optochin disk – “P” = pneumococcus Inhibition >=14mm NO Inhibition Colonies dissolved

33. Streptococcus pneumoniae Upper and Lower respiratory tract infection, sepsis and meningitis Multi-serotype vaccine aids 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 – in broth or Etest method Testing of CSF isolate a result of Penicillin = >2 mcg/ml is resistant Therapy – If susceptible 1 st line therapy is either Penicillin or 3 rd generation Cephalosporin MIC MIC

34. Viridans Streptococcus Several species of alpha hemolytic Streptococcus are NF in mouth and upper respiratory tract S. mutans S. salivarius S. sanguis S. mitis Bile esculin slant = negative Not bile soluble 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 especially in the immune suppressed host Variable susceptibility patterns, some isolates with elevated MICs to Penicillin Viridans Streptococcus

35. 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, perhaps due to capsule Cause deep tissue abscess, bacteremia, endocarditis, intra abdominal infections… Variable susceptibilities – so best to do susceptibility testing

36. Nutritionally Variant Streptococcus Vitamin B6 (pyridoxal) deficient – so will not grow on media without B6 supplementation Will grow in a patient blood culture bottle due to vit B6 in patient’s blood Will not subculture onto 5% Sheep’s blood agar plate ( no vit B6) Requires Staph aureus streak (supplies vitamin B6) Nutritionally variant Strep will satellite @ S. aureus streak (see pix) There are 2 species: Abiotrophia spp Granulicatella spp Endocarditis – More destructive to valve than than “regular” viridans Strep Higher MIC’s to Penicillin Satellite streptococcus

37. Gram Negative Cocci Neisseria Moraxella

38. Gram Negative Cocci Neisseria species and Moraxella catarrhalis Small kidney bean shaped cocci in pairs 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 + N. gonorrhoae will NOT grow on 5% Sheep’s blood agar N. meningitids will grow on 5% Sheep’s blood agar

39. Miniature CHO fermentation reactions for Neisseria and Moraxella catarrhalis 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/ blue control Full size tube CHO fermentation test

40. Neisseria meningitidis Meningitis in children and young adults Hallmark is petechia (organisms crowd into capillaries) tissue necrosis and DIC (endotoxin) /infection can be rapidly fatal Carriage in Nasopharynx/ Susceptible to Penicillin Capsular polysaccharide is the primary virulence factor Complement deficiencies in 7,8,and 9 can predispose to chronic type disease Adrenal necrosis is referred to Waterhouse Friderichsen syndrome Type C is the current endemic strain in the USA – 1*problem in college Freshmen living in dorm Immunization available for all serotypes except serotype B

41. Neisseria gonorrhoeae 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, however, cervix Gram stain not specific for females Transport for culture – charcoal swabs, No refrigeration Media: Selective Thayer Martin or Martin Lewis agar Amplification methods [PCR] increase sensitivity of detection and superior to culture Produces a beta lactamase enzyme and also Chromosomal resistance : Therapy: Ceftriaxone + Azithromycin or Doxycycline to prevent resistance

42. Neisseria species gram negative diplococcus – intra and extra cellular Acinetobacter species – Cocci are larger and more round than Neisseria, not usually in pairs – normal flora in female genital tract Primary reason why female Gram stains Cannot be trusted.

43. Moraxella catarrhalis Pneumonia, ocular, sinusitis, otitis media Gram stain of sputum can be helpful in diagnosis of Moraxella pneumonia Polys with gram negative diplo-cocci Hockey puck colony – able to push around on agar surface Oxidase enzyme positive DNA’ase enzyme positive Resistant to ampicillin by beta lactamase enzyme production – Cefinase test can be used Therapy: Augmentin or 2 nd or 3 rd generation Cephalosporin

44. Gram Positive Rods Corynebacteria Bacillus Listeria Erysipelothrix

45. Corynebacterium Over 20 species, most not pathogenic Gamma hemolytic gray colonies Catalase + Diphtheroid morphology – Gram positive Chinese letter forms N o spores produced

46. 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) Toxin detected by Elek immuno-diffusion test Grows well on 5% Sheep’s BAP Selective medium Cysteine Tellurite agar Produces black colonies with brown halos Grow on egg containing Loeffler medium then stain with methylene blue to observe metachromatic granules - colorful storage granules characteristic of C. diphtheria Elek plate Tellurite agar Metachromatic granules

47. Other Corynebacterium Corynebacterium jeikeium – normal skin flora bacteria / thrives on lipid Infects patients with plastic catheters and indwelling devices by tunneling into the device from the skin Biofilms are formed on the plastic, protecting the organism from antibiotic therapy Very resistant to most antibiotics Susceptible to vancomycin and tetracycline only Corynebacterium urealyticum – Rapidly urease positive diphtheroid Urinary tract infection in post renal transplants Resistant to many antibiotics – vancomycin susceptible Red is (+) for Urease reaction

48. Bacillus species Large gram positive rods boxcar shaped with square ends Can over-decolorize easily and appear reddish Spores produced – causes clearing in rod Catalase enzyme positive Most are motile

49. Bacillus anthracis Agent of Anthrax Natural infection of herbivores – infected animals can have fatal infection and contaminate the environment for years Virulence factors: anthrax toxin and capsular polypeptide Skin ( Woolsorters ’ disease) is the most common presentation Unique black eschar skin lesions Pneumonia, sepsis, and meningitis uncommon

50. Bacillus Bacillus anthracis culture: Medusa head colonies on BAP non-hemolytic on 5% Sheep’s blood agar Non-motile Penicillin susceptible With these reactions – Alert public health department for assistance Bacillus cereus – food poisoning with rapid onset (1 – 6 hr ) of vomiting Preformed emetic toxin in food Fried rice is one of the most common food sources Traumatic wound infection from contaminated soil Beta hemolytic colony on Sheep’s blood agar Motile

51. Listeria monocytogenes Small gram positive rod Catalase positive / No spores produced Subtle beta hemolysis on Blood agar Tumbling motility on wet mount Umbrella motility in tube media More motile at 25˚C than 35˚C Cold loving – Grows well at 4˚C and reason it is abundant in refrigerated foods Found in dairy products and deli case meats Infections: Bacteremia in pregnant women and can induce still births Infections: Bacteremia and CNS in immune suppressed Ampicillin is drug of choice/ resistant to Cephalosporins

52. Erysipelothrix rhusiopathiae Small gram positive rod Catalase enzyme negative Alpha hemolysis on blood Only G+R that produces hydrogen sulfide (H2S) in a TSI agar slant Human skin infection acquired from swine with erysipelas or poultry Infection most common in butchers Bacteremia uncommon most common in drug addict High % of endocarditis with extensive damage to valve Intrinsically resistant to Vancomycin H2S production on Triple sugar iron agar (TSI)

53. Gram negative bacilli Enterics Non fermenters Fastidious

54. Enterobacteriaceae Enteric Gram Negative Bacilli Ferment glucose Almost all are oxidase negative Nitrate reduced to nitrates Non fermenters Glucose is not fermented Oxidase positive and negative species

55. Enterics that Ferment Glucose Escherichia coli Major aerobic NF in intestine #1 cause of UTI [@80%] Bacteremia, neonatal meningitis Abdominal cavity infections Spot indole reaction = positive Green sheen produced on EMB agar Pathogen of diarrhea Traveler’s diarrhea - travel to developing countries Enterohemorrhagic E. coli [0157:H7 as well as other serotypes] Bloody diarrhea usually acquired from eating undercooked cow meat from an infected cow HUS – hemolytic uremic syndrome [hemolytic anemia, thrombocytopenia, and renal failure] particularly in children Does not ferment sorbitol – most other E. coli types ferment Sorbitol Green sheen on EMB agar Mucoid colony Indole positive = Robin’s egg blue

56. Enterics that ferment Glucose Enterobacter species Enterobacter cloacae and E. aerogenes most common Environmental organism with low pathogenicity Enterobacter (Cronobacter) sakazakii associated with neonatal meningitis Klebsiella species – K. pneumoniae most common Mucoid colony Currant jelly sputum in alcoholics due to blood mixed with capsular polysaccharide in sputum

57. Enterics that do NOT ferment lactose! No fermentation = colorless colony on Mac Proteus species Swarming colonies in layers on agar surface Indole positive – Proteus vulgaris Indole negative – Proteus mirabilis Serratia marcescens Produces a red pigment that intensifies at room temp Causes infections in immune suppressed Ventilator associated pneumonia Bacteremia

58. Gram negative bacillus MacConkey agar Lactose fermenter Lactose non fermenter Gram stain enteric bacilli – plump No lactose fermentation Lactose fermentation

59. Glu/lac/suc fermented with gas Glucose fermented only Glucose fermented with H2S No CHO fermentation Non fermenter Triple Sugar Iron Agar – Detect fermentation of glucose, lactose and/or Sucrose and the production of hydrogen sulfide [H2S] Fermentation= yellow Gas = Disruption of the agar H2S No fermentation = red

60. Extended Spectrum Beta Lactamase Enzymes – “ESBL” ESBL enzymes are produced by numerous species of gram negative rods These enzymes confer resistance to Cephalosporins and synthetic Penicillins Plasmid mediated Tem 1 beta lactamase is the most common ESBL enzyme produced Possible plasmid dissemination makes this an infection control issue ESBL producing GNR are usually treated with: Carbapenemases – Meropenem , & Imipenem most common

61. Carbapenamase Resistance (CRE) Resistance enzymes conferring resistance to carbapenamase antibiotics (meropenem and imipenem) Most common in Klebsiella pneumoniae – where this enzyme is known as “KPC” Cross resistance to virtually all classes of antibiotics – so very difficult to treat Treatment: Colistin or antibiotic combinations

62. Salmonella species Salmonella spp. Diarrhea with +/- fever – polys in the stool Infection from food - Eggs, meats and contaminated uncooked vegetables, must ingest large #’s of organisms to make you ill (1,00,000 bacteria), stomach acid protective Does not ferment lactose/ produces Hydrogen sulfide Selective agars for growth: SS and Hektoen Identification based on biochemical reactions & serologic typing Kaufman White serologic typing scheme O Somatic (cell wall) antigen – Salmonella group “B” H flagellar antigens – 2 phases [h1 & h2] aids in speciation Salmonella Vi capsular antigen – Found in S. typhi only

63. Salmonella Shigella Agar (SS agar) Salmonella and Shigella are colorless due to lactose not being fermented Hektoen agar – Salmonella produces H2S [Hydrogen sulfide] producing black colonies Shigella – green colonies Normal flora – orange colored due to fermentation of lactose

64. Salmonella typhi Typhoid fever – fever, sepsis, high fatality rate Vi capsular antigen surrounds the D cell wall antigen Boil solution of organism for 15 minutes to destroy the Vi capsular antigen and expose the cell wall D antigen Serotyping can then be performed for the D antigen Moustache of H2S in the TSI tube Carrier state in gallbladder Ingest organism, it clears the bowel then in @ 1 week enters Blood stream & Bone Marrow

65. Shigella Diarrhea, +/-vomiting, fluid loss, polys and blood in stool , tenesmus, usually treated, Quinolone therapy Human to human transmission /control with good hygeine Low #’s of organisms to make you ill [10 – 100 bacteria] Non motile and No H2S produced (differ from Salmonella) – Green colonies on Hektoen Does not ferment lactose 4 species based on somatic antigen S. boydii Group C S. dysenteriae Group A S. flexneri Group B S. sonnei Group D

66. Yersinia enterocolitica Diarrhea Major reservoir – swine Human usually infected by non pasteurized milk Causes septicemia in iron overload syndromes Unique infection Mesenteric adenitis – RLQ pain which mimics appendicitis Grows well at 4 °C (like Listeria) CIN agar (Cefsulodin-irgasan-novobiocin) is selective for Y. enterocolitica Has been associated banked blood infections related to transfusion of infected products

67. Yersinia pestis - Plague Category A agent of bioterrorism Obligate flea/ rodent/ flea cycle Infected Flea bite - leads to Bubonic form/ painful buboes (lymph node swelling) Pneumonic form +/- bacteremia spread Fatality >=50% Endemic Southwestern USA Grows on blood agar catalase +, oxidase - Bipolar staining “safety pin”

68. Glucose Fermenters Oxidase Positive Vibrio cholerae Rice water stool/ mucus flecks in diarrhea Natural environment is saltwater Virulence due to enterotoxin – receptor on epithelial cell –activates adenyl cyclase – increases cAMP with hyper secretion of NaCl and H20 – death from dehydration and metabolic acidosis Halophilic bacteria which means it is a salt loving / 1% salt actually enhances growth Curved appearance to gram negative rod Selective media – TCBS = thio citrate bile sucrose agar, colony turns yellow due to sucrose fermentation TCBS Agar

69. Other Vibrio species Vibrio parahaemolyticus Colony on TCBS medium is green = sucrose negative Diarrhea from ingestion of raw fish and shellfish Vibrio vulnificus Highly virulent Vibrio species – infection leads to formation of painful skin lesions on lower extremities with muscle necrosis Most prevalent in patients with pre existing liver disease 50% fatality rate Ingestion of raw oysters and shellfish from coastal waters Usuallly Green colonies TCBS

70. Classic Gram Negative Rod Gram Stains Campylobacter Vibrio species Enteric gram negative rod Sea gull wings C shaped Plumb and rectangular

71. Do not ferment Glucose Oxidase negative Acinetobacter species – isolated from a hospital environment and human skin, large gram negative cocco-bacilli Ac. baumannii is a major nosocomial pathogen Glucose oxidizer Acquires antibiotic resistance with pressure Ac. lwoffi – glucose non oxidizer Stenotrophomonas maltophilia Rapid maltose oxidizer Long Gram negative bacillus Naturally resistant to many antibiotics Gun metal gray pigment Nosocomial pathogen – super-colonizer when on long term Imipenem therapy due to natural resistance to Imipenem

72. Glucose non-fermenter Oxidase Positive Pseudomonas aeruginosa Fluorescent & blue-green pigment( pyocyanin ) Grape-like odor Growth at 42˚C Ps fluorescens / putida – no growth at 42°C Major pathogen in cystic fibrosis Mucoid strains [polysaccharide capsule] in combination with Burkholderia cepacia can cause major lung damage Nosocomial pathogen – associated with water & moisture Intrinsically resistant to many antibiotics

73. Glucose Non Fermenters/oxidase + Burkholderia cepacia Dry , yellow colony Oxidase negative Problem organism in cystic fibrosis colonization and infection with extensive lung damage Flavobacterium ( Chryseobacterium ) meningosepticum Associated with fatal septicemia in the neonate Low virulence Environmental source Yellow pigment

74. Haemophilus species Haemophilus influenza Variety of infections Transmission – close contact secretions Polysaccharide “B” capsule = virluence factor Requires 2 nutritional factors for growth X = hemin V= NAD (nicotinamide adenine dinucleotide) Demonstrate by satellite phenomenon or X/V strips Small pleomorphic GNR / Grows on chocolate agar Will not grow on 5% sheep’s blood agar Requires high level C0₂ [5 – 8%] for growth Vaccine targets the H. influenza type B Resistance to Ampicillin by beta lactamase production [15- 20 %], Cefotaxime becomes the antibiotic of choice +

75. Disk test for Beta lactamase Detection Add bacteria to filter paper impregnated with Nitrocefin or Cefinase test (yellow colored/chromogenic cephalosporin) Incubate at room temp for @ 1 minute Positive result is color change from yellow to red - beta lactamase enzyme breaks down beta lactam ring to form hydrolyzed red end product Detects resistance to Amp/Pen/Cephalosporin in Haemophilus species, N. gonorrhoea , Moraxella catarrhalis , and anaerobic gram negative rods Does NOT detect the ESBL enzyme

76. More Haemophilus species H. parainfluenza – requires V (NAD) factor only Usually normal flora in the upper respiratory tract One of the H ACEK organisms of endocarditis H. (Aggregatibacter) aphrophilus – (new taxonomy) No 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

77. Satellite phenomena Demonstrates need for X and V factor Small pleomorphic GNR

78. The HACEK group Oral flora can be pathogens of endocarditis caused by poor detention or dental procedures Requires 2-4 days to grow in patient blood cultures H aemophilus species = oxidase neg, catalase neg A ctinobacillus actinomycetom-comitans = oxidase neg, catalase pos C ardiobacterium hominis = oxidase pos E ikinella corrodens = oxidase pos, colony pits BAP K ingella kingii = oxidase pos, hemolytic on BAP , also cause infections in small children (septic joints)

79. 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 Toxin adheres to bronchial epithelial cells and cough continues until toxin wears off – can be months (3) Paroxysmal - recovery phase Gram stain = Tiny gram negative coccobacillus Inhabits Nasopharynx – specimen source PCR most sensitive and specific for diagnosis Charcoal containing media for transport and culture – Regan Lowe Charcoal agar Reservoir for infection – young adults due to waning immunity. Reason behind booster shots for young adults

80. Pasteurella multocida Primary cause of Cat and Dog bite wound infections Human pneumonia from close animal contact Infections can disseminate to blood stream Found as normal flora in animal’s mouth Small gram negative coccobacilli Growth on 5% Sheep’s blood agar Non hemolytic grey colony Will not grow MacConkey agar (big clue) Oxidase Positive One of very few Gram negative rods sensitive to penicillin

81. Capnocytophaga Fusiform shaped gram negative rods – very pleomorphic Gliding motility, fingerlike projections from colonies Oxidase negative, Catalase negative C. canimorsus – dog bites with bacteremia – high% of these infections lead to endocarditis Other Capnocytophaga species are normal flora in the human mouth and Can infect mouth ulcers induced by chemotherapy – Blood stream can be invaded from infected mouth ulcer

82. Brucella species Disease – Fever of unknown origin, significant joint pain Small gram negative coccobacilli – slow and difficult to grow from blood cultures – intracellular pathogen of the RES system Castaneda biphasic blood culture held for 21 days [old school] Current: Automated Blood culture systems with growth within 5 days Specimens: blood and bone marrow most profitable Granuloma formation in bone marrow Serology can be used for genera/species diagnosis Brucella species in clinical specimen related to animal species the patient was exposed: B. abortus – ingestion of raw cow milk B. melitensis – ingestion raw goat milk, feta cheese B. suis – contact with pigs B. canis - contact with dogs Granuloma in bone marrow

83. Campylobacter Small curved bacilli – shaped like sea gull wings C. jejuni – agent of diarrhea Related to undercooked poultry ingestion Requires selective media containing antibiotics - Skirrow’s blood agar Thrives at 42˚C - can use to selectively culture Campylobacter Requires microaerophilic atmosphere (high CO₂, low O₂) Significant % Guillain-Barre syndrome as sequelae of infection C fetus – Bacteremia in the immune suppressed host / does not cause diarrheal disease C. jejuni – grows at 37˚C and 42˚, hippurate positive C. fetus - grows at 37˚C and 25˚C hippurate negative

84. Reservoir – rabbits, rodents, ticks and flies. Humans infected by bug bites or directly from exposure to animal blood Strongly associated with 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 Fastidious small gram negative cocco-bacillus Culture media requires cysteine* Francisella tularensis

85. Helicobacter pylori Small curved Gram negative bacilli Cause of acute gastritis…..gastric adenocarcinoma Human to human transmission/ fecal oral route H. Pylori, Rapidly!! And strongly urease positive – used for detection directly from gastric biopsy tissue Difficult to grow in culture Stool antigen/diagnosis and test of cure ] Serum antibody for exposure Treatment – Antibiotics and acid suppression Histologic exam of gastric biopsy Hematoxylin-eosin stain or Warthin-Starry Silver stain

86. Legionella Legionella pneumophila most common species [6 serotypes] Requires cysteine in culture medium for growth Buffered Charcoal Yeast Extract agar -colonies form in 3-5 days Will not show on Gram stain of specimens Use silver impregnation stains in tissue Pulmonary disease associated with water, cooling towers, shower heads, stagnant water Urinary antigen test will detect L. pneumophila type I infection only Treatment: Erythromycin (macrolide) BCYE

87. Bacteria without cell walls Mycoplasma and Ureaplasma – have cell membranes only! Media and transport contain sterols to protect the membrane Do not form discreet colonies on agar plates – must read plates under a microscope to visualize organisms [up to 14 days of incubation] M. pneumoniae – community acquired pneumonia Serology and PCR for diagnosis Cold agglutinins produced Genital mycoplasmas M. hominis – fried egg colony, vaginitis, cervicitis, postpartum sepsis, neonatal infections , pre rupture of membranes Ureaplasma urealyticum – rapid urea hydrolysis in broth, NGU & upper genital tract infection, spontaneous abortion, neonatal infections Ureaplasma Mycoplasma hominis

88. Difficult to grow Bartonella henselae – Diagnose by PCR and/or serology 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 by Serology Chlamydia trachomatis Serovars L1,L2,& L3 agent of Lymphogranuloma venereum Serology / clinical findings – lymphatics and lymph nodes involved C. pneumoniae (TWAR agent)- Pneumonia, Diagnose by PCR C. psittaci - psittacosis, pneumonia, exotic parrot exposure/ Serology, PCR and epidemiology

89. Molecular Testing for Neisseria gonorrhoeae and Chlamydia trachomatis Amplification of DNA by Polymerase Chain Reaction (PCR) Urine, Cervix, and urethral most often tested More sensitive than any culture based system Sensitivity/Specificity @ 96%/99% Females most sensitive specimen = cervix Males good sensitivity with urine and urethral PCR replaced culture methods as “Gold” standard of detection Iodine staining of inclusions in McCoy Cell culture Elementary bodies in Fluorescent antibody stain

90. More difficult to grow Ehrlichiosis – Rickettsia bacteria Zoonotic intracellular pathogens Anaplasma spp, inclusion in the PMN Ehrlichia spp inclusion in the Monocytes Vector = Ixodes tick Fever, leukopenia, thrombocytopenia, Elevated serum aminotransferases, No rash (differs Rocky Mountain Spotted Fever) Found in the south central, southeast , midwest USA PCR and serology for diagnosis

91. Spirochetes Borrelia burgdorferi - Lyme’s disease Primarily found in NE part of USA Vector = Ixodes tick Serology and PCR Borrelia recurrentis - Relapsing fever Vector = human body louse Blood smear observe spirochete

92. 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 Spirochete – Shepherd’s crook

93. Unusual Bacterial diseases Granuloma inguinale Klebsiella (Calymmatobacterium) granulomatis Rare STD – causes ulcerative genital lesions Streptobacillus moniliformis Rat bite fever or Haverhill fever L form - cell wall deficient bacteria Inhibited by SPS in blood culture media Needs serum supplementation to grow, will not grow on routine bacteriology media Infection obtained from rat bite

94. Anaerobes Anaerobic infections can occur in virtually every organ and region of the body Polymicrobial – both aerobic and anaerobic species in the infections Endogenous organisms (commensal flora) cause most anaerobic infections Due to trauma, vascular or tissue necrosis cutting off the oxygen supply to the involved tissue Surgery plus antibiotics often necessary Anaerobic culture collection – must be oxygen free Gel containing swabs ESwab, Evacuated vials (port o cult)/ oxygen free collection Do not refrigerate specimens – absorb oxygen and kill anaerobes

95. Anaerobes PRAS media – pre reduced anaerobically sterile Media packaged in oxygen free environment Most common media used CDC anaerobic blood agar Kanamycin-vancomycin blood agar Bile Esculin agar Thioglycollate broth Chopped meat glucose broth Anaerobic gas pack jars – incubate cultures in anaerobic conditions Wet pack – add 10 ml water to hydrogen and CO2 generating envelope/ requires palladium coated catalysts – generate heat Dry pack – (Anaeropack) Absorbs O2 and generates CO2 Anaerobic bacteria can be poorly staining with bizarre & irregularly shape

96. Anaerobic Gram Negative Rods

97. Bacteroides fragilis group Pleomorphic irregular staining gram negative rod Grows in the presence of bile Esculin positive – turning media black Resistant to Penicillin and Kanamycin Common normal flora in GI tract Infections foul smelling/gas produced B. fragilis * - most common B. ovatus B. thetaiotamicron – indole positive B. uniformis B. vulgatus Resistant to Penicillin by beta lactamase enzyme Metronidazole is one antibiotic of choice Growth on bile Black pigment

98. Gram Negative Anaerobic Bacillus Prevotella and Porphyromonas species Will not grow in the presence of bile Will not turn black on esculin media Brick red fluorescence and black pigment Normal commensal flora of the upper respiratory tract Respiratory tract infections

99. Fusobacterium spp. Long thin gram negative bacilli – spindle shaped with pointed ends Normal flora in upper respiratory tract Associated with mouth and respiratory tract abscess formation and liver abscess Vincent’s angina – necrotizing oral infection caused by Fusobacterium species and spirochetes Lemierre’s syndrome - oropharyngeal infection that leads to thrombosis in jugular vein, septicemia, high fatality rate, caused by Fusobacterium necrophorum

100. Anaerobic Gram Positive Rods

101. Clostridium species Gram positive bacilli (boxcar shaped ) with spores Bacilli may over-decolorize and appear red Clostridium perfringens Predominate 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

102. Clostridium Clostridium botulinum – Adult disease – Preformed heat labile toxin ingested in mass produced or home-canned foods Infant disease - spore ingested from product produced in nature, neurotoxin produced in gut Begins with constipation and difficult sucking bottle Associated with Honey, soil, household dust Life threatening neuroparalytic disease Clostridium tetani Tetanus Gram stained cells appear like Tennis racket Toxin enters human due to penetrating skin injury Disease occurs in Non-immunized Spastic contractions of voluntary muscles, hyper-reflexia, lock jaw (trismus)

103. Clostridium C. septicum – Bacteremia or Gas Gangrene in patient with underlying malignancy C. difficile – Normal Colonization in 5% adults and 70% healthy infants Disease = Antibiotic associated colitis, pseudomembranous colitis due to toxin production [Virulence Factor] Toxin A – enterotoxin causing fluid accumulation Toxin B – potent cell cytotoxin Binary toxin (Nap1) strain is unique and produces larger amount of toxins A and B and is more virulence Diagnosis of infection: EIA methods [toxin A/B] are not sensitive and are discouraged PCR methods [toxin B] are more sensitive and for infection Culture – Cycloserine , Cefoxitin , Fructose Agar [CCFA]

104. Actinomyces Branching gram positive bacilli - do not form spores Normal flora in the mouth Capable of forming sulfur granules in tissue – often 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.

105. Branching Gram positive rods of Actinomyces – antler like Molar tooth colony Sulfur granule

106. Propionibacterium acnes Pleomorphic gram positive rod Catalase positive Indole positive Normal flora - skin, oral, GU and GI Potential contaminate in blood cultures Pathogen in cerebral shunt infections Firmly established as significant cause of prosthetic joint infection – particularly shoulder joints Cultures should be held up to 7- 14 days Therapy - Ampicillin

107. Bacterial vaginosis Clue cells are diagnostic Mixed anaerobic/aerobic bacterial infection - the two most common organisms in vaginosis are: Gardnerella vaginalis (aerobic gram variable rod) and Mobiluncus (anaerobic curved gram negative rod) Human blood agar used to culture Gardnerella, it is beta hemolytic and distinctive morphology Nugent score – gram stain scoring system to diagnose bacterial vaginosis [BV] Healthy = Lactobacillus-like Gram positive rods Intermediate = mixed bacterial types BV = Gardnerella and Mobiluncus on smear Clue Cell