Section 3 Flashcards
1
Q
Describe Biosafety level 1
A
- minimal risk
- type of agents = those not known to cause disease in healthy adults
- examples = bacillus subtilis, mycobacterium Gordon we, soil microbes
- precautions = standard microbiological practices. No special equipment
2
Q
Describe Biosafety level 2
A
- moderate risk
- type of agents = common human pathogens
- examples = Enterohemorrhagic E. Coli, salmonella, HIV, HBV, and influenza
- Precautions
— BSC level I or II
— PPE
— autoclave must be available
— limited access
— most micro labs fall in this category
3
Q
Describe Biosafety level 3
A
- high risk
- type of agents = those that may cause serious or lethal disease via inhalation. Effective treatment available
- examples = B. Anthracis, Francisella, Brucella, Mycobacterium TB, Rickettsia Rickettsii
- precautions = same as level 2 plus negative air flow and sealed windows
4
Q
Describe Biosafety level 4
A
- extreme risk
- type of agents = those that pose high risk of life-threatening disease
- may be transmitted by aerosols
- no vaccine or therapy
- examples = Ebola, Lassa virus
- precautions = requires BSC class III
- full bodied air-supplied positive pressure suit; independent unit with specialized ventilation and waste management to prevent release into environment
5
Q
Describe class A biological agents
A
- highest priority
- spread = easily disseminated or transmitted from person to person
- impact = high mortality, potential for major public heath impact
- example = B. Anthracis, Yersinia pestis, Francisella tularenisis, C. Botulinum toxin, smallpox, hemorrhagic fever viruses (Ebola, Marburg, Lassa, Machupo)
6
Q
Describe class B biological agents
A
- second highest priority
- spread = moderately easy to disseminate
- impact = moderate illness, low death rate
- example = Brucella, Salmonella, Shigella, E. Coli O157:H7
7
Q
Describe class C biological agents
A
- third highest priority
- spread = could be engineered for mass dissemination
- impact = high morbidity/mortality, major public health impact
- example = Nipah virus, hantavirus
8
Q
Describe Biosafety Cabinet Class I
A
- description = open front
- unsterilized room air enters
- Air passes through high-efficiency particulate air (HEPA) filter before being exhausted
- use = provides minimal personnel protection. Doesn’t protect work surfaces
9
Q
Describe Biosafety Cabinet class II
A
- laminar flow cabinets with variable sash opening.
- air passes through 1 HEPA filter before reaching work surface and second one before being exhausted
- use = type most commonly used in hospital micro labs. Provides protection for worker and work
10
Q
- Describe Biosafety Cabinet class III
A
- completely enclosed
- negative pressure
- air is filter sterilized coming in and going out
- gloves are attached to front
- use = provides maximum protection, used in labs that work with extremely hazardous organisms
11
Q
Describe autoclaving
A
- steam under pressure
- the most widely utilized method in clinical laboratories
- 15 lbs of pressure for 15 minute (for media) at 121C
- kills spores, may not kill prions
12
Q
Describe boiling used for sterilization
A
- not reliable
- spores may not be killed
13
Q
Describe pasteurization as sterilization
A
- used in food industry to kill food borne pathogens
- doesn’t sterilize
- liquid is heated to 71.7C for 15 seconds
14
Q
Describe hot air sterilization
A
- used when steam may damage or fail to penetrate
- 2 hours at 170C
- kills spores
15
Q
Describe incineration for sterilization
A
- used to sterilize inoculating loops and biomedical wastes
16
Q
Describe filtration of sterilization
A
- used to sterilize liquids that are thermolabile
- HEPA air filters are used in BSC
17
Q
Describe radiation of sterilization
A
- Ultraviolet (UV) light wavelengths in the 200 to 320 nm range used for surface disinfection both in the laboratory and patient rooms
18
Q
Descibe alcohols used in sterilization
A
- ethyl and isopropyl (70-80%) frequently used as antiseptics and disinfectant
- does not kill spores
19
Q
Describe chlorine compounds used in sterilization
A
- sodium hypochlorite (household bleach)
- one of the most effective agents HIV and HBV
- 10% solution
- can kill spores
20
Q
Describe glutaraldehyde in sterilization
A
- effective against most vegetative cells other than mycobacteria
- used for cold sterilization of items damaged by heat
21
Q
Describe hydrogen peroxide used for sterilization
A
- 3-6% solution used as disinfectant
22
Q
Describe iodine and iodophors used for sterilization
A
- iodophors release iodine slowly and are less irritating and nonstaining
- effective skin disinfectant, used with alcohol on skin prior to collection of blood for blood culture.
23
Q
Describe phenolic compounds used for sterilization
A
- 0.5-3% solution for disinfecting
- does not kill spores
24
Q
Describe quaternary ammonium compounds used for sterilization
A
- effective against wide range of vegetative bacteria
- not effective against spores, mycobacteria or nonenveloped viruses
- used to disinfect floors, walls and furniture
25
What is the gas used for sterilization? Describe
- ethylene oxide
- widely used in hospitals in sterilize material that can’t withstand steam
26
Describe preservation and storage of anaerobes
- aspirates preferred to swabs
- anaerobic transport systems to eliminate O2
- store at 25C. Do not refrigerate
27
Describe preservation and storage of CSF
- no preservation or transport device
- storage not recommended, process immediately
28
Describe preparation and storage of Neisseria Gonorrhoeae
- Amies transport medium with charcoal
- JEMBEC plates
- Gono-Pak
- BioBag
- best to inoculate immediately
- store at 25C. Do not refrigerate
29
Describe preparation and storage of sputum
- no preservation or transport device
- store at 4C
30
Describe preparation and storage of Stool for culture
- transport media
- Cary-Blair transport medium (has high pH and sodium thioglycollate added) if specimen can’t be cultured within 2 hours of collection
- store at 4C.
- best to process without delay
- refrigeration may kill Shigella
31
Describe preparation and storage of stool for ova and parasites
- depending on procedures to be performed: polyvinyl alcohol (PVA), 10% formalin, sodium acetate - acetic acid - formalin (SAF), merithiolate-iodine-formalin (MIF)
- can hold formed specimens at 4C
- soft or liquid specimens should be examined immediately or preserved
32
Describe preparation and storage of urine
- transport tubes with boric acid - glycerol if specimen can’t be processed within 2 hours of collection
- store at 4C for no more than 24 hours
33
Describe preparation and storage of viruses
- collect during acute phase of infection (2-3 days)
- use viral transport medium (VTM)
- store at 4C
- for >24 hour delay, freeze at -70C
34
What is the criteria for rejection of specimens?
- unlabeled or improperly labeled specimen
- improper collection site
- prolonged transit (over 2 hour without preservation)
- improper temperature during transport or storage
- leaking specimens
- specimens in non sterile containers
- improper transport medium
- dry swab
- improper swab
- syringes with needles attached
- culture for anaerobes requested on inappropriate sources or not received in anaerobic transport tube
- specimen received in formalin
- saliva instead of sputum
- Foley catheter tip
- insufficient quantity
- formed stool for C. Diff testing
- swab for acid-fast bacilli or fungal smear and culture
35
What organisms requiring special collection and processing?
- anaerobes
- chlamydia
- Haemophilus influenzas
- Neisseria gonorrhoeae
- Neisseria meningiditis
- salmonella
- shigella
- step. Pneumonia
- viruses
- parasites
36
What reagents/dyes are used in gram stain?
- crystal violet
- iodine
- 95% ethyl alcohol or acetone or combo
- safranin
37
Describe crystal violet in gram stain
- primary dye
- stains all bacteria purple
38
Describe iodine of gram stain
- mordant
- reacts with crystal violet to form a complex that incorporates into peptidoglycan
39
Describe 95% ethyl alcohol or acetone for gram stain
- decolorizer
- removes crystal violet from gram-negative bacterial cells
40
Describe safranin of gram stain
- counterstain
- Stains gram-negative bacterial cells pink
41
What are the properties of gram positive for bacteria?
- thick peptidoglycan layer with teichoic acid ad lipoteichoic acid
- teicholic acid cross-links prevent decolonization in gram stain
- stained by crystal violet
- purple
42
What are the properties of gram-negative bacteria?
- thin peptidoglycan layer covered with proteins, phospholipids, and lipopolysaccharides
- decolorizing agent causes increased permeability of lipid- rich cell wall
- primary stain (crystal violet) washes out
- stained by safranin
- pink
43
Describe nutritive media
- supports growth of most nonfastidious bacteria
- example = nutrient agar, trypticase soy agar
44
Descibe enrichment media
- contains added growth factors
- examples = Sheep BAP (SBA), CHOC, brain-heart infusion, buffered charcoal-yeast extract (BCYE) agar
45
Describe selective media
- contains additives such as dyes, bile salts, alcohols, acids, or antibiotics to inhibit growth of certain bacteria - example = CNA, EMB, MAC, HE, XLD, and Thayer-Martin
46
Describe differential media
- contains compounds that allow certian bacteria to be visually differentiated
- examples = EMB, MAC, HE and XLD
47
Describe SBA
- Sheep Blood Agar
- type = E, D
- for isolation = most nonfastidious bacteria
- tryptic soy agar with 5% sheep blood
- allows differentiation of hemolysis
48
Describe CHOC
- Choclate agar
- type = E
- for isolation = fastidious organisms including Haemphilus and Neisseria
- supplies X (hemin) and V (NAD) factors
- incubate in 5% CO2
49
Describe CNA agar
- Columbia colistin-nalidixic acid agar
- type = S
- for isolation = gram-positive cocci (GPC), especially from wound and stool culture
- colistin and nalidixic acid suppress most gram negative (GN)
- contains 5% sheep blood but should not be used to observe hemolytic reactions
50
Decribe PEA agar
- phenylethyl alcohol agar
- type = S
- for isolation = GPC ad anaerobic gram-negative rods (GNR)
- phenylethyl alcohol inhibits enteric GNR
- contains 5% sheep blood
51
Describe Brain heart infusion agar
- type = E, S
- for isolation = fastidious and nonfastidious organisms (anaerobes included)
- useful for culturing streptococci, pneumococci, and meningococci
- can add vancomycin-resistant enterococci (VRE)
52
Describe EMB
- eosin methylene blue
- type = S, D
- for isolation = enteric GNR
- eosin and methylene blue inhibit gram positive (GP)
- lactose fermenters are green-black or purple
- E. Coli produces green with metallic sheen
- nonlactose fermenters = colorless or light purple,
53
Describe MAC agar
- MacConkey
- type = S, D
- for isolation = enteric GNR
- bile salts and crystal violet inhibit GP
- LF will be pink
- NLF colorless
54
Describe SMAC
- Sorbitol MacConkey agar
- type = S
- for isolation = E.coli 0157:H7 doesn’t ferment sorbitol
- colorless colonies
- some labs have stopped using because non-0157 serotyping can be pathogens
55
Describe HE agar
- Hektoen enteric agar
- type = S, D
- for isolation = salmonella ad shigella in stool
- bile salts, bromothymol blue and acid fuchsia inhibit normal GI flora
- nonpathogens orange to salmon pink
- NLF is green to blue-green
- H2S-positive colonies have black precipitate
56
Describe XLD agar
- xylose lysine deoxychocolate agar
- type = S, D
- for isolation = salmonella and shigella in stool
- deoxychocolate inhibits many GNR and GP
- 4 types of colonies
—yellow (e. Coli)
—yellow with black centers (some proteus spp)
—colorless or red (shigella)
—red colonies with black center (salmonella)
- some shigella may be inhibited
- some salmonella may not produce H2S
57
Describe Salmonella-Shigella (SS) agar
- type = S
- for isolation = salmonella and shigella in stool
- brilliant green and bile salts inhibit other enterics
- salmonella and shigella don’t ferment lactose (colorless colonies)
- salmonella produces H2S (black center)
58
Describe Deoxychocolate-citrate agar
- type = S
- for isolation = salmonella and shigella
- other nonpathogenic enterics inhibited
59
Describe thioglycolate medium
- type = E
- for isolation = Campylobacter from stool
- subculture to campy-selective agar after overnight incubation at 4C
60
Describe Campylobacter blood agar (CAMPY BAP)
- type = E, S
- for isolation = campylobacter from stool
- incubate plates in 5% CO2 at 42C
61
Describe Modified-Thayer Martin (MTM)
- for isolation = Neisseria gonorrhoeae and Neisseria meningitidis
- vancomycin, colistin, nystatin and trimethoprim (TMP) inhibit growth of normal genital flora
- incubate in increased CO2
- some N. Gonorrhoeae may be inhibited
62
Describe Martin-Lewis agar
- for isolation = N. Gonorrhoeae and N. Meninigiditis
- similar to Thayer-martin, but different antibiotics
- inhibits yeast better
- incubate in increased CO2
63
Describe NYC agar
- New York City agar
- for isolation = N. Gonorrhoeae and N. Meningitidis
- incubate in increased CO2
- Some N. Gonorrhoeae are inhibited by antibiotics
- genital mycoplasmas will grow
64
Describe GC-LECT
- isolation for = N. Gonorrhoeae and N. Meningiditis
- antibiotics to inhibit GN and GP Bacteria and yeast
65
Describe JEMBEC plates
- for isolation = N. Gonorrhoeae and N. Meninigiditis
- for transportation and growth of N. Gonorrhoeae
- plates contain Neisseria- selective medium and come with resealable polyethylene bag and CO2 generating tablet
- no need to transfer to culture plate `
66
Describe cystine-telluride blood agar
- used for differential medium for isolation of corynebacterium diphtheria
- c. Diphtheria produces dark grey to black colonies
67
Describe Loeffler medium
- enrichment medium for C. Diphtheria’s
- promotes development for metachromatic granules
68
Describe TIndale agar
- use for selective differential medium got C. Diphtheriae
- corynebacterium spp. procure gray to black colonies due to reduction of tellurite
- C diphtheria colonies are surrounded by a brown halo
69
Describe Bismuth sulfite agar
- use for selective for salmonella
- bismuth sulfite and brilliant green inhibit most others
- S. Typhi colonies are black, surrounded by metallic green sheen. Others are light green
-
70
Descibe CIN agar
- Cefsulodin-lrgasan-novobiocin
- use for selective medium for Yersinia enterocolitica, aeromonas, and plesiomonas shigelloides
- crystal violet inhibits most GN
- Novobioicin inhibits GPC
- Cefsulodin inhibits most GP and GN
- Y. Enterocolitica ferments mannitol, appears as red “bulls eye” colonies surrounded by colorless halo
71
Describe alkaline peptide water
- use for enrichment medium for recover of vibrio from stool
- alkaline pH supresses commensals
- subcultured to thiosulfate citrate bile salts sucrose (TCBS)
72
Describe TCBS agar
- used for selective for vibrio
- high pH inhibits most bacteria
- V. Cholera ferments sucrose, produces yellow colonies
- V. Parahaemolyticus and V. Vulunificus do not ferment sucrose ; usually produces blue-green colonies
73
Describe Bordet-Gengou agar`
- used for selective enrichment medium for isolation of Bordetella Pertussis
- Potato-glycerol-based medium enriched with blood
- contaminants inhibited by methicillin
- “cough plate”
- Bordetella colonies resemble mercury droplets
74
Describe Regan-Lowe agar
- Used for selective for B. Pertussis
- charcoal agar supplemented with horse blood, cephalexin and amphotericin B
75
describe BYCE agar
- used for enrichment medium for isolation of legionella
- yeast extract and L-cysteine enhance growth of Legionella
- Charcoal absorbs toxic compounds
76
Describe V agar
- used for selective and differential for G. Vaginalis
- incubate in increased CO2 for 48 hours
- colonies are beta hemolytic
77
Describe CHROMagar or chromID
- detect emerging antibiotic-resistant pathogens such as extended-spectrum beta-lactamase (ESBL) -producing E. Coli, methocillian-resistant staph aureus and carbapenemase-producing enterobacteriaceae
- contains chromogens that are released if specific enzymes produced by target organism are present
78
Describe growth of aerobes
- BAP incubated aerobicaly = growth
- BAP incubated anaerobically = no growth
- CHOC incubated in CO2 = growth
79
Describe growth of capnophilic aerobes
- BAP incubated aerobically = no growth
- BAP incubated anaerobically = no growth
- CHOC in CO2 = growth
80
Describe growth of facultative anaerobes
- BAP incubated aerbically = growth
- BAP incubation anaerobically = growth
- CHOC in CO2 = growth
81
Describe growth for obligate anaerobe
- BAP incubated aerobically = no growth
- BAP incubated anaearobically = growth
- CHOC in CO2 = no growth
82
Describe growth of aero tolerant anaerobe
- BAP incubated aerobically = no growth
- BAP incubated anaerobically = growth
- CHOC in CO2 = growth
83
What are the organisms that require incubation in increased CO2?
- Campylobacter (10-15% CO2)
- Haemophlilus (5-10%)
- Helicobacter (5-10%)
- Moraxella catarrhalis (5% CO2)
- Mycobacterium (5-10%)
- Pathogenic Neisseria (5-10%)
84
Describe Alpha hemolysis
- green zone around colony
- may be narrow or wide
- partial lysis of RBCs
- examples = strep. Pneumonia and viridans streptococci
85
Describe Beta hemolysis
- clear zone around colony
- may be narrow or wide
- complete lysis of RBCs
- examples = strep A (GAS), group B strep (GBS), an listeria monocytogenes
86
Describe Gamma hemolysis
- nonhemolytic
- no zone of hemolysis
- no lysis of RBCs
- example = enterococcus faecalis
87
Describe Staphylococcus spp.
- GPC, usually clusters
- on SBA = 1-3 mm round, smooth, convex, glistening, opaque, entire edge butter like
- Catalase = POS
- fermentive
- Microdase = NEG
- Resistant to bacitracin
- matrix-assisted laser desorption ionization-time of flight (MALDI) mass spectrometry routinely used for ID of isolates
- Normal flora of skin, mouth, pharynx, vagina, urethra, GI tract
- facultative anaerobes
- grows on most non selective media.
- salt tolerant
88
Describe Staph. Aureus
- pathogenicity = causes suppurative cutaneous infections, toxic shock syndrome, food poisoning
- GPC, usually is clusters
- most are beta hemolytic with small zone. May be golden
- Coagulase = POS
- ferments mannitol
- usually DNase = POS
- second leading cause of health-care associated infections, spread by direct contact with personnel and contaminated objects.
- 85-90% resistant to penicillin MRSA strains are very common
- methicillian resistant is mediated by the mecA green or it’s homologous
- most important resistant mechanism
89
Describe Staph. Epidermidis
- pathogenicity = opportunistic pathogen. Causes nosocomial infections he to contamination of inserted or implanted medical devices
- GPC. Usually in clusters
- on SBA = white to slightly yellow, usually nonhemolytic
- Coagulase = NEG
- grows on MSA but does not ferment mannitol
- sensitive to novobiocin
- frequently recovered from blood cultures but not usually clinically significant in adults
90
Describe Staph. Saprophyticus
- pathogenicity = UTI in young sexually active females, urethritis and prostatitis in males
- GPC, usually in clusters
- on SBA = white to slightly yellow
- non hemolytic
- Coagulase = NEG
- Resistant to novobiocin
- may ferment mannitol
- Novobiocin only performed when Coagulase-negative staph (CNS) isolated from urine of female
91
Describe Micrococcus
- pathogenicity = usually nonpathogenic
- found in environment and on skin, mucous membranes
- large GPC, in pairs or tetrads
- on SBA = often pigmented (bright yellow, orange, pink, tan)
- High-domed colonies
- Catalase = POS
- Coagulase = NEG
- Microdase = POS
- Urease = mostly POS
- resistant to furazolidone and lysostaphin
- not commonly isolated
- Must differentiate from staph.
- usually only grow aerobically
- micrococcus Lylae included in this genus
92
What are the tests used to ID staphylococcus?
- Catalase
- Coagulase
- Slide test for s. Aureus
- Microdase
- rapid latex and hemagglutination assays
- Matrix-assisted laser desportion/ionization-time of flight mass spectrometry
93
Describe the catalase test
- enzyme catalase converts 3% hydrogen peroxide to O2 and water
- immediate bubbling = POS
- key reactions = staphylococci and micrococcius POS
- Catalase from RBCs in blood agar may produce weak bubbles
94
Describe the Coagulase test
- enzyme Coagulase causes coagulation (tube test) or agglutination (slide test) in plasma
- key reactions = S. Aureus POS
- slide test is screening test
- detects bound Coagulase (clumping factor)
- if neg, tube test should be performed
- largely replaced by latex agglutination tests
-
95
Describe Slide agglutination tests for S. Aureus
- agglutination of latex beads coated with fibrinogen and antibodies to protein A
- key reaction = s. Aureus POS
- most labs report POS organism as s. Aureus
96
Describe MIcrodase (MSA)
- fermentation of mannitol results in color change from pink to yellow
- key reactions = s. Aureus POS
- 7.5% salt inhibits most organisms other than staph.
- All staph can grow on MSA
- rarely used for ID of s. Aureus today because other species ferment mannitol
97
Describe Rapid latex and hemagglutination assays
- later agglutination slide-card tests to detect Coagulase and/or protein A associated with Staph. Aureus and MRSA
- key reaction = s. Aureus POS
- high sensitivity but relatively low specificity
- may be false-pos. With CNS
98
Describe Matrix-assisted laser desportion/ionization-time of flight (MALDI-TOF) mass spectrometry
- a type of MS good for analyzing bio molecules like peptides, lipids, and saccharides
- key reaction = rapid and specific ID of staphylococci and other catalase Positive cocci
- ID of staph at species level is >97% but lower for Coagulase-negative staph.
99
Describe the streptococcus spp.
- oval GPC in chains or pairs
- on SBA = <1 mm, white to gray translucent or semi opaque
- variable hemolysis
- catalase NEG
- faculatative anaerobes
- require enriched media
- chaining best in broth cultures
100
Describe Group A Strep (GAS)
- pathogenicity = causes 90% of strep infections
- strep sore throat, rheumatic fever, glomerulonephritis, scarlet fever, erysipelas, Puebla sepsis, impetigo
- oval GPC in chains
- on SBA = pinpoint, grayish white, translucent. Usually beta hemolytic (wide zone)
- sensitive to bacitracin
- PYR = POS
- most common s. Pyogenes
- hemolysis due to O2-stable streptolysin S and labile streptolysin O
- definitive diagnosis includes immunoassay for Lancefield group A or MALDI-TOF
101
Describe group B strep (GBS)
- also called S. Agalactiae
- pathogenicity = normal flora of female genital tract
- most common cause of neonatal septicemia and meningitis
-oval GPC in chains
- on SBA = slightly larger that GAS. Gray-white, narrow zone of diffuse beta hemolysis
- may be non-hemolytic
- resistant bacitracin
- sodium hippurate POS
- CAMP POS
- vaginal and rectal swaps collected from pregnant women at 35-37 weeks gestation
- inoculated in selective broth
- nucleic acid amplifications (NAATs) recommended for ID after culture enrichment
102
Describe Strep. Pneumonia
- pathogenicity = colnizes upper respiratory tract of some children
- most common cause of community acquired pneumonia
- major cause of otitis media and meningitis in adults
- infects sinuses and eyes
- football shaped (lancet shaped) GPC
- usually in pairs
- may be single or in short chains
- frequently encapsulated
- on SBA = round, translucent, glistening, dome-shaped when young, central depression with age due to autolysis. Encapsulated strains are mucoid
- alpha hemolytic
- no Lancefeild group MALDI-TOF can be used for ID
103
Describe Strep C/G Streptococci
- also called S. Dysgalactiae
- normal flora upper respiratory tract but can cause pharyngitis
- oval GP in pairs and chains
- on SBA = beta- hemolytic
- resistant to bacitracin
- PYR = POS
- indistinguishable from pharyngitis caued by S. Pyogenes
104
Describe Viridans Strep.
- includes = s. Mitis group, s. Aginosus group, S. Mutans group, S. Salivarius group, and S. Bovis group
- Normal in oral respiratory, GI mucosa
- oppotunistic pathogens
- frequent cause of subacute bacterial endocarditis, bacteremia, sepsis, and abscesses
- GPC in chains
- On SBA = alpha hemolytic (S. Mitis group), non-hemolytic (S. Salivarius and S. Bovis), and beta-hemolytic (S. Anginosus group)
- S. Mutans group ferment sorbitol.
- Bile solubility = NEG
- S. Bovis is esculin POS
105
Describe E. Faecium and E. Faecalis
- normal in mouth, GI tract, and male genital tract
- causes nosocomial UTI, wound infections, endocarditis, and bacteremia
- oval GPC in pair, chains
- ON SBA = E. Epidermidis is usually alpha or nonhemolytic
- some stains of E. Faecalis may be beta hemolytic
- catalase NEG
- hydrolyes esculin
- grows in 6.5% NaCl broth
- PYR = POS
- E. Faecalis is often resistant to vancomycin
- Vancomycin-resistant enterococci (VRE) are a significant public health concern.
- VRE fecal surveillance cultures are often done for early detection
- chromogenic agar can be used for ID or VRE
106
Describe the Bacitracin disk test
- zone of inhibition after overnight incubation = susceptibility to bacitracin
- GAS susceptible
- GBS resistant
- Also know as A disk
- performed on SBA
- no longer recommended because some groups C ad G are susceptible
- replaced by PYR and serogrouping by latex agglutination
107
What are the tests used for ID of Stretococci spp?
- bacitracin
- MALDI-TOF
- PYR test
- CAMP test
- Hippurate hydrolysis
- Slide agglutination tests
- optochin disk
- Bile solubility
108
Describe MALDI-TOF in Strep. Ida
- a type of mass spectrometry good for ID biomolucles like peptides, lipids and saccharides
- reliable for S. Pyogenes, S. Agalactiae, and S. Pneumonia
109
Describe the PYR test
- if PYR is hydrolysis, red color after addition of color developer
- GAS POS.
- more specific than bacitracin for GAS.
- GAS is only beta hemolytic strep that’s PYR POS.
- disks test only takes minutes
110
Describe CAMP test
- GBS produces extracellular protien that enhances hemolysis of beta-hemolytic S. Aureus on SBA
- GBS POS.
- Classic method: unknown streaked perpendicular to streak of Beta-lysin producing S. Aureus
- incubated in ambient air overnight
- false POS in CO2
- Arrowhead hemolysis where inoculum lines meet
- DIsks containing Beta-lysin can be used instead of S. Aureus
- rapid test: drop of beta-lysin on colonies on SBA
- only requires 20-minutes incubation
111
Describe hippurate hydrolysis
- organisms the produce hippuricase hydrolyze sodium hippurate to benzoate and glycine
- GBS POS
- alternative to CAMP
- 2 hour test available
112
Describe Slide agglutination tests for ID of Strep.
- latex particles coated with group-specific antibodies agglutinate in presence of bacterial antigens
- organisms agglutinate in corresponding antisera
- tests commercially available for rapid ID of groups: A, B, C, D, F, G strep and S. Pneumonia
113
Describe Optochin disk
- zone of inhibition greater than or equal to 14 mm with 6mm disk or greater than or equal to 16 mm with 10 mm disk - susceptibility to optochin
- S. Pneumo susceptible
- Viridans strep is resistant
- also know as P disk
- contains ethylhydrocuperine hydrochloride
- placed on lawn of inoculum on SBA
- plates incubated over in CO2-
- if zone of inhibition <14 mm, ID organism as S. Pneumonia on if bile soluble
114
Describe bile solubility test
- biles salts cause lysis of some organisms
- S. Pneumonia POS
- can be performed in broth or on colony
- POS = clearing of broth of disappearance of colony
- results in 30 minutes or less
115
Describe methicillin-sensitive staphylococcus aureus of antibiograms of GPC
- generally resistant to penicillin, susceptible to cephalosporins and vancomycin
- has routine susceptibility testing
116
Describe MRSA of antibiograms of GPCs
- resistant to all beta-lactams (pencilling, ampicillin, and cephalosporin)
- has susceptibility testing
117
Describe GAS of antibiograms of GPCs
- universally susceptible to penicillin, cephalosporins, vancomycin
- no routine susceptibility tests
118
Describe GBS of antibiograms of GPC
- susceptible to penicillin, vancomycin, cephalosporins
- routine susceptibility test can only be done when isolated from baby
119
Describe S. Pneumonia of antibiograms of GPCs
- increasingly resitant to penicillin
- susceptible to vancomycin
- has routine susceptibility testing
120
Describe enterococcus of antibiograms of GPCs
- E. Faecium is usually resistant to penicillin and several other commonly used antibiotics
- most are susceptible to vancomycin, but VRE, especially E. Faecium, are becoming more common in hospitals
- has routine susceptibility testing
121
What organisms are aerobic spore-forming GPC?
- Bacillus spp.
- Bacillus Anthracis
- bacillus cereus
122
Describe Bacillus Spp.
- usually contaminants
- large spreading beta-hemolytic colonies with irregular edges (Medusa heads). Whitish gray
- may be pigmented
- Catalase POS
- most are motile
- must rule out C. Anthracis (often done via PCR
123
Describe bacillus Anthracis
- one of most highly pathogenic microorganisms
- causes anthrax
- contracted from contaminated hides, wool and meat
- large with square ends
- may be in chains
- oval, central to sub terminal spores that agents swollen
- looks like bamboo
- spores may not be seen in direct smear
- large adherent, nonhemolytic, flat to slightly convex, irregular border, ground-glass appearance, comma-shaped projections
- stands up like beaten egg white when touched with loop
- non-hemolytic
- Catalase POS
- non-motile
- capsules seen in CSF and blood smears
- potential bioterrorism organism
- handle in BSC
- submit to public health lab for confirmation
- most strains are susceptible to penicillin
124
Describe Bacillus Cereus
- often environmental contaminant
- can cause local and systemic infections, food poisoning
- large with square ends
- may be in chains
- oval, central to sub terminal spores that aren’t swollen
- looks like bamboo
- spores mat not be seen in direct smear
- large adherent, flat to slightly convex, irregular border, ground-glass appearance, comma-shaped projections
- stands in like beaten egg white when touched with loop
- hemolytic
- motile
- second most important pathogen in genus
125
What organisms are aerobic non-spore-forming GPC?
- Corynebacterium spp
- Corynebacterium diphtheria
- Corynebacterium jeikeium
- Listeria monocytogenes
- Nocardia
126
Describe Corynebacterium spp.
- normal skin and mucous membrane flora
- opportunistic pathogens
- gram stain = irregular, slightly curved, non-parallel sides, slub shaped ends. It taken from fluid, they can arrange in palisades or clusters with angular V-shaped forms
- culture = facultative anaerobes. Grows on most media
- catalase POS
- nonmotile
- called diphtheroids because resembles C. Diphtheria
127
Describe Corynebacterium diphtheria
- pathogenicity = diphtheria
- gram stain = irregular, slightly curved, non-parallel sides, club-shaped ends, if taken from fluid, the can arrange in palisades or clusters with angular V-shaped forms
- culture = Gray-black colonies with brown halos on TIndale agar. Black colonies on cystine tellurite agar
- Catalase POS
- non-motile
- toxin producing
- ID requires demonstration of toxin production
- PCR or MALDI-TOF are used for ID but must be followed up with Elek immunoprecipitation test to confirm toxins production
- C. Ulcerans which is closely related to C. Diphtheria, also contains the TOX gene and can cause diphtheria
128
Describe Corynebacterium Jeikeium
- hospital-acquired pathogen
- gram stain = irregular, slightly curved, non-parallel sides, slub shaped ends. It taken from fluid, they can arrange in palisades or clusters with angular V-shaped forms
- culture = Gray-black colonies with brown halos on TIndale agar. Black colonies on cystine tellurite agar
- Catalase POS
- Rapid sucrose urea (RSU) NEG
- most commonly isolated diphtheroid
- multi-Drug resistant (MDR)
129
Describe Listeria Monocytogenes
- able to cross the placenta and can infect the fetus causing preterm labor and other complications resulting in fetal loss up to 24%
- meningitis and septicemia in newborns and immunocompromised
- food poisoning with mortality ranges up to 50%
- gram stain = parallel sides, rounded ends, coccobacillary. Singles or short chain arrangement
- culture = tiny colonies with narrow zone of indistinct beta hemolysis. Translucent, gray
- Catalse POS
- hippurate hydrolysis POS
- Esculin POS
- CAMP POS
- hemolysis shaped like shovel
- tumbling motility in wet mount
- umbrella growth in motility agar at RT but not at 35C
- grows from 0.5-45C
- cold enrichment may be used
- catalase differentiates from GBS
- motility differentiates from diphtheroids
- MALDI-TOF can be used for ID
130
Describe Nocardia
- immunocompotent: skin infections
- immunocompromised: invasive pulmonary and disseminated infections
- aerobic actinomycetes, fine, branching filaments with fragmentation, often beaded
- slow-growing
- on SBA wrinkled, dry, crumbly, chalky white to orange-tan, beta hemolytic
- catalase POS
- partially acid fast
- Sulfur granules
- Nocardia isolates may be ID and reported at “complex” or “group” level.
- N. Brasiliensis, N. Pseudobrasiliensis and N. Abscesses are some more frequently isolated
131
Describe Neisseria Spp
- gram-negative diplococci or single coffee bean shaped
- aerobic or facultative
- pathogens are capnophilic
- Catalase POS
- Oxidase POS
132
Describe N. Gonorrhoeae
- gonorrhea, salpingistis, ophthalmia of the newborn
- intracellular and extracellular GNDC
- diagnostic in utheral discharge from symptomatic males
- culture confirmation required for females
- requires increased CO2
- usually does not grow on SBA
- grows on CHOC and Neisseria selective media
- colonies are small, grayish white or tan
- may look like mixed culture
- oxidase POS
- carbohydrate utilization: glucose only
- susceptible to drying and cold
- culture used from presumptive ID followed by definitive ID such as monoclonal antibodies
- NAATs can be used on male urine specimens with high sensitivity but should not be used with oropharyngeal, rectal, ocular or pediatric specimens
133
Describe N. Meninigiditis
- can be normal flora in upper respiratory tract, but always a pathogen when recovered from usually sterile body fluids such as blood or CSF
- common cause of Meninigiditis in young adults
- intracellular and extracellular GNDC
- gram stain of CSF is required for all cases of suspected bacterial meningitis sent to the lab
- grow on SBA, CHOC, and Neisseria selective media
- colonies are gray, convex, glistening, and occasionally mucoid
- carbohydrate utilization: glucose and maltose
- ONPG NEG
- do not refrigerate
- Handle in BSC
- vaccine is available
134
Describe Commensal Neisseria spp.
- normal in upper respiratory tract
- rarely cause disease
- GNDC
- grow on SBA and CHOC at RT, nutrient agar at 35C
- some grow on Neisseria selective media
- do not require increased in CO2
- may be pigmented
- not speciated from respiratory specimens
- must differentiate from pathogenic Neisseria when isolated on selective media or from normally sterile body site
- N. Lactamica is easily confused with N. Meninigiditis but N lactamica acidifies lactose in addition to glucose
135
Describe Moraxella Catarrhalis
- normal in upper respiratory tract
- causes respiratory infections in young, old, and compromised; otitis media and sinusitis in children
- GNDC
- grows on SBA and CHOC
- Some may grow at RT and/or on Neisseria-selective media
- “hockey pluck” colonies, can be pushed over agar surface with loop
- catalase POS
- oxidase POS
- carbohydrate utilization: NEG for all sugars and ONPG
- DNase POS
- Butyrate esterase POS.
- DNase and butyrate esterase differentiates from Neisseria Spp.
136
What are characteristics of Enterobacterales?
- gram stain = non-spore forming GNRs
- facultative anaerobes
- BAP = most are large, dull, gray, nonhemolytic
- MAC = lactose fermenters are pink. No lactose fermenters are colorless
- ferment glucose
- Oxidase NEG
- most reduce nitrates to nitrites
- most are catalase POS
137
What are the tests run for ID of Enterobaterales?
- Oxidase
- nitrate reduction
- carbohydrate fermentation
- ONPG
- H2S production
- TSI agar
- Indole
- MR
- VP
- citrate
- urease
- PD
- decarboxylase reactions
- motility
138
Describe the oxidase test
- tetramethyl-para-phenylene-diamond dihydrochloride reacts with cytochrome C to produce blue or purple color
- POS = blue or purple
- good test to differentiate Enterbacterales from nonfermenters
- false POS from iron containing wire
- use platinum wire or wooden stick
139
Descibe Nitrate reduction
- if organism reduces nitrates to nitrites, red colo develops when sulfanilic acid and N, N-dimethyl-L-naphtylamine added
- POS = red or no color after addition of zinc dust
- if no color develops , zinc dust added
- reduced nitrates
- red color after zinc dust indicates presence of residual nitrates (NEG)
- no color after zinc dust means nitrates reduced to N2 or NO2 (POS)
140
Descibe carbohydrate fermentation
- when carbohydrate fermented acidic end products cause color change in pH indicator
- with phenol red indicator, change from red to yellow
- frequently tested carbohydrates are glucose, lactose, sucrose, mannose, sorbitol, mannitol, xylose, adonitol, cellobiose, dulcitol, trehalose
- all enterobacterales ferment glucose
141
Describe ONPG
- ONPG is changed to orthonitro-phenol by beta-galactosidase
- POS = yellow
- test for slow lactose fermentation
- helpful in differentiating citrobacter (POS) from most salmonella’s (NEG)
142
Describe H2S production
- organisms that possess H2S-producing enzymes produce colorless H2S reacts with iron salt in medium to form black ferrous sulfide
- black precipitate
- sulfur-containing compounds = sodium thiosulfate, cystine, methionine
- good test to differentiate salmonella (POS) from shigella (NEG)
143
Describe Triple Sugar Iron agar (TSI)
- sugar fermentation produces acid, changes color of pH indicator
- Yellow = acid (A)
- pink = alkaline (K)
- pink butt = glucose not fermented
- yellow butt = glucose fermented
- yellow slant = lactose and/or sucrose fermented
- black precipitate = H2S production
- bubbles = gas production
- contains 0.1% glucose, 1% lactose, 1% sucrose, phenol red, sodium thiosulfate, iron salt.
- leave cap slightly loose
- record as slant/butt
- Kligler iron agar (KIA) is same except no sucrose
144
Describe Indole test
- tryptophanase delaminates tryptophan
- Indole produced
- Kovacs reagent forms pink-colored complex with indole
- pink = POS
- spot Indole test commercially available
- need source of tryptophan
- use colonies from BAP or CHOC agar, not MAC
- blue = POS for e.coli and proteus vulgaris are POS
145
Describe Methyl Red (MR) test
- acid products formed when glucose metabolized by mixed acid fermentation pathway
- color changes pH indicator.
- POS = red color after addition of MR (pH indicator)
- test becomes POST at pH of 4.5
146
Describe Voges-Proskauer (VP) test
- acetoin is produced from alternate pathway for glucose metabolism
- POS = red color where alpha-naphthalene and KOH added
- little acid produced that are VP POS are usually MR NEG and vice versa
147
Descibe citrate test
- if organism can use citrate as sole source of carbon, pH increase, pH indicator changes color
- POS = green to blue or growth
- False NEG if cap not loose
- klebsiella and enterobacter POS
148
Describe urease test
- urease breaks for urea
- ammonia released, pH inscreased, pH indicator changes color
- POS = yellow to pink
- proteus and Morganellla are rapid urease producers
149
Describe Phenylalanine delaminate (PD) test?
- PD delaminates phenylalanine to phenylpyruvic acid, which reacts with ferric chloride to produce green color
- POS = green color after addition of ferric chloride
- Proteus, Proidencia, Morganella = POS
149
Describe decarboxylase reactions
- if organism has enzyme to decarboxylate amino acid, pH increased, pH indicator changes color
- POS = yellow to purple
- ornithine decarboxylase (ODC)
149
Describe Phenylalanine delaminate (PD) test?
- PD delaminates phenylalanine to phenylpyruvic acid, which reacts with ferric chloride to produce green color
- POS = green color after addition of ferric chloride
- Proteus, Proidencia, Morganella = POS
150
Describe motility of enterobacterales ID
- motile organisms grow away from stable line in motility medium
- POS = movement away from stable line or hazy appearance throughout medium after overnight incubation
- most eneterobacterales are POS, except Klebsiella and Shigella
- differentiates Klebsiella (NEG) and enterobacter (POS)
- differentiates Shigella (NEG) and Salmonella (POS)
151
What are the antigens of enterobacterales?
- O
- K
- H
152
Describe the O antigen
- somatic antigen
- location = cell wall
- lipopolysaccharide
- heat stable
- used for serological grouping of Salmonella and Shigella
153
Describe the H antigen
- flagellar antigen
- location = flagella
- protiens, heat labile
- used to stereotype salmonella
154
Describe the K antigen
- capsule antigen
- location = capsule
- polysaccharide, heat labile
- may mask O antigen
- removed by heating
- role in preventing phagocytosis
- increased virulence
- Vi antigen is K antigen produced by S. Typhi
155
Describe Escherichia Coli characteristics
- UTI, septicemia, neonatal sepsis and meningitis, diarrhea (some)
- POS = lactose, gas, Indole, MR, motility
- NEG = H2S, VP, citrate, PD, urease
- predominant aerobe in GI tract
- most common cause of UTI
- green metallic sheen on EMB
- presumptive ID: lactose POS, dry colony MAC, oxidase NEG, indole POS
- E.coli 0157:H7 doesn’t ferment sorbitol; colorless colonies SMAC
156
Describe Shigella characteristics
- dysentery
- most communicable of bacterial diarrheas
- found primarily in crowded or substandard conditions
- POS = MR
- NEG = lactose, gas, H2S, VP, citrate, PD, urease, motility
- blood mucous, neutrophils in stool
- rarely disseminates
- fragile organism
- S. Dysenteriae most severe
- Shigella Sandi most common in US.
- Serogrouped by O antigens
- Serogroups A, B, C, D.
- closely related to Escherichia on molecular basis
157
Describe Salmonella characteristics
- typhoid (enteric) fever, bacteremia, enterocolitis
- POS = H2S, MR, motility, LDC
- NEG = lactose’s indole, VP, PD, urease, ONPG
- found in poultry
- may be transmitted by reptiles
- S. Typhi has Vi antigen, only trace H2S, citrate NEG
- grouped by O antigens
- serotyped by H antigens
158
What are the types of diarrheagenic E. Coli?
- EHEC
- ETEC
- EIEC
- EPEC
- EAEC
- DAEC
159
Describe Enterohemorrhagic (EHEC)
- also known as Shiga toxin-producing (STEC) or verotoxin- producing (VTEC)
- diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS)
- most common cause of renal failure in children in US.
- maybe fatal, especially in young or elderly
- transmission = undercooked meat, raw milk, apple cider
- toxins is disease mechanism
- gram stain of stool = RBCs but usually no segemented neutrophils
- E. Coli 0157:H7 is most common isolate of group of pathogen most often isolated from bloody stools
- non 0157:H7 STEC also cause disease
- DNA probes can ID genes that code for toxins
- report to public health
160
Describe Escherichia Coli characteristics
- UTI, septicemia, neonatal sepsis and meningitis, diarrhea (some)
- POS = lactose, gas, Indole, MR, motility
- NEG = H2S, VP, citrate, PD, urease
- predominant aerobe in GI tract
- most common cause of UTI
- green metallic sheen on EMB
- presumptive ID: lactose POS, dry colony MAC, oxidase NEG, indole POS
- E.coli 0157:H7 doesn’t ferment sorbitol; colorless colonies SMAC
161
Describe enterotoxigenic (ETEC)
- traveler diarrhea, diarrhea in infants
- transmission = contaminated food or water
- disease mechanism are toxins
- gram stain of stool = no segemented neutrophils or RBCs
- profuse watery stool, DNA probes to detect toxins or toxins genes
162
Describe enteroinvasive (EIEC)
- blood diarrhea, dysentery-like, Usually in young children in areas of poor sanitation
- transmission = contaminated food or water
- disease mechanism is invasiveness
- gram stain of stool = segemented neutrophils, RBCs, mucus
163
Describe enteropathogenic (EPEC)
- diarrhea in infants. Major pathogen in infants in developing countries
- transmission = formula and food contaminated with fecal material
- disease mechanism is adherence attachment
- gram stain of stool = no segemented neutrophils or RBCs
- watery diarrhea with mucus
164
Describe enteroaggregative (EAEC)
- diarrhea in developing counties. Chronic diarrhea in HIV-infected patients
- transmission = nosocomial and community acquire
- disease mechanism is adherence attachment
- most labs can not detect
165
Describe Diffusley Adherent of diarrheagenic e. Coil
- diarrhea and UTI
- most common in children in developing countries
- transmission = little known about epidemiology
- disease mechanism is adherence attachment
- not well studied
166
Describe Klebsiella pneumonia
- pneumonia, UTI, septicemia
- carbapenem-resistant K. Pneumonia is significant pathogen is both community and hospital settings
- POS = lactose, gas, VP, citrate, urease (slow)
- NEG = H2S, indole, MR, PD, motility, ODC
- encapsulated
- colonies usually mucoid
- some strains hydrolyze urea slowly
- K. Oxytocin is similar to K. Pneumonia except indole POS
- motility and ODC differentiates from enterobacter
167
Describe citrobacter
- nosocomial infections, urinary tract is most common site of infection
- POS = gas, H2S, MR, citrate, motility, ONPG
- NEG = VP, PD, LDC
- lactose variable
- ONPG and LDC differentiate from salmonella
-
168
Describe Edwardsiella Tarda
- oppotunistic, can cause bacteremia, wound, and GI infections
- environmental risk factors include exposure to brackish water or undercooked fish
- POS = gas, H2S, indole, MR, motility
- NEG = lactose, VP, citrate, PD, and urease
- primarily a fish pathogen
- POS. Indole differentiates from salmonella
169
Describe Klebsiella aerogenes
- opportunistic and nosocomial infections, UTI, respiratory tract infection (RTI), and wound infections
- POS = VP, ONPG, ODC, citrate, motility, gas
- NEG = H2S, indole, MR, PD
- colonies may be mucoid
170
Describe enterobacter cloacae
- nosocomial infections from contaminated medical devices ad instruments
- POS = ODC, VP, sucrose
- NEG = Oxidase, H2S, indole
- frequently antibiotic resistant (intrinsic and plasma-based)
171
Describe serratia marcescens
- opportunistic pathogen
- UTI (indwelling catheter), ocular, and GI tract in children
- POS = VP, citrate motility
- NEG = lactose, H2S, indole, PD, urease
- some produce red pigment when incubated at RT
172
Describe Proteus vulgaris and mirabilis
- UTI, would infections and septicemia
- POS = H2S, MR, PD, urease, motility
- NEG = Lactose
- swarming
- burned chocolate odor
- p. Mirabilis is most common and indole NEG
- P. Vulgaris indole POS, A/A on TSI because of sucrose fermentation
173
Describe Morganella Morganii
- mainly nosocomial infections, UTI, wound infections
- POS = Indole, MR, PD, urease (weak), motility, ODC
- NEG = lactose, H2S, VP, citrate
174
Describe Providencia
- UTI, diarrhea
- POS = indole, MR, citrate, PD, and motility
- NEG = lactose, H2S, VP
- P. Rettgeri is urease POS
- P. Stuartii is most commonly isolated
175
Describe Yersinia enterocolitica
- diarrhea
- POS = MR, urease, ONDC
- NEG = lactose, H2S, VP, citrate, PD
- GN coccobacilli
- bipolar staining
- optimal temperature 25-30C
- motile at 25C but not 35C
- CIN agar is selective
- Incubate 48 hours
- red “bulls eye” colonies surrounded by colorless halo
- Y. Pestis causes the plague
176
Which Enterobaterales are lactose NEG?
- shigella
- edwardsiella
- salmonella
- citrobacter (some)
- serratia
- proteus
- Morganella
- Providencia
- Yersinia
177
What enterobacterales are H2S POS?
- Edwardsiella
- salmonella
- citrobacter
- proteus
178
What enterobacterales are VP POS?
- Klebsiella
- Enterobacter
- Serratia
179
What enterobacterales are PD POS?
- Proteus
- Morganella
- Providencia
- Klebsiella (slow)
180
What enterobacterales are urease POS?
- proteus
- Morganella
- Providencia rettgeri
- some citrobacter
181
What enterobacterales are nonmotile at 35C?
- shigella
- klebsiella
- yersinia (motile at 22C)
182
What is the appearance of E. Col on selected media?
- TSI = A/A, gas
- MAC = flat, dry pink colony with darker pink halo
- HE = yellow
- XLD =yellow
183
What is the appearance of Shigella on selective media?
- TSI = K/A
- MAC = colorless
- HE = green
- XLD = colorless
184
What is the appearance of Edwardsiella on selected media?
- TSI = K/A, gas, H2S
- MAC = colorless
- HE = colorless
- XLD = red, yellow, or colorless with or without black centers
185
What is the appearance of citrobacter on selected media?
- TSI = A/A or K/A, gas, with or without H2S
- MAC = colorless at 24 hours. May become pink at 48 hours
- HE = colorless
- XLD = red, yellow, or colorless with or without black centers
186
What is the appearance of Salmonella on selected media?
- TSI = K/A, gas, H2S
- MAC = colorless
- HE = green
- XLD = red, with black center
187
What is the appearance of Klebsiella on selected media?
- TSI = A/A. Gas
- MAC = pink, mucoid
- HE = yellow
- XLD = yellow
188
What is the appearance of enterobacter on selected media?
- TSI = A/A, gas
- MAC = pink, may be around
- HE = yellow
- XLD = yellow
189
What is the appearance of Serratia on selected media?
- TSI = K/A
- MAC = colorless at first, turning pink. S. Mercescens may have red pigment at RT
- HE = colorless
- XLD = yellow or colorless
190
What is the appearance of proteus on selected media>
- TSI = K/A (Mirabilis), A/A (vulgaris), gas, H2S
- MAC = colorless, may swarm
- HE = colorless
- XLD = yellow or colorless, with or without black centers
191
What is the appearance of Morganella on selected media?
- TSI = K/A, gas
- MAC = colorless
- HE = colorless
- XLD = red or colorless
192
What is the appearance of Providencia on selected media?
- TSI = K/A
- MAC = colorless
- HE = colorless
- XLD = yellow or colorless
193
What is the appearance of Yersinia on selected media?
- TSI = yellow/orange
- MAC = colorless to peach
- HE = Salmon
- XLD = yellow or colorless
194
What are characteristics of non fermenting gram-negative rods?
- obligate anaerobes
- do not ferment carbs
- K/K on TSI
- may oxidizers or nonoxidizers (asaccharolytic)
- oxidation-fermentation (OF) medium: either open tube POS/closed tube NEG (oxidizer) or open tube NEG/closed tube NEG (nonoxidizers)
- grow on BAP and CHOC in 24-48 hours
- most grow are oxidase POS
- differentiates from enterobacterales
- resistant to a variety of antibiotics
195
Describe Psuedomonas Aeruginosa
- usually not normal flora
- important cause of nosocomial infections i.e. burn wound, RTI, UTI, bacteremia
- causes swimmer’s ear and contact lens keratitis
-predominant respiratory pathogen is patients with cystic fibrosis
- gram stain = Long, thin, pale-staining GNR. Slightly pointed or rounded ends
- culture = on BAP: flat, spreading colonies. Usually beta hemolytic. Dull gray or blue-green. Metallic sheen. Grows on MAC and EMB (lactose NEG)
- Oxidase POS
- catalase POS
- motile
- grows at 42C
- grape like odor
- only nonfermenters to produce pyocyanin (4% do not)
- also produces pyoverdin (fluroescent pigment)
- resistant to many antibiotics
196
Describe Acinetobacter spp
- part of normal flora of skin, pharynx in some
- opportunistic pathogen
- Nosocomial infections i.e. UTI, pneumonia, septicemia, meningitis
- 2nd to P. Aeruginosa in frequency
- A. Batmannii is most commmon
- gram stain = pleomorphic gram-negative coccobacilli (GNCB) in singles, pairs, short chains. Can be confused with N. Gonorrhea, Moraxella. May retain crystal violet in broths and direct smears and be confused with GPC
- culture = can grow on most media, including MAC. Some produce purplish colonies (might be mistaken for lactose fermenter)
- oxidase NEG (differentiates from N. Gonorrhoeae)
- Catalase POS
- nonmotile
- resistant to many antibiotics
197
Describe Stenotrophomonas maltophillia
- not part of normal flora
- common cause of wound infections from agricultural machinery
- colonizes immunocompromised patients.
- common in hospital
- nosocomial infections i.e. pneumonia
- gram stain = straight or slightly curved slender GNR in single or pairs
- culture = on BAP: large, non-hemolytic. May be light yellow. Agar may have lavender-green discoloration in areas of heavy growth. Grows on MAC
- most often Oxidase NEG
- Catalase POS
- motile
- rapid oxidation of maltose, weaker oxidation of maltose, weaker oxidation of glucose
- Ammonia odor
- resistant to many antibiotics
- disk diffusion can give false results
- both dilution recommended
198
Describe Campylobacter Jejuni
- most common cause of bacteria diarrhea
- sources of infection: chickens, raw milk, pets
- gram stain = curved, slender, GNR. “Gull wing-shaped,” loose spirals and S shaped. Stain faintly
- culture = microaerophilic and capnophilic. Grown on campy-BAP at 42C in increased CO2 (can grow at 37C; normal enteric flora inhibited by 42C incubation). Slow growing. Hold plate 3 days. NAATs can be used for faster ID
- Darting corkscrew motility
- Oxidase, catalase and hippurate hydrolysis POS.
199
Describe campylobacter Coli
- similar to C.jejuni, but less severe.
- usually food-borne
- gram stain = curved, slender, GNR. “Gull wing-shaped,” loose spirals and S shaped. Stain faintly
- culture = microaerophilic and capnophilic. Grown on campy-BAP at 42C in increased CO2 (can grow at 37C; normal enteric flora inhibited by 42C incubation). Slow growing. Hold plate 3 days. NAATs can be used for faster ID
- hippurate hydrolysis NEG
- A MALDI-TOF can be used to differentiate from C. Jejuni
200
Describe Campylobacter fetus
- causes bacteremia in immunocompromised and elderly
- uncommon stool isolate
- gram stain = curved, slender, GNR. “Gull wing-shaped,” loose spirals and S shaped. Stain faintly
- culture = most often isolated in blood cultures. Hold for 2 weeks. Inihbited on campy agar. Grows on routine media at 37C, not at 42C
- oxidase POS
- catalase POS
- hippurate hydrolysis NEG
201
Describe Helicobacter pylori
- gastritis, duodenal and peptic ulcers
- possible risk factor for gastric carcinoma
- gram stain = curved, slender, GNR
- culture = grows on nonselective media incubated at 37C in same atmosphere as Campy. Does not grow at 42C. Slow growing
- rarely cultured
- rapid urease tests on gastric biopsy, urea breath test, histology, PCR, serology, and ELISA all can be used for ID
202
Describe Vibrio cholerae
- cholera (acute diarrhea, dehydration, electrolyte imbalance)
- transmitted by contaminated water, seafood
- uncommon in US but may be seen in coastal areas
- gram stain = small comma-shaped GNR in direct smears; straight pleomorphic GNR in culture
- culture = nonhalophilic (does not require NaCl for growth). Grows on BAP, CHOC, MAC (NLF). Large yellow colonies on thiosulfate citrate bile salts sucrose (TCBS) (ferments sucrose). Alkaline peptide water (APW) can be used for enrichment
- “rice water” stools
- oxidase POS
- motile
- serological ID with antisera to 0 ag. 01 and 0139 strains cause epidemic
- 01 strains are divided into two serotypes and two bio types
203
Describe vibrio vulnificus
- 2nd most serious type of vibrio infection
- causes primary septicemia and wound infections most often following consumption of raw oysters
- patients usually have pre-existing conditions such as liver disease or immunosuppression
- seen in US
- gram stain = straight or curved GNR
- culture = halophilic (salt lovin, requires addition of Na+). Most are green on TCBS; some are yellow. May look like enteric on MAC because some are lactose POSSIBLE
- oxidase POS
204
Describe vibiro parahaemolyticus
- most common cause of bacterial foodborne intestinal infection in Asia
- contaminated seafood.
- water-associated would infections
- seen in U.S.
- gram stain = straight or curved GNR
- cultures = requires 1% NaCl for growth. Browns on BAP, MAC(NLF). Blue-green colonies on TCBS (does not ferment sucrose)
- oxidase POS
- motile
205
Describe aeromonas spp
- gastroenteritis and wound infections, often related to aquatic exposure. Septicemia, and meningitis
- gram stain = straight or curved GNR with rounded ends
- culture = Grows on routine medi. Most are beta hemolytic on BAP, NLF on MAC. Doesn’t grown on TCBS. CIN agar and APW can be used for selective isolation
- oxidase POS
- motile
- NAATS and MALDI-TOF can be used for ID
206
Descibe plesiomonas shigelloides
- Gastroenteritis from contaminated water or seafood
- bacteremia and meningitis in immunocompromised and neonates
- gram stain = pleomorphic GNR in singles, pairs, short chains, or long filaments
- culture = grows on BAP, CHOC. Most grow on MAC, appear as NLF. Does not grow on TCBS
- Biochemical and antigenic similarities to Shigella
- oxidase POS
- motile
207
Describe H. Influenza
- normal flora of upper respiratory tract.
- causes sinusitis, otitis media, pnuemonia, bronchitis, often elderly or compromised.
- type B common cause of pneumonia and meningitis in children where Hib vaccine not available
- gram stain = small, pleomorphic GNCB to long filaments. Capsules may be seen
- culture = grow on CHOC in 5-10% CO2. Media for optimum recovery should contain at least 10 mcg/mL f free X and V factors. Translucent, moist, tannish colonies. Encapsulated stains form larger and more mucoid colonies. Mousy or bleach-like odor. May demonstrate satellitism with staph on BAP
- porphyrins and porphobilinogen NEG
- NAATs can be used to detect from CSF, plasma, serum and whole blood
208
Describe H. Influenzae biotype aegyptius and H. Aegyptius
- both cause conjunctivitis (pink eye).
- H. Infleunza bio-type aegyptius also causes Brazilian purpuric fever
- gram stain = small, pleomorphic GNCB to long filaments. Capsules may be seen
- culture = grow on CHOC in 5-10% CO2. Media for optimum recovery should contain at least 10 mcg/mL f free X and V factors. Translucent, moist, tannish colonies. Encapsulated stains form larger and more mucoid colonies. Mousy or bleach-like odor. May demonstrate satellitism with staph on BAP
- characteristics are similar to H. Influenzae
- difficult to differentiate unless molecular methods are employed
209
Describe H. Parainfluenza , H. Haemolyticus and H. Parahaemolyticus
- normal flora of upper respiratory tract
- low incidence of pathogenicity
- gram stain = small, pleomorphic GNCB to long filaments
- culture = are larger, dry and tannish
- H. Parainfluenza is porphyrins POS
210
Describe H. Ducreyi
- never normal fora
- cause chancroid (sexually transmitted disease), rare
- gram stain = small GNCB, bipolar staining. May resemble schools of fish or railroad tracks
- culture = difficult to culture without the use of many growth factors in the media
- can ID by PCR
211
What is the speciation of H. Influenzae and H. Aeqyptius?
- requires X and V factor
212
What is the speciation of H. Parainfluenzae?
- requires V factor and porphyrin/delta-aminolevulinice acid (ALA)
213
What is the speciation of H. Haemolyticus?
- requires X factors, V factors, and hemolysis on rabbit or horse blood agar
214
What is the speciation of H. Parahaemolyticus?
- requires V factors, hemolysis on rabbit or horse blood agar, and porphyrin/delta-aminolevulinic acid (ALA
215
What is the speciation of H. Ducreyi?
- requires X factor only
216
Describe Bartonella
- trench fever, relapsing fever, bacteremia, endocarditis, cat-scratch disease
- gram stain = GNR pleomorphic rods
- culture = not practical, takes 9-40 days to grow. Requires heme for growth
- ID by nucleic acid technique
217
Describe Bordetella pertussis
- whooping cough in children and adults
- DTaP vaccine (diphtheria, tetanus, and pertussis)
- gram stain = small GNCB
- grows on Bordet-Gengou and Regan-Lowe (charcoal, horse’s blood) after 3-7 days
- MALDI or agglutination with specific antibodies used for ID
218
Describe Brucella
- Brucellosis (undulant fever).
- from unpasteurized milk or contact with infected goats, cows, hogs, dogs
- gram stain = tiny, faintly staining GNBC
- culture = most often isolated from blood cultures or biopsies of reticuloendothelial (RE) tissue. Blood cultures incubated in increased CO2 for 3 week. Culture not sensitive
- oxidase POS
- catalase POS
- urease POS
- level 3 pathogen
- potential bioterrorism agent
- reportable disease
- slide agglutination test with specific antibodies used for ID
219
Describe Francisella
- Tularemia (rabbit fever).
- human infected by tick or handling infected animal
- gram stain = pale-staining, small, pleomorphic, intracellular GNCB with bipolar staining
- culture = grows on special media enriched with glucose and cystine, Thayer-Martin (TM), and BCYE. Small, transparent colonies after 3 days
- level 3 pathogen.
- direct fluorescent ab methods for ID.
- send to public health lab for confirmation
220
Describe Gardnerella
- Normal flora of female genital tract
- associated with BV when it and anaerobics GNR are predominant and Lactobacillus is absent
- linked to maternal and neonatal infections, bacteremia, rarely UTI
- gram stain = small pleomorphic gram-variable rods
- cultures = not recommended for diagnosis of BV. Grows on BAP, PEA, CNA, HBT, and V agar. Produces diffuse beta hemolysis only on media containing human blood. Requires increased CO2 and 48-72 hour incubation
- catalase NEG
- oxidase NEG
- tests for diagnosis of BV: clue cells (vaginal epithelial cells covered with GNR
- whiff test (10% KOH added to vaginal secretion = fish-like odor
221
Describe Legionella
- legionnaire disease, Pontiac fever
- found in water (air conditioners, shower heads, whirlpools)
- gram stain = small, pleomorphic, weakly staining GNR (sometimes filamentous)
- culture = grows on BCYE in 3-4 days. Pale yellow-green fluorescence with Wood lamp
- oxidase POS
- ID by immunofluorescent stain or agglutination methods
- MALDI can also be used
222
Describe Pasterurella Multocida
- contracted by bite or scratch of cat or dog or contact with infected carcass. Wound and soft-tissue infection
- gram-stain = small, pleomorphic GNBC with bipolar staining
- culture = grows on BAP and CHOC but not MAC. Musty odor
- oxidase POS
- catalase POS
- indole POS
- weak oxidation reaction after 30 minutes being characteristic of Pasteurella
- MALDI can also be used for ID
223
What are acceptable specimens for an anaerobic culture?
- bile
- blood
- body fluids
- bone marrow
- percutaneous lung aspirate or biopsy
- suprapubic bladder aspirates
- tissue
- transtracheal aspirate
- wound
224
What are unacceptable specimens for anaerobic culture?
- expectorated sputum
- feces
- gastric juice
- swabs
- voided or Catherized urine
- bronchial washings
225
What are types of media used for culture of anaerobes?
- anaerobic blood agar-CDC
- Bacteroides bile - esculin (BBE) agar
- Brucella blood agar
- CNA blood agar
- cycloserine cefoxitin fructose egg yolk (CCFA) agar
- Kanamycin-vancomycin lakes blood (KVLB) agar
- egg-yolk agar (EYA)
- PEA agar
- THIO broth
226
Describe anaerobic blood agar-CDC
- nonselective enrichment medium that grows obligate and facultative anaerobes
- contains yeast extract, L-cysteine, hemin, and vitamin K
227
Describe Bacteroides bile - esculin (BBE) agar
- selective differential medium for Bacteroides fragilis
- bile salts and gentamicin act as inhibitors
- B. Fragilis colonies black with dark halos due to esculin hydrolysis
228
Describe Brucella blood agar
- enriched medium that grows obligate and facultative anaerobes
229
Describe CNA blood agar
- selective medium that grows obligate anaerobes and GP facultative anaerobes
230
Describe CCFA agar
- cycloserorine cefoxitin fructose egg yolk
- selective and differential for Clostridium difficile
- colonies are yellow due to fermentation of fructose.
- chartreuse fluorescence
231
Describe EYA agar
- egg yolk agar
- for determination of lecithinase and lipase production by clostridia and fusobactria
232
Describe KVLB agar
- kanamycin-vancomycin lacked blood agar
- most commonly used selective medium for isolation of anaerobic GNRs, especially Bacteroides and Prevotella
233
Describe PEA agar
- selective medium that inhibits enteric GNRs and grows obligate anaerobes and GP facultative anaerobes
234
Describe THIO broth
- thioglycolate broth
- all-purpose medium that supports growth of most aerobes and anaerobes
- can be used as backup broth to detect organisms present in small numbers or anaerobes
- THIO acts as a reducing agent
- Aerobes grow at top, strict anaerobes at bottom, facultative anaerobes throughout
- store at RT
- boil and cool before use
235
What are the ideal anaerobic atmosphere?
- 80-90% N2
- 5% H
- 5-10% CO2
236
What methods are used to create an anaerobic environment?
- anaerobic transport media
- self-contained anaerobic agar
- anaerobic generating systems
- anaerobic chamber
237
Describe anaerobic transport media
- tubes of O2-reduced agar, tubes with anaerobic environment inside, or gas-impermeable bags with gas-generating environment
- most have indicator that changes color in presence of O2 can maintain anaerobes for 24-72 hours
238
Describe self-contained aerobic agar
- Brucella-based blood and LKV agars contain enzyme that reduces O2
- lids create airtight seal
239
Descibe anaerobic generating system
- plates in bag or container
- packet or ampule added
- chemical reaction removes O2
240
Describe anaerobic chamber
- anaerobic atmosphere created/maintained by gas tank, palladium catalysts, and desiccants
- work done through glove or gloveless openings
241
What is used as quality control for anaerobic environment?
- methylene blue strip
- resazurin
- oxygen analyzers
242
Describe methylene blue strip
- blue = O2 present, white or colorless = no O2
243
Descibe resazurin
- pink = O2 present
- colorless = no O2
244
Describe oxygen analyzers as quality control of anaerobic environment
- more expensive
- real time monitoring
245
What are the methods for IDing Anaerobes?
- gram stain
- growth on media
- special-potency antimicrobial disks
- rapid tests
- conventional tubed biochemical
- biochemical multi test systems
- preformed enzyme-based systems
- Gas-liquid chromatography (GLC)
- 16S ribosomal ribonucleic acid (RNA) gene sequencing
- MALDI
246
Describe gram stain for IDing anaerobes
- gram reaction; morphology; presence, location and shape of spores provides clues to ID
- some GN anaerobes stain faintly with safranin
- recommended to extend time for counterstaining to 3-5 min or use 0.1% basic fuchsia
- some GP anaerobes
- clostridium may stain pink
247
Describe growth on media to ID anaerobes
- which media organism grows on, pigmentation, hemolysis and colonial morphology provide clues to ID
248
Describe special-potency antimicrobial disks
- kanamycin, vancomycin, and colistin disks can be used to differentiate anaerobes and ensure that over-decolonized Clostridium is not misidentified as GNR
- disks placed on 1st quadrant of plate
- after incubation, observe if organism is susceptible or resistant
249
Describe rapid tests for IDing Anaerobes
- for presumptive ID i.e. fluorescence; catalase; spot idole; urease; motility; sodium polyanethanol sulfonate (SPS), nitrate, and bile disks; lecithinase, lipase, and proteolytic reaction on EYA
250
Describe conventional tubed biochemicals for IDing anaerobes
- test tubes contain in variety of media inoculated and incubated in anaerobic environment
- reaction leads to change in pH
- expensive and time consuming
- largely replaced by multi test systems
251
Describe biochemicals multi test systems for IDing anaerobes
- trays or strips are inoculated and read after 24-48 hour incubation in anaerobic environment
- code number is obtained and ID determined from codebook
- only contains codes for most commonly isolated anaerobes
252
Describe performed enzyme-based systems for IDing anaerobes
- detect pre-existing exymes
- panels or cards are inoculated and incubated in room air.
- color changes are read in 4 hours
- code number obtained and ID determined from codebook
- only contains codes for most commonly isolated anaerobes
253
Describe Gas-liquid chromatography for IDing anaerobes
- analysis of metabolic end products or cellular fatty acids
- not frequently used
254
Describe 16S ribosomal ribonucleic acid gene sequencing of IDing anaerobes
- DNA extracted from organism, amplified by PCR, sequenced on automated sequencer
- nucleotide sequence compared with known sequences in database
255
Describe MALDI of IDIng anaerobes
- extremely sensitive method requiring little sample volume for ID
- the mass spectra generated are compared to to known databases
256
Describe Finegoldia magna
- normal flora on skin and in mouth, intestines, female genital tract
- most commonly isolated and most pathogenic anaerobic GP
- associated with skin infections, decubitus ulcers, septic arthritis, bone infection following orthopedic surgery, oral and female genital tract infections, bacteremia
- gram stain = GPC in singles, pairs, tetrads, clusters. Resembles staph.
- small colonies
- may take 48 hour to grow
- catalase and indole NEG
- resistant to SPS
257
Describe peptostreptococcus anaerobius
- normal on skin and in mouth, GI, and genitourinary (GU) tracts
- mixed infections on skin, soft tissues, GI tract, female genital tract, bones, joints, lungs, brain
- gram stain = tiny GPC in chains. Resembles strep.
- small gray-white colonies in 24-48 hours
- sweet odor
- sensitive to SPS
258
Describe peptoniphilus asaccharolyticus
- normal on skin and in GI and GU tracts
- associate with obstetric and gynecological infections
- gram stain = GPC in pairs, short chains, tetrads, clusters.
- some strains decolonize easily and can be confused with gram-negative anaerobes
- yellow colonies
- musty odor
- resistant to SPS
- Indole POS
259
Descibe Actinomyces
- normal inhabitants of mucosal surfaces throughout the body
- infects brain, face, lungs, genitals
- gram stain = short or long gram-Positive rods (GPR). Branched or unbranched. Banded appearance. Can break into club-shaped rods resembling diphthroids
- sulfur granules may be seen in discharge
- crush and stain to revel characteristics Gram-stain morphology
- “Molar tooth” colonies
260
Descibe Clostridium botulinum
- Botulism due to ingestion of toxin in inadequately cooked or improperly canned foods
- infant botulism due to ingestion of spores from honey
- wound botulism from injection drug use
- GPR with oval subterminal spores
- reportable disease
- toxin testing at public health labs
261
Describe Clostridioides difficile
- antibiotic-associated diarrhea, pseudomembranous colitis
- gram stain = thin GPR. May form chains. Rare oval subterminal spores
- yellow ground glass colonies on CCFA
- usually not cultured
- must demonstrate toxin production
- toxins A and B
- tissue culture molecular methods or enzyme immunoassay (EIA) to confirm
262
Decribe Clostridium perfringens
- normal in GI tract
- causes gas gangrene
- most common bacteria cause of food poisoning in US
- can produce more than 16 different toxins and is classified by toxinotype
- gram stain = large GPR with blunt ends in chains. “Box-cars”. Tendency to stain gram NEG. Usually no spores
- most commonly isolated clostridium
- double zone of beta hemolysis on SBA
263
Describe Clostridium tetani
- causes tetanus
- deep wounds infected with soil
- gram stain = GPR with swollen terminal spores. “Drumstick” “tennis racquet”. Becomes gram negative after 24 hours
- rare in US because of DTaP vaccine but common worldwide
- lab confirmation rarely required
264
Describe cutibacterium
- normal flora of skin
- common contaminant of blood cultures
- causes acne; infections associated with artificial joints, catheters, shunts, artificial heart valves; keratitis; bacteremia; endocarditis
- gram stain = club shaped, pleomorphic GPR. Diphtheroid-like
- most common anaerobic GPR
- catalase POS
- indole POS
265
266
Describe lactobacillus
- normal in the mouth,
- predominant and beneficial flora of vagina during reproductive years
- produces lactic acid, maintains vaginal pH
- absence n vagina predisposes to BV and yeast infections
- rare cause of endocarditis, septicemia in immunocompromised
- gram-stain = pleomorphic, long, thin, non-spore-forming GPRm often in chains. Chains of rods in THIO
- aerotolerant anaerobes
- grow better under anaerobic conditions
- colonies vary greatly
- may resemble S. Viridans
- catalase NEG
267
Describe veillonella
- normal flora of upper respiratory tract, GI and GU tracts
- usually in mixed culture
- can cause infections in immunocompromised
- gram stain = tiny GNDC in pairs, clusters, short chains. Can resemble Neisseria
- may show weak red fluorescence under UV light
- does not grow on KVLB
- usually does not reduce nitrates
268
Describe Bacteroides fragilis
- main anaerobic organism recovered from intraabominal infections
- also causes endocarditis
- more virulent and antibiotic resistant than most other anaerobes
- gram stain = pleomorphic, pale, irregularly staining GNR
- resistant to bile
- hydrolyze esculin
- turns BBE brown
269
Describe Fusobacterium
- pulmonary infections, brain abscesses, oral lesions
- GNR. F. Nucleated is long and thin with tapered ends (spindle shaped)
- Indole POS
- most fluoresce chartreuse (green-yellow) under UV light
270
Describe porphyromonas
- head, neck, and pleuropulmonary infections
- gram stain = GNCB
- slow growing
- brown to black on BAP
- some fluoresce brick-red to orange under UV light
- does not grow on KVLB
271
Describe Prevotella
- head, neck, nad pleuropulmonary infections
- gram stain = GNCB
- slow growing
- some are pigmented
- brown to black on BAP and KVLB
- some fluoresce brick-red to orange under UV light
- Does not grow on BBE
272
Describe lab safety for Mycobactrium tuberculosis
- transmitted by inhalation of aerosols
- low infective dose
- separate room w/ non circulating ventilation system and negative air pressure
- properly functioning BSC is crucial
- BS-2 practices suggested only for non-aerosol generating activities and BSL-3 for manipulation of cultures and susceptibility testing
- electric incinerators instead of flames to sterilize wire loops
- slide-warming trays instead of flames to fix slides
- tuberculocial disinfectants to clean equipment and BSC: 10% bleach solution
- UV light in BSC
- annual testing of employees for exposure
273
What mycobacteria specimens require digestion/decontamication
- sputum and other specimens with normal flora such as gastric lavage
274
What mycobacteria specimen does not require disgeston/decontamination ?
- tissue of body fluids collected aseptically
275
What are digestion/decontamination methods for mycobacteria?
- Sodium Hydroxide (NaOH), N-acetylene-L-Cysteine (NALC)/NaOH, benzalkonium chloride, oxalic acid
- NaOH digest at and decontaminating agent.
- NALC is liquifying agent
276
Describe staining to ID mycobacteria
- cell walls have high lipid content (mycolic acid)
- difficult to stain
- resist decolorization by acid alcohol (acid-fast)
- gram stains poorly
- use carbofuchsin or fluorochrome acid-fast stains
277
What media is used to ID Mycobacteria?
- Middlebrooke 7H10 & 7H11
- Löwenstein-Jensen
- Middlebrooke 7H9
- combo of solid based and liquid based medium recommended for primary isolation
278
Describe incubation to ID mycobacteria
- 35C in 5-10% CO2
- exception: M. Marinum, M. Ulcerans, or M. Haemophilum suspected, incubate at 25-30%
279
Describe rate of growth for mycobacteria
- slow
- on solid media, most require 2-6 weeks incubation
- rapid growers 2-3 days
- more rapid growth in liquid media
280
What methods are used to ID mycobacteria?
- colony morphology
- growth rate
- optimum temp for growth
- photoreactivity
- biochemical tests
- smear microscopy
- nucleic acid hybridization
- PCR
- automated DNA sequencing
- MALDI
281
What are acid-fast stains?
- Ziegler-Neelsen
- Kinyoun
- Fluorochrome
282
Describe Zeihl-Neelson Stain
- primary stain = carbolfuchsin
- decolorizer = acid alcohol
- counterstain = methylene blue
- appearance of AFB = red, slightly curved beaded rods (2-8 um). Blue background
- requires heat
- examine at least 300 OI fields
- wipe lens after POS smear to avoid cross-contamination and false POS
- can be modified for improved detection by adding cytospin and Triton processing
283
Describe Kinyoun stain
- primary stain = carbolfuchsin
- decolonizer = acidic alcohol
- counterstain = methylene blue
- appearance of AFB = red, slightly curved beaded rods (2-8 um). Blue background
- cold stain
- examine at least 300 OI fields
- wipe lens after POS smear to avoid cross-contamination and false POS
284
Describe fluorochrome stain
- primary stain = auramine-rhodamine
- decolorizer - acid alcohol
- couter stain = potassium permangnate or Acridine orange
- appearance of AFB = yellow-orange rods against dark backgrounds
- more sensitive and faster than carbolfuchsin
- can examine at 250x
- read immediately or store at 2-8C in dark to avoid fading
- examine a least 300 fields
- POS should be confirmed with carbolfuchsin stain
- rapid 2-minute stain now available
285
What are medically important mycobacteria’s?
- M. Tuberculosis
- M. Bovis
- M. Africanum
- M. Avium complex
- M. Haemophilium
- M. Ulcerans
- M. Kansasii
- M. Marinum
- M. Simiae
- M. Gordonae
- M. Abscessus
- M. Chelonae
- M. Fortuitum
- M. Leprae
286
Describe M. Tuberculosis
- TB
- slow growth
- average recovery time by conventional methods 21 days
- rough, dry, buff- colored colonies
- serpentine cording on smear from culture
- Niacin POS
- nitrate POS
- 68C catalase NEG
287
Describe M. Bovis
- causes diseases similar to M. Tuberculosis and is treated accordingly
- resistant to pyrazinamide
- Bacille Calmette-Guerin (BCG) is attenuated strain used in vaccine in some parts of world
- cause false-POS purified protein derivative (PPD) test
288
Describe M. Africanum
- causes lung infection
- disseminated disease in immunocompromised
- spread by respiratory droplets
- endemic in equatorial Africa
- rare in the US
289
Describe M. Avium complex
- most common NTM to cause lung disease in US
- most common systemic bacterial infection in AIDS patients
- cervical lymphadenitis in children
- M. Avium and M. Intracellulare most commonly cause lung disease
- contracted from environment
290
Describe M. Haemophilum
- skin, joint, bone, lung, infections, in immunocompromised.
- lymphadentitis in children
- requires hemin for growth
- grows best at 28-30C
291
Describe M. Ulcerans
- chronic infection of skin and subcutaneous tissue. Buruli ulcers
- endemic in tropics
- 3rd most common mycobacterial disease after TB and leprosy
- grows best at 30C
- molecular techniques aid ID
292
Describe M. Kansasii
- 2nd most common NTM to cause lung disease
- causes skin and soft tissue infections, lymphadenitis
- can disseminate in immunocompromised
- often found in tap water
- photochromogens
- can ID by 16sRNA probe
293
Describe M. Marinum
- skin infections
- contracted from swimming pools, aquariums.
- Grows best at 28-30C
- photochromogens
294
Describe M. Simiae
- rare but associated with chronic respiratory infection
- difficult to treat due to multidrug resistance (MDR)
- high mortality and morbidity
295
Describe M. Gordonae
- Rarely cause infection (laboratory contaminant)
- “tap-water bacillus”
- scotochromogen (does not require light to produce pigment)
296
Describe M. Abscessus
- in water, soil, dust.
- contaminant of mecical devices
- skin and soft tissue infections
- lung infections in those with chronic lung disease (cystic fibrosis)
- disseminated infection in immunocompromised
- rapid grower
- tap water is reservoir
297
Describe M. Chelonae -
- disseminated cutaneous infections in immunocompromised, infections of lungs, bone, central nervous system, prosthetic heart valves
- rapid grower
298
Describe M. Fortuitum
- infections of skin, soft tissues, IV and infection sites, surgical wounds
- rapid grower
- common environment
- weakly gram POS
- weakly acid fast
- stains with carbolfuchsin but may not with fluorescent stain
299
Describe M. Leprae
- leprosy (Hansen disease)
- endemic in southern hemisphere
- <100 cases per year in the US
- Most in Texas, California, Louisiana, Hawaii, Puerto Rico
- armadillos may be reservoir
- doesn’t grow on artificial media
- can be grown in footpads of mice and armadillos
- diagnose by acid-fast stain of tissue
- less acid fast than M. Tuberculosis
300
Describe Chlamydia trachomatis
- most common STI (bacterial) in US
- trachoma, lymphogranuloma venereum, non- gonococcal urethritis, pelvic inflammatory disease
- pneumonia and conjunctivitis in newborns
- Geimsa stain
- direct fluorescent antibody stain, and most commonly NAATs
- obligate intracellular parasite
- need epithelial cells for culture
- wooden swabs are toxic
- NAAT preferred for genital and first-catch urine specimens
- commercial systems available for simultaneous detection of N. Gonorrhoeae
301
Describe chlamydia pneumonia
- 3rd most common cause of acute RTI
- serological or molecular tests are method of choice
- olbligate intracellular parasite
- risk factor for Guillain-Barré syndrome
302
Describe chlamydia Psittaci
- psittacosis (“parrot fever”)
- rare in the US
- serological or molecular tests
- obligate intracellular parasite
- spread by birds
303
What organisms are spirochetes?
- Borrelia recurrent is
- Borrelia burgdorferi sensu lato
- treponema pallidum
- leptospira interrogens
304
Describe Borrelia recurrentis
- relapsing fever
- lab diagnosis = geimsa or wright stained blood smear. Thrombocytopenia is common
- transmitted by ticks and lice
- there are over 20 species in the relapsing fever Borrelia group but B. Recurrentis causes the most cases
305
Describe Borrelia burgdorferi sensu lato
- Lyme disease (erythema chronicum migrans, neurological and cardiac abnormalities, arthritis
- lab diagnosis = grows on modified Kelly medium. ELISA is most common method
- most commonly reported tick-borne infection in US
- vector = Ixodes tick
306
Describe Treponema pallidum subspecies pallidum
- syphilis
- lab diagnosis = doest grow on artificial media. Darkfield microscopy. Fluorescent stain. Serology method (EIA) is most common method
- found worldwide
- 3 stages if left untreated
- can also cause congenital syphilis
307
Describe leptospira interrogates
- Weil disease (infectious jaundice, leptospirosis)
- lab diagnosis = PCR or serological tests. Can culture, growth = turbidity below surface of semisolid media such as EMJH. Hold culture for 13 weeks
- Zoonotic disease
- transmitted by urine of infected animal
- organism is blood and CSF during first week, then urine.
- common in tropical regions
308
Descibe Mycoplasma pneumonia
- primary atypical pneumonia (walking pneumonia)
- smallest free-living cells
- lack cell walls
- pleomorphic
- not visible by gram staining
- difficult to grow
- culture rare performed
- may take 21 days or more
- usually diagnosed by serology
309
Describe mycoplasma Hominis
- urogenital tract disease
- only species that will grow on SBA and CHOC but may require 4 days
- pinpoint translucent colonies easily overlooked.
- “fried-egg” colonies seen with stereomicroscope after staining weigh methylene blue
- serological methods available
310
Describe ureaplasma urealyticum
- urogenital tract disease
- granular brown appearance on AB agar because of urease production
- serological methods available
311
Describe Rickettsia
- Rocky Mountain Spotted Fever, rickettsial pox, epidemic typhus, murine typhus, scrub typhus
- lab diagnosis = don’t grow on artificial media. grow in lice, ticks, tissue culture, eggs.
- usually diagnosis by serology. Immunohistologic and molecular methods available
- obligate intracellular parasite
- transmitted by ticks, mites, lice, fleas.
- RMSP is most common rickettsial infection in US
- caused by R. Rickettsii
- BSL-3 biohazard
312
Describe Coxiella
- Q fever
- lab diagnosis = usual diagnosis by NAATs and serology. IFA and imaging available
- obligate intercellular parasite
- zoonotic disease
- found in cattle, sheep, goats
- highly contagious
- BSL - 3 biohazard
- reportable disease
- potential bioterrorism agent q
313
Describe Ehrlichia
- ehrlichiosis
- lab diagnosis = modular (clusters of organisms that resemble blackberries) in WBCs. Usual diagnosis by serology, molecular amplification, and blood smear examination
- obligate intracellular parasite
- infects WBCs
- transmitted by ticks
- disease similar to RMSF
314
Describe culture setup and interpretation of throat/nasopharynx specimen
- media = BAP, CHOC, strep. Selective agar may be used
- normal flora = alpha and gamma strep, commensal Neisseria, CNS, diphtheroids, S. Pneumonia, candida
- common pathogens = GAS (S. Pyogenes)
- stab streaking to detect beta strep that only produce streptolysin O
- direct antigens tests for GAS
- Negative result should be cultured
315
Describe culuture setup and interpretation of sputum, bronchial washings/aspirates, transtracheall aspirate specimens
- media = BAP, CHOC, MAC
- normal flora = CNS non-beta hemolytic strep, diphtheroids, commensal Neisseria, Haemophilus, yeast. Aspirates: none
- common pathogens = S. Pneumonia, H. Influenza, S. Aureus, enterobacterales, psuedomonas M. Catarrhalis, C. Albicans
- gram stain
- acceptable sputum: <10 epithelia cells/lpf, >25 segemented neutrophils/lpf
- neutropenic patients may not produce segemented neutrophils
316
Describe culture setup and interpretation of urine specimens
- media = BAP, MAC. Use 0.01 mL or 0.001 mL calibrated loop
- normal flora = clean catch: few skin flora. Catheterized or aspirate: none
- common pathogens = E. Coli, Klebsiella, enterobacter, proteus, enterococcus, pseudomonas. S. Aureus, S. Saprophyticus, CNS, GBS
- rapid screen for UTI:1 or more bacteria/OI field on Gram stain of uncentrifuged urine
- interpretive guidelines vary.
- low counts are significant in catheterized urines and aspirates
- 3 or more organisms with none predominant = probable contamination
317
Describe culture setup and interpretation of stool specimens
- media = BAP, MAC, and EMB, XLD or HE, campy blood
- normal flora = anaerobes, enterobacterales, enterococci, strep, yeast
- common pathogens = salmonella, shigella, campy, diarrheagenic E. Coli, aeromonas, plesiomonas, Y. Enterocolitica, vibrio
- segemented neutrophil = invasive diarrhea (salmonella, shigella, campy, EIEC, C. Diff.
- No segemented neutrophil = enterotoxin-mediated diarrhea (ETEC, vibrio. RBCs with EHEC)
318
Describe culture setup and interpretation of genital specimens
- media = BAP, CHOC, MAC, Thayer-Martin. Add THIO and anaerobic media for tissue and aspirates
- normal flora = urethra: CNS, diphtheroids. Vagina prepubescent/postmenopausal: yeast, GNR, GPC. Vagina reproductive years: lactobacillus, GPC. Surgical/aspirates: none
- common pathogens = N. Gonorrhea, C. Albicans, GBS, S. Aureus. Surgical/aspirates: also anaerobes
- gram stains
319
Describe the setup and interpretation of CSF specimen
- Media = BAP, CHOC, MAC, some add THIO
- normal flora = none
- common pathogens = GBS (infants), Listeria (infants,elderly), H influenza (unimmunized children), N. Meningiditis, S. Pneumonia, GNR. Any isolate is significant
- use tube 2
- handle stat
- do not refrigerate
- use cytocentrifuge to concentrate specimen increases sensitivity of gram stain.
- ID all isolates
320
Describe setup and interpretation for blood specimens
- media = most labs inoculate an aerobic and anaerobic bottle. Some use 2 aerobic bottles, no anaerobic
- normal flora = none
- common contaminants = CNS, micrococcus, alpha strep, diphtheroids, cutibacterium
- common pathogens = any isolate potentially significant. GPC are most common
- skin prep: 80-95% ethanol or isopropyl alcohol followed by povidone-idone or tincture of iodine
- optimum volume: 20 mL for adult; child 1 mL per year of life
- blood to blood ratio = 1:5 to 1:10
- usually draw 2 sets in 24 hours
- automated systems used for monitoring
- growth indicated by consumption of O2 or production of CO2
- gram stain and subculture required for ID
321
Describe culture setup and interpretation of wound specimens
- media = BAP, CHOC, MAC. Surgical, aspirate or tissue: add anaerobic blood, KVLB, THIO
- normal flora = CNS, diphtheroid, cutibacterium. Surgical, aspirate or tissue: none
- common pathogens = S. Aureus, beta hemolytic strep, Enterobacterales, P. Aeruginosa. Surgical, aspirate, or tissue: also anaerobes. Human bite: alpha hemolytic strep, S. Aureus, S. Pyogene, eikenella corrodens, anaerobes
- gram stain
322
Describe culture setup and interpretation of Eye specimen
- MEdia = BAP and CHOC
- normal flora = CNS, diphtheroids, cutibacterium
- common pathogens = H. Influenza, S. Pneumonia, S. Aureus, enterbacterales, pseudomonas
- gram stain
- if POS, call
- Keratitis is emergency
- loss of eye can occur
323
Describe culture setup and interpretation of ear specimens
- media = BAP, CHOC, MAC
- normal flora = normal skin flora
- common pathogens = S. Pneumonia, H. Influenza, GAS, M. Catarrhalis, S. Aureus, psuedomonas
- gram stain
324
Which fecal pathogens are oxidase POS?
- Campylobacter
- Vibrio
- Aeromonas
- Plesiomonas
325
Which fecal pathogens are oxidase NEG?
- E. Coli
- salmonella
- shigella
- yersinia
326
250
0
327
Describe Natural penicillin
- penicillin V and G
- mode of action = inhibit cell wall synthesis
- spectrum of activity = narrow. GPS other than staph, some GN
- beta-lactam, bactericidal, allergic reaction summon side effect
328
Describe synthetic penicillins
- methicillin, oxacillin, ampicillin, carbenicillin, piperacillin
- mode actions = inhibit cell wall synthesis
- spectrum of activity = broader spectrum of activity than natural penicillins. GP’s (not MRSA) and GNs
- beta-lactam, bactericidial, penicillinase-resistant penicillin = oxacillin, methicillin, nafcillin, cloxacillin, dial oxacillin.
- drug class of choice of for staph. Infections
329
Describe cephalosporins
- cephalexin, ce foot in, cleft RIA one, cefepime
- mode of action = inhibit cell wall synthesis
- spectrum of activity = GP’s and GNs
- each generation has broader spectrum of activity than previous
- B-lactam. Bactericidal
330
Describe Carbapenems
- imipenem
- inhibit cell wall synthesis
- widest spectrum. GPS and GNs, including P. Aeruginosa and anaerobes, but not MRSA or VRE
- B-lactam, bactericidal.
- reserved for IV use in serious infections
331
Describe monobactams
- aztreonam
- mode of action = inhibit cell wall synthesis
- spectrum of activity = P. Aeruginosa and other GNR. Low activity against GPs and anaerobes
- B-lactam. Bactericidal
332
Describe glycopeptide anitbiotics
- vancomycin
- mode of action = inhibit cell wall synthesis
- spectrum of activity = GPs. Drug of choice for MRSA. Some strain of enterococcus are now reisistant (VRE)
- bactericidal. Used for serious infections
333
Describe macrolides
- erythromycin, Clarithromycin, azithromycin
- mode of action = inhibit RNA-dependent protein synthesis
- spectrum of activity = GPs and some GNs, intracellular organisms, atypical mycobacteria - bacteriostatic
334
Describe tetracyclines
- tetracycline
- doxycycline
- mode of action = inhibit protein synthesis
- GPs and GNs, intracellular organisms, spirochetes, MRSA, VRE
- Bacteriostatic.
- used less often than Beta-lactams.
- not given to children or pregnant women due to staining of teeth, abnormal bone growth `
335
Describe aminoglycosides
- gentamicin, tobramycin, amikacin
- mode of action = inhibit protein synthesis by binding irreversibly to 30S ribosomal subunit
