Growth Requirements

• temperature
Microbiology Review – psychrophiles - 15C
– mesophiles - 37C
– thermophiles - 50-60C
Dr. Hassan Aziz ©
• pH
Armstrong Atlantic State University – bacteria: 6.5-7.5
– fungi: 5.0-6.0

Growth Requirements Growth Requirements

• osmotic pressure • other
– isotonic – microaerophiles
– halophiles • low oxygen

• oxygen – capnophiles
• CO2
– aerobes
– facultative anaerobes
– obligate anaerobes

3 4

Media Media
• agar • anaerobic media
– polysaccharide from marine algae – reducing agents (thio, cysteine)
– melts at 100C, solidify at 40C – heat broth tubes
• complex media – Gas pak envelopes
– peptone and extracts • sodium bicarbonate
• water ==> hydrogen + CO2
• palladium (catalyst)

5 6

1
 Media Media
• enriched • BAP
– most fastidious bacteria
• selective – hemolysis
• differential • CHOC
– Haemophilus, Neisseria
• 80C
• MAC, EMB
– selective, differential
• (crystal violet, bile, eosin, methylene blue)
– GNR
7 8

Media Media
• phenylethyl Alcohol • CAMPY-BAP
– GPC – Campylobacter
– selective • 10% + antibiotics
• Columbia colistin nalidixic acid • THIO broth
– GPC – general
• MTM – anaerobes
– N. gonorrhoeae, N. meningitidis • Lowenstein-Jensen, Middlebrook
• Vancomycin, colistin, nystatin, trimethoprim – Mycobacterium

9 10

Thioglycolate Broth Anaerobic Media

• Anaerobic BAP
• Bacteroides Bile Esculin Agar (BBE)
• Kanamycin-vancomycin laked blood
• Cycloserine-cefoxitin fructose agar (C.
difficile)

11 12

2
 Incubation Sterilization
• 5% CO2 • heat - denatures protein
• 35-37C – autoclave
• 15lb, 121C, 15 minutes
• 50-70% humidity
– dry heat
• flame, incinerator
– pasteurization
• 72C, 15 seconds

13 14

Sterilization Sterilization vs. Disinfection

• filtration • sterilization
– pore size
– kills microorganisms
• refrigeration - slow growth
– autoclave
• desiccation - remain viable, can not multiply
• radiation • disinfection
• disinfection – inactivate or inhibit microorganisms
– phenol: damage cytoplasmic membrane – bleach
– halogens: oxidizers
– alcohol: denature protein & dissolve lipids

15 16

Specimen Collection & Handling Criteria for Rejection

• time of collection • preservative
• quantity • QNS
• appropriate collection device • dry swab
• prior to antibiotic therapy • leaky containers
• within 2 hours of collection
• NEVER refrigerate CSF, anaerobes,
GC
17 18

3
 Blood Culture Collection Culture Conditions
• prep skin with alcohol and iodine • most at 35-37C
• prior to fever spike • Campylobacter: 42C
• Yersinia: 25-30C
• 2-3 cultures within 24 hours
• Campy - microaerophilic: 5-10% CO2
• 1:5 - 1:10 blood to broth dilution
• 50-70% humidity
• 10 ml for adults, 1-5 for infants and • anaerobes:
children – THIO broth
– 10% H2, 5% CO2, 85% N2, palladium

19 20

Inoculation Stains
• chrome, platinum, plastic loop • gram stain
• calibrated (0.01ml or 0.001 ml) – crystal violet, iodine, alcohol, safranin
• number of colonies x 100 (or 1000) • kinyoun & ZN
– carbol fuchsin, acid alcohol, methelene
blue

21 22

Bacterial Structure: Introduction Bacterial Structure: Introduction

• Animal • Prokaryotes
• Plant – Primitive nucleus
• Protista • True bacteria.
"eubacteria”
– Eukaryotes
– Prokaryotes
Prokaryotic cell
– Archaebacteria

23 24

4
 Bacterial Structure: Introduction
The Bacterial Cell: The Cell Wall
• Functions
• Eukaryotes – Provides strength
– True nucleus bound – Protects the internal contents
by a nuclear
membrane – Determines the shape
• Fungi, protozoan

Eukaryotic cell

25 26

The Bacterial Cell: The Cell Wall The Gram-Positive vs.

The Gram-Negative Cell Wall
• Peptidoglycan:A network
of N-acetyl Glucosamine
(NAG) and N-
acetylmuramic acid (NAM)
connected by peptide
bonds

27 28

The Gram-Positive vs. Morphology of Bacterial Species

The Gram-Negative Cell Wall

Cocci in clusters

Cocci in chains

Gram-positive bacteria Gram-negative bacteria

Large spore-forming bacilli

Small non–spore-
29 30
forming bacilli

5
 Other Optional Parts Special Stains
• Capsule
• Flagella
• Pili
• Endospores

Acridine orange
Lactophenol cotton blue

Streptococcus pneumoniae with capsules

Clostridium sp. with endospores 31 32

Acid-fast India Ink

Microbial Growth and Nutrition Gram Positive Cocci

• Staphylococcus
• Streptococcus

33 34

Staphytoccus aureus
Staphylococcus
• Habitat: anterior nares (carriers)
• grape-like clusters
• Primary pathogen of the genus
• S. aureus
• Produce superficial to systemic infections
– coagulase positive
• Mode of transmission: traumatic introduction
– boils & carbuncles
• Predisposing conditions
– impetigo
– Chronic infections
– post surgical wounds
– Indwelling devices
– toxigenic – Skin injuries
• scalded skin syndrome
– Immune response defects
• TSS
• food poisoning 35 36

6
Virulence Factors: Extracellular Enzymes Virulence Factors: Extracellular Enzymes
• Hemolysins: hemolyze RBCs • Exfoliatin
• Leukocidin (Panton-Valentine): kill – Epidermolytic toxin
PMNs – Phage group II staphylococci
• Enterotoxins – Ritters Disease

– A/D: food poisoning • TSST-1: Toxic shock syndrome toxin-1

– Multisystem disease
– F: TSSAT
– High fever
– B: pseudomembranous enterocolitis – Hypotension
– Shock
37 38

Virulence Factors: Extracellular Enzymes

Clinical Infections
• Skin and wound
• Hyaluronidase: connective tissue – Impetigo
– Furuncles
• Staphylokinase: fibrinolysin – Carbuncles
– Boils
• Coagulase: virulence marker – Surgical wound infections
• Food poisoning
• Lipase: allows colonization
• Scalded skin syndrome
• Penicillinase: confers resistance
Bullous impetigo

39 40

Clinical Infections Staphylococcus

• Toxic shock syndrome • coagulase negative
• Other infections – opportunistic infections
– Respiratory (less often)
– S. epidermidis
– Bacteremia • endocarditis, prosthetic device infections
– Osteomyelitis – S. saprophyticus
• UTI in young women

41 42

7
 Laboratory Diagnosis: Laboratory Diagnosis:
Direct Smear Examination Cultural Characteristics
Microscopic Examination • Colony morphology
 Gram-positive cocci – Smooth, butyrous,
 pairs and clusters
Insert Figure 10-1 white to yellow,
creamy
 Numerous polymorphonuclear
– S. aureus may
cells (PMNs) produce hemolysis on
blood agar

S. aureus

43 44

Cultural Characteristics
Catalase
• Coagulase-negative
staphylococci • 3% H2O2 == catalase ==> H2O + O2
– Smooth, creamy, (bubbles)
white
– Small-to medium-
sized, usually non-
hemolytic
• S. saprophyticus
– Smooth, creamy, may
produce a yellow
pigment
45 46

Identification Tests: Coagulase

Coagulase Test Test
• Detects enzyme
coagulase
– Cell-bound “clumping Tube test detects the
factor” extracellular enzyme
– Extracellular enzyme “free coagulase”
“free coagulase”
• Two methods
– Slide test
– Tube test
Slide coagulase test detects clumping factor
47 48

8
 Novobiocin Susceptibility Test Schematic Diagram
• Test to differentiate
coagulase-negative
staphylococci from
S.saprophyticus from
urine samples
– S. saprophyticus is
resistant (top)
– Other CNS are susceptible

49 50

Antimicrobial Susceptibility
Streptococcus
• For non–beta-lactamase producing S. aureus
(methicillin-susceptible) • GPC in chains
– Penicillinase-resistant synthetic penicillins (methicillin, • Lancfield grouping based on C
nafcillin, oxacillin, dicloxacillin) carbohydrate
• For methicillin -resistant S. aureus (MRSA) and
methicillin-resistant S. epidermidis (MRSE)
– Vancomycin combined with rifampin or gentamicin
• Emergence of vancomycin resistance

51 52

Cell Wall Structure Beta (ß) Hemolysis

• Thick peptidoglycan
layer
• Teichoic acid
• C=carbohydrate layer
present except in
viridans group
• Capsule in S.
pneumoniae and in
young cultures of most
species 53 54

9
 Alpha (a) Hemolysis No hemolysis (gamma) Hemolysis

55 56

Species Hemolysis Group Common Disease Association(s)

S.pyogenes ß
Antigen
A
Terms
Group A
streptococci
Pharyngitis; scarlet fever
pyoderma; rheumatic
fever; AGN
S. pyogenes
S.agalactiae ß B Group B Neonatal sepsis;
streptococci puerperal fever;
pyogenic infections;

S. equisimilis ß C Group C
streptococci
pneumonia; meningitis
Pharyngitis; impetigo;
pyogenic infections
• beta hemolysis
E. faecalis Alpha or no D Enterococci Urinary tract infections
E. faecium
E. durans
hemolysis
( rarely ß )
Wound infections
Bacteremia;
Endocarditis
• streptolysin S
S. bovis Alpha (α)or D Nonenterococci Urinary tract; pyogenic
S. equinus none
(rarely ß)
infections;
Endocarditis infections
– stable in oxygen
S. pneumoniae Alpha (α)
hemolysis
Pneumococcus Bacteremia;
pneumonia; – non-antigenic
meningitis;

Viridans and
Nonhemolytic
Alpha (α)
hemolysis or
- Viridans strep Endocarditis
• streptolysin O
S. sanguis no
S. salivarius
S. mitis or
nonhemolytic
hemolysis
– oxygen labile
S. milleri
S. mutans Dental caries
– antigenic
Other species

57 58

Laboratory Diagnosis
S. pyogenes
• Identification
• erythrogenic toxin – Catalase-negative
– scarlet fever – Bacitracin-
• sensitive to penicillin susceptible
• pharyngitis
• impetigo
• wounds (burns)
• rheumatic fever (autoimmune sequalae)
• bacitracin positive
59 60

10
 Laboratory Diagnosis
S. agalactiae
• CAMP-test–positive
• narrow beta hemolysis zone
• neonatal sepsis, meningitis, UTI,
vaginal infections
• CAMP, sodium hippurate positive

61 62

S. bovis Enterococcus
• group D • growth in 40% bile and 6.5% NaCl
• bile esculin positive
• no growth in 6.5% NaCl

63 64

Laboratory Diagnosis
S. pneumonae
• diplococci, lancet shaped • Identification
– Catalase negative
• alpha hemolysis (mucoid)
– Optochin-
• lobar pneumonia susceptibility-
test–susceptible
• meningitis
– Bile-solubility-
• bacteremia test–positive
• otitis media
• sensitive to optichin, bile soluble
65 66

11
 Viridans GPC, Catalase Negative
Beta Hemolysis

• alpha or gamma A Disk

• endocarditis suscpetible resistant

S. pyogenes CAMP or
PYR + Hippurate

positive negative

S. agalactiae bile esculin

positive negative

enterococcus group D not A, B, or D

PYR & NaCl + PYR & NaCl -ve
67 68

GPC, Catalase Negative GPC, Catalase Negative

alpha hem olysis gam m a
hem olysis

optichin (P) disk

CAMP
(hippurate)
susceptible resistant
positive negative

S. pneum oniae bile esculin

S. agalactiae bile esculin

positive negative
positive negative

PYR viridans
6.5% salt viridans
6.5% NaCl PYR

positive negative positive negative

enterococcus group D enterococci group D

69 70

Gram Negative Cocci Laboratory Diagnosis

• Identification
• Neisseria – Morphology
• Gram-negative, kidney-
• Moraxella bean–shaped
diplococci
• diplococci (kidney bean shape)
• oxidase positive

71 72

12
 N. gonorrhoeae N. meningitidis
• CHOC and MTM • BAP, CHOC, MTM
• requires CO2
• CO2
• ferments glucose
• gonorrhea • ferments glucose and maltose
– STD • transmitted by respiratory droplets
– gram stain sensitive for males (close contact)
– no refrigeration
– penicillin sensitive - perform beta lactamase to • meningitis
determine penicillin sensitivity – children <3 years old
73 74

Moraxella catarrhalis Bacterial Meningitis

• respiratory infection • high neutrophils
• BAP, CHOC • low glucose
• hard colonies (move over) • high protein
• beta lactamase positive

75 76

Identification
Gram Positive Rods

Species Growth Acid production

• Corynebacterium diphtheriae
BAP R.T T/M Gluc Mal Lac Suc
• Listeria monocytogenes
N. gonorrhoeae =/+ = + + = = =

N. meningitidis
• Erysipelothrix
+ = + + + = =

N. lactamica + v + + + + =
• Bacillus
N. sicca + + = + + = +

M. catarrhalis + + = = = = =

77 78

13
 Corynebacterium diphtheriae Listeria monocytogenes
• pleomorphic with clubbed ends • small colonies with narrow zone of
• Chinese letters hemolysis
• catalase positive • catalase positive
• metachromatic granules • tumbling motility
• tinsdale agar: black colonies due to tellurite – umbrella motility at RT
hydrolysis
• bile esculin positive
• Elek test to determine toxin production
• cause diphtheria
• neonatal meningitis, sepsis
79 80

Erysipelothrix Bacillus
• non-motile • large ground glass colonies
• catalase negative • beta hemolysis
• H2S positive in TSI • catalase positive
• occupational infection for fishermen, • large GPR
butchers, rose growers • spore formers

81 82

B. anthracis Significant Bacillus Species

• Bacillus anthracis
• bamboo shoots – Agent of anthrax, a disease in livestock
– Humans acquire infection by contamination of
• Medusa head colonies wound or ingestion or inhalation of spores
• non-motile, non-hemolytic – Medusa head colonies
• anthrax – non-motile, non-hemolytic
• Bacillus cereus
• B. cereus
– Causes food poisoning
– food poisoning (fried rice) - enterotoxin – An opportunist
• Bacillus subtilis
– Common laboratory contaminant
83 84

14
 Enterobacteriaceae E. coli
• peritrichous flagella when motile • indole and lactose positive
• ferment glucose • MR positive, VP negative, citrate negative
• reduce nitrate • UTI
• oxidase negative • intestinal infections
• antigens – enterotoxigenic
– H: flagella – enteroinvasive
– enterohemorrhagic (E. coli O157:H7)
– K: envelope
– enteropathogenic
– O: cell wall
– all posses endotoxin, some exotoxin 85 86

Shigella Klebsiellae
• lactose negative • opportunist, UTI, pneumonia
• non-motile • Klebsiella
• bacillary dysentery (<100 organism for – non-motile, capsule
disease) – urea positive, ornithine negative
• S. dysenteriae - group A • Enterobacter
• S. flexneri - group B – motile, ornithine positive
• S. boydii - group C • Serratia
• S. sonnei - group D – red pigments, DNAse positive
87 88

Salmonella Citrobacter
• >10,000 organisms for disease • lysine negative (similar to Salmonella)
• H2S & lysine positive, indole & urea
negative
• S. typhi: typhoid fever
– blood positive early, stool positive in 2-3
weeks

89 90

15
 Proteus Yersinia
• urea positive, deaminase positive • Y. enterocolitica
• P. mirabilis – optimal growth: RT
– indole negative – invasive & toxigenic
• P. vulgaris • Y. pestis
– indole positive – plague

91 92

Oxidase Test
• p-phenylenediamine dihydrochloride
• blue when positive

93 94

Lactose ONPG
• lactose == permease ==> intracellular • orthonitrophenylgalactopyranoside ==
lactose galactosidase ==> orthonitrophenol
• lactose == galactosidase ==> glucose + (yellow) + galactose
galactose

95 96

16
 Nitrate Test TSI
• napthylamine + sulfanilic acid • 0.1% glucose, 1% sucrose, 1% lactose
• positive: pink • yellow butt: glucose fermented
• zinc dust to confirm • yellow slant: lactose or sucrose
• red slant: neither
• black butt: H2S (acid)
• KIA: same as TSI w/o sucrose

97 98

citrate Decarboxylase Tests

• citrate as source of carbon • ability to decarboxylate amino acids
• green media • lysine ==> cadaverine
• blue when positive • ornithine ==> putrescine
• arginine ==> putrescine
• indicator dye: bromocresol purple

99 100

Indole Urease
• tryptophan == tryptophanase ==> indole • urea == urease ==> ammonia
+ end products • phenol red turns pink
• indole + aldehyde ==> colored complex
• reagent: Kovac’s reagent

101 102

17
 MR-VP Phenyl Deaminase
• MR: organism ability to produce and • phenylalanine == deaminase ==>
maintain acid environment phenylpyruvic acid
– red color: acidic • phenyl pyruvic acid + ferric chloride ==>
• VP: green color
– acetooin +40% KOH + O2 ==> diacetyl
– diacetyl + peptone ==alpha naphthol ==>
red complex

103 104

105 106

Common GNR Media Nonfermentative Gram Negative

Rods
• glucose not fermented
• TSI: K/K or K/NC
• oxidase positive

107 108

18
 Pseudomonas aeruginosa Vibrio
• lactose negative • curved rods
• pyocyanin (green), fluorescein (yellow - • TCBS selective for Vibrio
IV), pyorubin (rust), pyoverdin (yellow)
• BAP: hemolytic colonies
• mucoid strains in CF cases
• lactose negative, nitrate positive,
• treated with 3rd generation
oxidase positive
cephalosporins or aminoglycosides
• burns, pneumonia, swimmer’s ear, eye
infections, UTI
109 110

V. cholera Campylobacter
• gastroenteritis, rice water diarrhea • C. jejuni
• yellow on TCBS – seagull appearance
• V. parahaemolyticus – microaerophilic
– green on TCBS – CAMPY-BAP
– enteritis – found in raw poultry and contaminated
• V. vulnificus water
– green on TCBS – catalase, oxidase, hippurate positive
– septicemia
111 112

Other Campylobacter Gardnerella vaginalis

• H. pylori • bacterial vaginosis
– gastric and duodenal ulcers • clue cells
– urease positive
• 10% KOH ==> fishy smell
• Aeromonas
– indole positive
– cellulitis, wound infections
• Chromobacter violaceum
– violet color
113 114

19
 Bordetella pertussis Haemophilus
• whopping cough • growth factors
• special media (Bordet-Gengou or – X: hemin, V: NAD
Regan and Lowe) – CHOC
• satellitism
• mercury drops colonies
– S. aureus produces V factor and release X
factor by hemolyzing blood
– grow in hemolytic zones
– horse or rabbit blood for hemolysis

115 116

Haemophilus Legionella pneumophila

• meningitis (1 month - 2 years) • legionnaires’ disease - severe
• epiglottitis (2-4 years) pneumonia
• conjunctivitis - pink eye by H. aegyptius • milder case: Pontiac fever
• chancroid by H. ducreyi • potable water, air condition vents,
– school of fish • sputum, pleural fluid, lung aspirate
• growth on BCYE

117 118

Mycoplasma & Ureaplasma Anaerobes

• smallest free living microorganisms • foul odor
• lack cell wall • close to mucous membrane
• fried egg colonies • animal or human bite
• M. pneumoniae: walking pneumonia • gas in specimen
• U. urealyticum: urethritis, stones • black discoloration (sulfur granules)
• M. hominis: NF GU tract, post partum • does not grow aerobically
fever • acceptable vs. unacceptable specimens
119 120

20
 Specimen Collection & Culture Examination of Primary Plates
• aspirate with syringe under reduced • pitting - Bacteroides ureolyticus
conditions – could be Eikenella
• jar technique • large colonies with double zone of
– catalyst: palladium crystals hemolysis - C. perfringes
– envelope generates H2 and CO2 when water
added
• bread crumb colonies - Fusobacterium
– methyelene blue indicator
nucleatum
• other methods • molar tooth - Actinomyces
– anaerobic bags, chamber • dark colonies on BBE - B. fragilis
121 122

Clostridium tetani Spirochetes

• terminal spores • Treponema pallidum
• subterminal spores – stained with silver
– darkfield
– sensitive to penicillin, tetracycline,
erythromycin
– detected serologically
– primary, secondary, latent, tertiary syphilis

123 124

Spirochetes Chlamydia
• Leptospira • obligate intracellular parasite
– animal pathogen passed to human via • GN cell wall, no peptidoglycan
contaminated water and animal urine
– Weil’s disease • dependent on host for ATP
• Borrelia • diagnosed by Geimsa stain, DFA, tissue
– B. recurrentis - relapsing fever from ticks or culture
lice • grow in yolk sac
– B. burgdorferi - lyme disease frm Ixodes
tick
125 126

21
 Chlamydia Rickettsiae
• C. psittaci • small GN coccobacilli
– parrot fever (poultry workers) • obligate intracellular parasite
• C. trachomatis • spread by insect vector
– eye infections (trachoma) • Q fever
– conjunctivitis
– STD (PID)

127 128

Zoonotic Infections Zoonotic Infections

• Brucella • Borrelia burgdorferi -
– Brucellosis (un dulant fever, Malta fever) Lyme Disease
– B. abortus (cow), B. suis (pigs), B. melentensis – ticks
(goats)
• Borrelia recurrentis -
• Francisella tularensis - rabbit fever (tick bite)
Relapsing Fever
• Yersinia pestis – plague
– Bubonic and pneumonic (black death)
• Pasturella multicidia - cat scratches
• Bartonella hensleae – cat scratch fever
• Streptobacillus moniliformis - rat bite fever
129 130

Fungus Like Bacteria Mycobacteria

• Nocardia • high lipid content in cell wall (does not
• partially acid fast properly gram stain)
– N. asteroides: casein, tyrosine, xanthine • acid fast:
negative – ZN: hot
– N. brasiliensis: casein & tyrosine positive, – kinyoun: cold stain
xanthine negative • auramine-rhodamine: fluorescent stain
• all stains based on presence of mycolic
acid
131 132

22
 Growth Requirement Specimen Collection
• Lowenstein-Jensen • first morning on 3 consecutive days
• Tween 80 • may refrigerate
• high CO2 – neutralize gastrics and urine if holding
overnight
• 3-6 weeks to grow
• 36C, some require 30C

133 134

Specimen Decontamination MTB

• N-acetyl-L-cysteine (NALC), NaOH • > 7 days for growth
• centrifuge for 20 minutes at 3000 rpm • rough & buff serpentine cording on
• sediment for smear and culture culture
• niacin and nitrate positive
• no growth on MAC
• tellurite negative

135 136

Antimicrobial Therapy Synergy Tests

• Effects of cell wall integrity

• Interruption of cell membrane Structure and
function
• Inhibition of protein synthesis
• Inhibition of essential metabolites
• Interference with nucleic acid metabolism

137 138

23
139 140

Virology
• RNA or DNA not both
• replicate in cells of another species
• serum shipped on ice
• Lab: ELISA, culture, EM, DNA probe,
PCR

141 142

143 144

24
 Growth Requirements
Microbiology Review • temperature
– psychrophiles - 15C
– mesophiles - 37C
– thermophiles - 50-60C
• pH
– bacteria: 6.5-7.5
– fungi: 5.0-6.0

Growth Requirements Growth Requirements

• osmotic pressure • other

– isotonic – microaerophiles
– halophiles • low oxygen

• oxygen – capnophiles
• CO2
– aerobes
– facultative anaerobes
– obligate anaerobes

Media Media

• agar • anaerobic media

– polysaccharide from marine algae – reducing agents (thio, cysteine)
– melts at 100C, solidify at 40C – heat broth tubes
• complex media – Gas pak envelopes
– peptone and extracts • sodium bicarbonate
• water ==> hydrogen + CO2
• palladium (catalyst)

1
 Media Media

• enriched • BAP
• selective – most fastidious bacteria
• differential – hemolysis
• CHOC
– Haemophilus, Neisseria
• MAC, EMC
– selective, differential
– GNR

Media Media

• phenylethyl Alcohol • CAMPY-BAP

– GPC – Campylobacter
– selective • THIO broth
• Columbia colistin nalidixic acid – general
– GPC – anaerobes
• MTM • Lowenstein-Jensen, Middlebrook
– N. gonorrhoeae, N. meningitidis – Mycobacterium

Anaerobic Media Incubation

• Bacteroides Bile Esculin Agar (BBE) • 5% CO2

• Kanamycin-vancomycin laked blood • 35-37C
• Cycloserine-cefoxitin fructose agar (C. • 50-70% humidity
difficile)

2
 Growth Curve Sterilization

• lag phase 120 • heat - denatures protein

• log phase
100
– autoclave
80
• 15lb, 121C, 15 minutes
• stationary phase 60

40 – dry heat
• death phase 20
• flame, incinerator
0

– pasteurization
Lag

Log

Stationary

Death
• 72C, 15 seconds

Sterilization Sterilization vs. Disinfection

• filtration • sterilization
– pore size – kills microorganisms
• refrigeration - slow growth – autoclave
• desiccation - remain viable, can not multiply • disinfection
• radiation – inactivate or inhibit microorganisms
• disinfection – bleach
– phenol: damage cytoplasmic membrane
– halogens: oxidizers
– alcohol: denature protein & dissolve lipids

Specimen Collection & Handling Criteria for Rejection

• time of collection • preservative

• quantity • QNS
• appropriate collection device • dry swab
• prior to antibiotic therapy • leaky containers
• within 2 hours of collection
• NEVER refrigerate CSF, anaerobes, GC

3
 Blood Culture Collection Culture Conditions

• prep skin with alcohol and iodine • most at 35-37C

• prior to fever spike • Campylobacter: 42C
• 2-3 cultures within 24 hours • Yersinia: 25-30C
• 1:5 - 1:10 blood to broth dilution • Campy - microaerophilic: 5-10% CO2
• 10 ml for adults, 1-5 for infants and children • 50-70% humidity
• anaerobes:
– THIO broth
– 10% H2, 5% CO2, 85% N2, palladium

Inoculation Stains

• chrome, platinum, plastic loop • gram stain

• calibrated (0.01ml or 0.001 ml) – crystal violet, iodine, alcohol, safranin
• number of colonies x 100 (or 1000) • kinyoun & ZN
– carbol fuchsin, acid alcohol, methelene blue

Gram Positive Cocci Staphylococcus

• Staphylococcus • grape-like clusters

• Streptococcus • S. aureus
– coagulase positive
– boils & carbuncles
– impetigo
– post surgical wounds
– toxigenic
• scalded skin syndrome
• TSS
• food poisoning

4
 S. aureus Staphylococcus

• most resistant to penicillin (β-lactamase) • coagulase negative

• most sensitive to penicillinase resistant – opportunistic infections
penicillins (methicillin, oxacillin) – S. epidermidis
• endocarditis, prosthetic device infections
• MRSA: vancomycin
– S. saprophyticus
• UTI in young women

Laboratory Diagnosis Catalase

• opaque regular colonies (some are β- • 3% H2O2 == catalase ==> H2O + O2

hemolytic) (bubbles)
• catalase positive
• growth in 7.5% NaCl
• ferment mannitol

Coagulase Streptococcus

• bound coagulase - clumping on slide • GPC in chains

• free coagulase - clot in tube • Lancfield grouping based on C
carbohydrate

5
 S. pyogenes S. pyogenes

• beta hemolysis • erythrogenic toxin

• streptolysin S – scarlet fever
– stable in oxygen • sensitive to penicillin
– non-antigenic • pharyngitis
• streptolysin O • impetigo
– oxygen labile • wounds (burns)
– antigenic
• rheumatic fever (autoimmune sequalae)
• bacitracin positive

S. agalactiae S. bovis

• narrow beta hemolysis zone • group D

• neonatal sepsis, meningitis, UTI, vaginal • bile esculin positive
infections • no growth in 6.5% NaCl
• CAMP, sodium hippurate positive

Enterococcus S. pneumonae

• growth in 40% bile and 6.5% NaCl • diplococci, lancet shaped

• alpha hemolysis (mucoid)
• lobar pneumonia
• meningitis
• bacteremia
• otitis media
• sensitive to optichin, bile soluble

6
 Viridans Gram Negative Cocci

• alpha or gamma • Neisseria

• endocarditis • Moraxella
• diplococci (kidney bean shape)
• oxidase positive

N. gonorrhoeae N. meningitidis

• CHOC and MTM • BAP, CHOC, MTM

• requires CO2 • CO2
• ferments glucose • ferments glucose and maltose
• gonorrhea • transmitted by respiratory droplets (close
– STD contact)
– gram stain sensitive for males • meningitis
– no refrigeration – children <3 years old
– penicillin sensitive - perform beta lactamase to
determine penicillin sensitivity

Moraxella catarrhalis Bacterial Meningitis

• respiratory infection • high neutrophils

• BAP, CHOC • low glucose
• hard colonies (move over) • high protein
• beta lactamase positive

7
 Gram Positive Rods Corynebacterium diphtheriae

• Corynebacterium diphtheriae • pleomorphic with clubbed ends

• Listeria monocytogenes • Chinese letters
• Erysipelothrix • catalase positive
• Bacillus • metachromatic granules
• tinsdale agar: black colonies due to tellurite
hydrolysis
• Elek test to determine toxin production
• cause diphtheria

Listeria monocytogenes Erysipelothrix

• small colonies with narrow zone of • non-motile

hemolysis • catalase negative
• catalase positive • H2S positive in TSI
• tumbling motility • occupational infection for fishermen,
– umbrella motility at RT not in 37C butchers, rose growers
• bile esculin positive
• neonatal meningitis, sepsis

Bacillus B. anthracis

• large ground glass colonies • bamboo shoots

• beta hemolysis • Medusa head colonies
• catalase positive • non-motile, non-hemolytic
• large GPR • anthrax
• spore formers • B. cereus
– food poisoning (fried rice) - enterotoxin

8
 Enterobacteriaceae E. coli

• peritrichous flagella when motile • indole and lactose positive

• ferment glucose • MR positive, VP negative, citrate negative
• reduce nitrate • UTI
• oxidase negative • intestinal infections
• antigens – enterotoxigenic
– H: flagella – enteroinvasive
– K: envelope – enterohemorrhagic (E. coli O157:H7)
– O: cell wall – enteropathogenic
– all posses endotoxin, some exotoxin

Shigella Klebsiellae

• lactose negative • opportunist, UTI, pneumonia

• non-motile • Klebsiella
• bacillary dysentery (<100 organism for – non-motile, capsule
disease) – urea positive, ornithine negative
• S. dysenteriae - group A • Enterobacter
• S. flexneri - group B – motile, ornithine positive
• S. boydii - group C • Serratia
• S. sonnei - group D – red pigments, DNAse positive

Salmonella Citrobacter

• >10,000 organisms for disease • lysine negative (similar to Salmonella)

• H2S & lysine positive, indole & urea
negative
• S. typhi: typhoid fever
– blood positive early, stool positive in 2-3 weeks

9
 Proteus Yersinia

• urea positive, deaminase positive • Y. enterocolitica

• P. mirabilis – optimal growth: RT
– indole negative – invasive & toxigenic
• P. vulgaris • Y. pestis
– indole positive – plague

Oxidase Test

• p-phenylenediamine dihydrochloride
• blue when positive

Lactose ONPG

• lactose == permease ==> intracellular • orthonitrophenylgalactopyranoside ==

lactose galactosidase ==> orthonitrophenol
• lactose == galactosidase ==> glucose + (yellow) + galactose
galactose

10
 Nitrate Test TSI

• napthylamine + sulfanilic acid • 0.1% glucose, 1% sucrose, 1% lactose

• positive: pink • yellow butt: glucose fermented
• zinc dust to confirm • yellow slant: lactose or sucrose
• red slant: neither
• black butt: H2S (acid)
• KIA: same as TSI w/o sucrose

citrate Decarboxylase Tests

• citrate as source of carbon • ability to decarboxylate amino acids

• green media • lysine ==> cadaverine
• blue when positive • ornithine ==> putrescine
• arginine ==> putrescine
• indicator dye: bromocresol purple

Indole Urease

• tryptophan == tryptophanase ==> indole + • urea == urease ==> ammonia

end products • phenol red turns pink
• indole + aldehyde ==> colored complex
• reagent: Kovac’s reagent

11
 MR-VP Phenyl Deaminase

• MR: organism ability to produce and • phenylalanine == deaminase ==>

maintain acid environment phenylpyruvic acid
– red color: acidic • phenyl pyruvic acid + ferric chloride ==>
• VP: green color
– acetooin +40% KOH + O2 ==> diacetyl
– diacetyl + peptone ==alpha naphthol ==> red
complex

Common GNR Media Nonfermentative Gram Negative

Rods

• glucose not fermented

• TSI: K/K or K/NC
• oxidase positive

12
 Pseudomonas aeruginosa Vibrio

• lactose negative • curved rods

• pyocyanin (green), fluorescein (yellow - • TCBS selective for Vibrio
IV), pyorubin (rust), pyoverdin (yellow) • BAP: hemolytic colonies
• mucoid strains in CF cases • lactose negative, nitrate positive, oxidase
• treated with 3rd generation cephalosporins positive
or aminoglycosides
• burns, pneumonia, swimmer’s ear, eye
infections, UTI

V. cholera Campylobacter

• gastroenteritis, rice water diarrhea • C. jejuni

• yellow on TCBS – seagull appearance
• V. parahaemolyticus – microaerophilic
– green on TCBS – CAMPY-BAP
– enteritis – found in raw poultry and contaminated water
– catalase, oxidase, hippurate positive
• V. vulnificus
– green on TCBS
– septicemia

Other Campylobacter Gardnerella vaginalis

• H. pylori • bacterial vaginosis

– gastric and duodenal ulcers • clue cells
– urease positive • 10% KOH ==> fishy smell
• Aeromonas
– indole positive
– cellulitis, wound infections
• Chromobacter violaceum
– violet color

13
 Bordetella pertussis Haemophilus

• whopping cough • growth factors

• special media (Bordet-Gengou or Regan – X: hemin, V: NAD
and Lowe) – CHOC
• mercury drops colonies • satellitism
– S. aureus produces V factor and release X
factor by hemolyzing blood
– grow in hemolytic zones
– horse or rabbit blood for hemolysis

Haemophilus Legionella pneumophila

• meningitis (1 month - 2 years) • legionnaires’ disease - severe pneumonia

• epiglottitis (2-4 years) • milder case: Pontiac fever
• conjunctivitis - pink eye by H. aegyptius • potable water, air condition vents,
• chancroid by H. ducreyi • sputum, pleural fluid, lung aspirate
– school of fish • growth on BCYE

Mycoplasma & Ureaplasma Zoonotic Infections

• smallest free living microorganisms • Brucella

• lack cell wall – brucellosis
• fried egg colonies – B. abortus (cow), B. suis (pigs), B. melentensis
(goats)
• M. pneumoniae: walking pneumonia
• Francisella tularensis - rabbit fever (tick
• U. urealyticum: urethritis, stones bite)
• M. hominis: NF GU tract, post partum fever • Yersinia pestis - plague
• Pasturella multicidia - cat scratches
• Streptobacillus moniliformis - rat bite fever

14
 Anaerobes Specimen Collection & Culture

• foul odor • aspirate with syringe under reduced

• close to mucous membrane conditions
• animal or human bite • jar technique
• gas in specimen – catalyst: palladium crystals
– envelope generates H2 and CO2 when water
• black discoloration (sulfur granules)
added
• does not grow aerobically – methyelene blue indicator
• other methods
– anaerobic bags, chamber

Examination of Primary Plates Spirochetes

• pitting - Bacteroides ureolyticus • Treponema pallidum

– could be Eikenella – stained with silver
• large colonies with double zone of – darkfield
hemolysis - C. perfringes – sensitive to penicillin, tetracycline,
erythromycin
• bread crumb colonies - Fusobacterium
nucleatum – detected serologically
– primary, secondary, latent, tertiary syphilis
• molar tooth - Actinomyces
• dark colonies on BBE - B. fragilis

Spirochetes Chlamydia

• Leptospira • obligate intracellular parasite

– animal pathogen passed to human via • GN cell wall, no peptidoglycan
contaminated water and animal urine
• dependent on host for ATP
– Weil’s disease
• diagnosed by Geimsa stain, DFA, tissue
• Borrelia culture
– B. recurrentis - relapsing fever from ticks or
lice • grow in yolk sac
– B. burgdorferi - lyme disease frm Ixodes tick

15
 Chlamydia Rickettsiae

• C. psittaci • small GN coccobacilli

– parrot fever (poultry workers) • obligate intracellular parasite
• C. trachomatis • spread by insect vector
– eye infections (trachoma) • Q fever
– conjunctivitis
– STD (PID)

Fungus Like Bacteria Mycobacteria

• Nocardia • high lipid content in cell wall (does not

• partially acid fast properly gram stain)
– N. asteroides: casein, tyrosine, xanthine • acid fast:
negative – ZN: hot
– N. brasiliensis: casein & tyrosine positive, – kinyoun: cold stain
xanthine negative
• auramine-rhodamine: fluorescent stain
• all stains based on presence of mycolic acid

Growth Requirement Specimen Collection

• Lowenstein-Jensen • first morning on 3 consecutive days

• Tween 80 • may refrigerate
• high CO2 – neutralize gastrics and urine if holding
overnight
• 3-6 weeks to grow
• 36C, some require 30C

16
 Specimen Decontamination MTB

• N-acetyl-L-cysteine (NALC), NaOH • > 7 days for growth

• centrifuge for 20 minutes at 3000 rpm • rough & buff serpentine cording on culture
• sediment for smear and culture • niacin and nitrate positive
• no growth on MAC
• tellurite negative

Virology

• RNA or DNA not both

• replicate in cells of another species
• serum shipped on ice
• Lab: ELISA, culture, EM, DNA probe, PCR

17
18
 Terms
Antimicrobial Agents
antibiotics
produced by microorganism that kill or inhibit
other organisms
antimicrobial agent
kills or inhibit organisms
natural: antibiotic
semisynthetic: chemically modified antibiotics
synthetic: man-made

Terms Terms

antibacterial agent spectrum of activity

affect bacteria range of organisms adversely affected by
chemotherapeutic agent antimicrobial agent
substance used to treat disease narrow vs. broad
antimicrobial or anticancer mechanism of action
bactericidal way agent harms organism
kills bacteria plasmid
bacteriostatic extrachromosomal DNA (replicate)
carry antimicrobial resistance gene - transferred
inhibit bacteria

Drug Interactions Cell Wall Characteristics

additive GP: thick peptidoglycan cell wall

effect is sum of activity of individual GN: thin peptidoglycan surrounded by
antimicrobial agents outer membrane
synergy many substances cannot diffuse across
effect is amplified membrane
antagonism actively transported into cell
one agent interferes with activity of other
indifferent
independent effects

1
Bacterial Targets Bacterial Resistance

cell wall intrinsic

protein & nucleic acid synthesis inherent
cell metabolism integral characteristic of species
all GN resistant to vancomycin
cell membrane functions
acquired resistance
change in susceptibility
gene mutation or transfer of resistance gene
S. aureus becoming resistant to penicillin

Resistance Mechanisms Resistance Mechanisms

enzyme inactivation low-affinity target sites

penicillinase drug binds poorly (or not at all) to target site
permeability barriers bypass mechanisms
agent unable to reach target organism circumvent metabolic block caused
drug efflux by antibiotic
energy-dependent system to pump antibiotic
out

Cross Resistance Resistance Expression

resistance to several antimicrobial agents constitutive

simultaneously with single change in organism constantly expressing resistance
bacterial cell mechanism
GN producing β-lactamase (inactivate
penicillin)
inducible
resistance only when exposed to agent
constitutive-inducible
constant expression at low levels

2
 β-Lactam Antimicrobial
Resistance Expression Agents

homogenous contain β-Lactam ring

entire bacterial population expressing penicillin
resistance cephems
heterogeneous carbapenems
some in population express resistance monobactams
MRSA

Mechanism of Action Resistance Mechanisms

inhibit cell wall synthesis β-Lactamase

bactericidal, stimulate autolytic enzymes cleaves ring
bind to penicillin-binding-proteins (PBP) penicillinase, cephalosporinase

PBP involved in formation of low-affinity PBP

peptidoglycan cross-links intrinsic or acquired
MRSA
permeability barriers

Penicillins Penicillins

from Penicillium spp extended spectrum penicillins

natural penicillin developed to treat GN infections
Strep, Treponema, GNR, anO2 ampicillin,
penicillinase-resistant penicillins carboxypenicillin, P. aeruginosa
semi-synthetic ureidopenicillin P. aeruginosa, anO2
Staph & Strep

3
Penicillins Other β-Lactams

β-Lactam and β-Lactamase inhibitor Cephems

combinations cephalosporins
penicllin + β-Lactamase inhibitor ⌧first generation - narrow spectrum
⌧inhibitor function is to bind to β-Lactamase • cephalothin
• many GP and GN
⌧binding prevent inactivation of penicillin
⌧second generation - extended spectrum
⌧ampicilin and sulbactam, amoxicillin and
• cefamandole
clavulanate
• many + Haemophilus & Neisseria
⌧most bacteria
⌧third or forth generation - broad spectrum
• cefepime
• most bacteria

Protein Synthesis
Cephems Inhibitors

carbapenems aminoglycosides
⌧imipenem gentamicin and amikacin
⌧most GP, Enterobacteriaceae, P. aeruginosa, anO2
bactericidal - inactivate enzymes
monobactams
synergistic with cell wall synthesis inhibitor
⌧aztreonam
⌧most GN
anO2: resistant
side effects: kidney and ear damage

Protein Synthesis Protein Synthesis

Inhibitors Inhibitors

aminocyclitols tetracyclines
related to aminoglycosides broad spectrum
spectinomycin diarrhea, discolored teeth
macrolides chloramphenicol
erythromycin broad spectrum
bacteriostatic BM suppression, aplastic anemia, gray baby
broad spectrum - few side effects syndrome (damage liver)
clindamycin
bacteriostatic or bactericidal

4
Glycopeptides Quinolones

vancomycin inhibits DNA synthesis (bind to DNA

inhibits cell wall synthesis gyrase)
GN: resistant narrow spectrum
acquired resistance nalidixic acid - UTI
vancomycin-resistant enterococci most Enterobacteriaceae
vancomycin intermediate S. aureus (VISA) broad spectrum
ciprofloxacin
Enterobacteriaceae + P. aeruginosa, other
GN, Staph

Sulfonamides Rifampin

inhibits folic acid synthesis (DNA inhibits RNC synthesis (binds to RNA
synthesis) polymerase)
humans do not synthesize folic acid! prophylactic treatment
some GP, some GN, actinomycetes Hib & N. meningitidis meningitis
trimethoprim: many GP, most GN red-orange BF
often combined for synergistic effect
(SXT)
enterococci: resistant

Metronidazole Nitrofurantoin

disrupts DNA damages DNA and bacterial enzymes

nitroreductase (anO2 enzyme) covert it to many GP and some GN
toxic compounds
treat UTI
anO2 and some protozoa

5
Bacitracin Polymyxins

acts on cytoplasmic membrane disrupt cytoplasmic membrane

topical colistin
some GP topical
some GN (p.aeruginosa)
combined with bacitracin

Mycobacterial Antimicrobial
Chemotherapy Susceptibility Tests (AST)

isoniazid (INH) National Committee for Clinical Laboratory

inhibits synthesis of mycolic acid (cell wall Standards (NCCLS)
component) develops standards
form free toxic radicals tests categories
other drugs (streptomycin, ethambutol, susceptible (S)
pyrazinamide, rifampin, kanamycin,…) resistant (R)
Therapeutic Considerations intermediate (I)
multiple drugs used
mutations are common (drug resistance)

Test Performance Selection of Antimicrobial

Indications Agents to Test

performed only when standardized NCCLS guidelines

method is available vary with organism, test method, body
factors site
identity and quantity of organism
body site
presence of other organisms
patient factors

6
Mueller-Hinton (MH) Agar
or Broth Other Media

standard medium for testing most Haemophilus test medium

bacteria supplemented GC agar
composition of MH BHI medium (enterococci)
cation concentration (Ca & Mg) anaerobic media
⌧affects tetracycline & aminoglycosides
BHI, Wilkens-Chlgren, Schaedler, Brucella
pH 7.2-7.4 BAP
thymidine
⌧interferes with TMP, sulfa, SXT
blood
agar depth, inoculm

McFarland Standards Inoculum

turbidity standards to prepare require pure culture

suspensions log-phase growth method
numbered 0.5, 1, 2, 3, … broth tube inoculated and incubated until 0.5
0.5 = 1.5 x 108 CFU/ml direct-colony suspension method
broth inoculated to produce 0.5
stationary-phase method
broth inoculated and incubated until >4.0
commercial systems
no incubation

Incubation Conditions Broth Dilution Tests

35C semi-quantitative results

atmosphere and time depend on test special antibiotic powders
organism test methods
macrodilution
⌧test panel
⌧tubes with 1 ml broth
microdilution
⌧microtiter trays (0.05 - 0.1 ml)

7
Macrodilution Microdilution

Inoculation Test Examination

standard suspension prepared purity plate, growth well, sterility well

wells or tubes inoculated within 15 checked first
minutes minimum inhibitory concentration (MIC)
purity check - lowest drug concentration with no visible
inoculum subcultured onto BAP growth

growth control - positive growth (no minimum bactericidal concentration (MBC)

antibiotics) lowest concentration results in >99.9%
killing
sterility check - no inoculation - no growth
interpretation: S, R, I based on amount of
microtiter trays covered to prevent drying drug safely achieved in patient (mcg/ml)

Test Interpretation MBC

performed in limited situations

log-phase growth method
aliquot removed from growth tube,
inoculated onto BAP, incubated
# organisms per ml calculated on each
tube with no visible growth
colony count compared to inoculum
colony count
MBC = 99.9%

8
Schlichter Test Agar Dilution Tests

serum bactericidal assay semi-quantitative

tests patient serum (containing antimicrobial agents incorporated into
antibiotics) against infecting organism agar medium
peak and trough specimens series of agar plates with different
trough: jest before drug administered concentrations
peak: 30-90 minutes after standardized suspension inoculated
incubated and examined for growth
MIC determined

Disk Diffusion Tests (Kirby

Breakpoint Tests Bauer)

qualitative results (S, R, I) qualitative results

paper disks impregnated with
antimicrobial agents placed on previously
inoculated plates
incubated and zone of inhibition
measured
agent diffuses through agar ==> zone of
inhibition
concentration decreases as distance
i

Disk Diffusion Tests Test Procedure

factors inoculation
ability to diffuse in agar standardized suspension (log-phase or direct
susceptibility of organism colony)
agar depth cotton swab streaked over plate
amount of drug in disk rotate 60 degrees, rotate again, rubbed
against agar edges
plates
diameter of 150 or 100 mm
disk application
depth: 4 mm within 15 minutes of inoculation
⌧false resistant if deeper plates used 12 disks on large plates, 5 on small plates
(avoid overlap of zones)

9
Plate Examination Disk Diffusion

inoculation check
growth and circular zones
reading
against black surface
zone measurement
ruler or template

Correlation of Disk
Test Interpretation Diffusion and MIC

S, I, R based on size of zone

susceptible isolate: large zone and low
MIC
resistant: small zone and high MIC

E-Test E-Test

agar plates inoculated in same manner as

disk diffusion
plastic strip containing gradient of
antimicrobial agent placed on surface
incubated and examined for elliptical zone

10
Automated Systems Vitek

Vitek
WalkAway (Dade)

Inducible vs. Constitutive

β-Lactamase Tests β-Lactamase

clinical applications constant production by: H. influenzae, N.

detect resistance to antimicrobial agents gonorroheae, M. catarrhalis, enterococci,
H. influenzae: ampicillin and amoxicillin Bacteroides
N. gonorrhoeae: ampicillin, amoxicillin, inducible: Staphylococci
penicillin
M. catarrhalis: ampicillin, amoxicillin,
penicillin
Staph: ampicillin, amoxicillin, penicillin,
others
enterococci: penicillin and ampicillin

Test Methods β-Lactamase

nitrocefin
chromogenic cephalosporin
intact ring == lactamase ==> cleaved ring
(red)
most sensitive
acidimetric
phenol red pH indicator
penicillin == lactamase ==> penicilloic acid
(yellow)

11
Test Methods Clinical Considerations

iodometric Salmonella & Shigella should not be

starch-iodine indicator reported as susceptible to
penicillin == lactamase ==> penicilloic acid aminoglycosides or first- and second-
blue (reduce iodine - ca not complex with generation cephalosporins
starch) MRSA: resistant to methicillin, oxacillin,
all other β-lactams
should not be reported susceptible to any β-
lactam regardless of lab results

Clinical Considerations Quality Control

enterococci organisms
high-level aminoglycoside resistance (HLAR) American Type Culture Collection (ATCC)
⌧resistance to gentamicin, tobramycin, amikacin, different strains used for specific tests
kanamycin,
properly stored and maintained
vancomycin-resistant enterococci (VRE)
stock organisms: frozen or lyophilized
Do not report as susceptible to TMP, sulfa,
working organisms: replaced monthly
cephalosporins, clindamycin, aminoglycosides
(weekly subculture)
N. gonorrhoeae
AST not routinely performed

QC

daily QC
each day AST performed
weekly
labs with documented AST proficiency
performed for 30 consecutive days
no more than three values for each organism
are out of control
antibiograms
pattern produced periodically (annual)

12
 Introduction
Body Fluids
asterile
aamniotic fluid
`surrounds fetus
apericardial fluid
other than CSF, blood, urine
`heart space fluid
aperitoneal fluid
`abdominal cavity
apleural fluid
`cover lungs lining chest cavity

More BF Amnionitis

adialysis fluid (dialysate) `women with prolonged fetal membrane

`used in chronic ambulatory peritoneal dialysis rupture
(CAPD) `happen also when membrane is still intact
`treatment for patients with end-stage renal `group B streptococci, anaerobes, E. coli,
failure Gardnerella vaginalis, Ureaplasma
`induced into peritoneal cavity urealyticum
`metabolic waste removed with dialysate
asynovial fluid
`joint fluid

Empyema Pericarditis

aassociated with pneumonia ausually caused by virus

abacteria recovered from pleural fluids aS. aureus, S. pneumoniae, group A
asame organisms causing pneumonia streptococci, Enterobacteriaceae

1
 CAPD-Associated
Peritonitis Peritonitis

aprimary peritonitis astaphylococci

`spontaneous bacterial peritonitis astreptococci
`no known source of infection aEnterobacteriaceae
`caused by Enterobacteriaceae, enterococci,
aNFGNR
pneumococci
asecondary peritonitis
`known source of infection - rupture appendix
`caused by Enterobacteriaceae, enterococci,
Bacteroides, P. aeruginosa

Septic Arthritis Specimen Collection

ainfectious arthritis acollected by percutaneous aspiration

acaused by staphylococci, streptococci, aas much fluid as possible is collected
enterococci, N. gonorrhoeae, H. aamniocentesis, arthrocentesis,
influenzae, Enterobacteriaceae pericardiocentesis, paracentesis,
aS. aureus is most common pathogen thoracentesis

Transport Processing

asterile screw-cap tubes aphysical appearance noted

aanaerobe transport tubes acencentrated
ablood collection tubes asome lab filter non-viscous fluids and
acapped syringes culture membrane filter
atransported at RT aclotted specimens should be homogenized
ain lab within 15 minutes of collection to release organisms entrapped in clots

2
Microscopic Examination Cultures

agram-stained smears examined for host aBAP, CHOC

cells and bacteria aTHIO or BHI
asmears prepared from specimen sediment ablood culture bottles
or by centrifugation aanaBAP recommended for amniotic,
pleural, peritoneal fluids
aincubate at 35-37C in CO2
aspecial conditions if MTB suspected

Reporting Results

apositive cultures reported ASAP

3
 Terms
Blood Cultures
bacteremia
bacteria in bloodstream
septicemia
bacteremia with clinical signs and symptoms
⌧fever
⌧chills
⌧hypothermia
⌧hyperventilation
⌧septic shock (mortality rate >50%)

Terms Terms

primary bacteremia pseudobacteremia

bacteremia with no other known infected site false bacteremia
secondary bacteremia contaminated materials are source of
bacteremia associated with an infected body organisms in blood culture media
site
occult bacteremia
bacteremia with no known cause
with or without symptoms
mainly in children

Bacteremia Sources Bacteremia Patterns

intravascular transient
associated with vascular system minutes to hours in bloodstream
infected heart valves, catheters, veins body site with organisms traumatized
extravascular ⌧mucous membrane or skin

outside vascular system mild (teeth cleaning), severe (surgery)

lymphatic vessels carry organisms to early stages of some diseases
bloodstream ⌧meningitis, osteomyelitis, infectious arthritis

GI, RT, abscesses

1
Bacteremia Patterns Causative Agents

intermittent Staphylococci
organisms periodically released into S. aureus and coagulase negative
bloodstream Streptococci
caused by un-drained abscess group A, B, pneumococci, viridans,
continuous enterococci
organism present constantly in bloodstream GPR
individuals with infected intravascular sites Listeria, Corynebacterium, Bacillus
Neisseria
N. gonorrhoeae, N. meningitidis

Causative Agents Specimen Collection

Haemophilus Anticoagulants
H. influenzae difficult to recover organisms in clotted
Enterics specimens
E. coli, Klebsiella, Salmonella Sodium polyanethol Sulfonate (SPS)
⌧prevents clotting
Anaerobes ⌧inhibits phagocytosis
Bacteroides, Clostridium ⌧inactivates complement
NFGNR ⌧neutralizes some antimicrobial agents
Pseudomonas ⌧inhibits some organisms (N. gonorrhoeae,
N. meningitidis)
other GNR

Anticoagulants Collection Sites

SPS venipuncture
0.025% concentration in blood culture media indwelling intravascular catheters not
minimize its antibacterial effects recommended - contamination
SPS blood collection tubes available but not used if it is only way to collect blood
recommended patient evaluated for catheter-related
inappropriate anticoagulants bacteremia
citrate, heparin, oxalate, EDTA
toxic to some organisms

2
Collection Methods Collection Procedure

needle & syringe specimen container preparation

in syringe then injected in blood culture site preparation
bottles
specimen collection
no need to change needle
site care
transfer set
two connected needles
⌧one for venipuncture
⌧the other inserted into vacuum blood tube

Specimen Container
Preparation Site Preparation

tops of culture bottles and tubes to avoid contamination with NF

disinfected with 70% alcohol or iodine some NF can cause significant diseases
clean skin with alcohol to remove debris
and oil
skin swabbed with iodine from inside out
in concentric circles
allow iodine to dry to ensure proper
disinfection

Specimen Collection Site Care

venipuncture iodine can cause skin irritation - should be

blood collected in tube or syringe removed with alcohol
blood from syringe must immediately
inoculated into blood culture bottles
invert bottles or tubes gently
mix blood with broth media or anticoagulant

3
NOTE Blood Volume

blood collected from one venipuncture is more likely to be positive when large volume
considered ONE blood culture of blood collected
even if blood is divided into several tubes recommended amount varies with patient
age
children have high level of bacteremia
adults: <10 organisms/ml, children: 100-
1000 organisms/ml
infants & children: 1-5 ml with each
venipuncture
adults: minimum of 10 ml

Timing and Number of

Blood-to-Broth Ratio Cultures

1:5 to 1:10 2-3 blood cultures should be collected

prevention of clot formation during bacteremic episode
dilution of inhibitory factors before antimicrobial therapy
traditional recommendation:
acutely ill: 2-3 from separate sites within 10
minutes
endocarditis: 3 cultures over 1-2 hours, 3
more 24 hours later if first 3 were negative
fever of unknown origin: 2-3 cultures 1 hour
apart, 2-3 more 24-36 hours later

Newer Recommendation Transport

three blood cultures collected collection can be STAT

simultaneously high priority transportation
three venipunctures one right after the other RT, at 37C incubator if delayed
-- OUCH
not the tubes
do not refrigerate

4
Culture Media - Broth Culture Media - Agar

BHI, Brucella, Columbia, thioglycollate, some systems use agar media

trypticase soy, special formulations biphasic culture bottles (agar and broth)
key components
SPS
nutrients
headspace contains CO2
antimicrobial neutralizers
hypersomatic contains sucrose
two culture bottles: aerobic and anaerobic

Incubation Conditions Subcultures

temperature: 35-37C bottles subcultured into enriched agar

time: varies, 5-7 days medium
longer incubation required for some plates incubated at 35-37C for 18-48
venting: aerobic bottles in some systems hours
vented before incubation blind subculture - negative bottles
insert needle into septum not required for instrumentation
release vacuum, oxygen in
agitation: shaking enhance growth by
increasing oxygen (automated)

Manual Culture Methods Manual Culture Methods

conventional broth Biphasic Methods

bottles examined daily for growth castaneda bottle: tipped to cover agar
⌧turbidity, gas, discoloration, (inoculated - colonies after incubation)
subcultre newer versions:
smears (gram stain) ⌧blood added
⌧paddle device attached to aerobic bottle
• contains e.g. CHOC & MAC
⌧paddle periodically bathed
⌧paddle and broth examined daily for growth

5
Manual Culture Methods Instrumentation

lysis-centrifugation method semiautomated systems

blood collected in special isolator tube BACTEC 460
blood anticoagulated and lysed Nonradiometric BACTEC
tube centrifuged Continuous-Monitoring Systems
sediment inoculated onto agar plates BacT/Alert
agar plate incubated and examined daily BACTEC 9000
advantage: early appearance of isolated ESP
colonies
disadvantage: contamination

BACTEC 460 Nonradiometric BACTEC

radioactive carbon measures CO2 production with infrared

microorganisms metabolize carbon and spectrophotometer
produce radioactive CO2
amount of radioactive CO2 in headspace
determined

Continuous Monitoring
Systems BacT/Alert

special bottles colorimetric

incubator monitors Co2 production
agitator bottles with gas-permeable sensors
detection system sensor changes from green to yellow as
monitors bottles for sign of growth (pressure gas is generated
change)
computer

6
BACTEC 9000 ESP

fluorescent sensor in base of bottles manometric system

fluorescence increases as CO2 is bottles attached to pressure transducer
generated measuring pressure inside bottle

Positive Blood Cultures Gram-Stained Smears

critical values first step of work-up

detected by determine morphology and gram stain
appearance of broth reaction
colonies on subculture plates
instrumentation
identification and antimicrobial
susceptibility to follow

Subculture Identification

positive cultures subcultured onto agar to gram stained smears determines which
isolate colonies tests are performed
media selection depends on sitting and use colonies from agar media (mixed
gram stain reaction growth)
aerobic: BAP, CHOC, or both in CO2 direct tests form broth
MAC, EMB can be added
anaerobic: anaBAP
bottle positive, smear negative

7
Results Reporting Contaminants

notify physician immediately determine clinical significance of isolate!

number of positive cultures Factors to consider:
gram stained smear results identity of isolate
⌧GPC in chains number of positive cultures
other tests performed collection method
document report (name, date and time) patient history
preliminary report after 48 hours, final to Bacillus, Corynebacterium,
follow Propionibacterium acnes, coagulase
“No growth after 7 days of incubation” negative staph

Intravascular Catheter
Special Cultures Cultures

prolonged incubation semiquantitative culture method

Brucella (30 days) catheter aseptically removed
special culture conditions tip cut off and sent to lab in dry sterile
satelliting streptococci (enriched blood container
culture bottles) tip rolled over surface of BAP
suspected when GPC on smear with no BAP incubated at 35C in CO2
growth on subcultured plates > 15 colonies: significant growth

Intravascular Catheter
Cultures

other methods
broth cultures
skin culture - at catheter site
quantitative blood cultures
⌧lysis-centrifugation method
⌧CFU/ml determined
direct microscopy
⌧catheters Gram stained and examined
microscopically

8
 Introduction
Central Nervous System
CNS
brain
spinal cord
meninges
strile

Terms Terms

cerebrospinal fluid (CSF) aseptic meningitis

bathes brain and spinal cord nonpyogenic
test: culture, cell count, glucose & protein levels usually caused by virus and some bacteria
meningitis (leptospires)
inflammation of meninges encephalitis
purulent meningitis inflammation of brain - caused by virus
pyogenic meningitis meningoencephalitis
pus in meninges inflammation of brain and meninges
bacteria, fungi, protozoa

Routes of Infection Routes of Infection

hematogenous trauma
most common route breach CNS protective barrier
bloodstream carrying organisms from ⌧skull fracture
colonized or infected site to meninges surgery
⌧N. meningitidis colonize nasopharynx, enter blood microbial contamination
==> meningitis (or harmless)
shunts
contiguous spread
placed to remove fluid
organism spreading from infected adjacent
site (sinusitis) portal of entry

1
Acute bacterial Meningitis Causative Agents

symptoms neonates (through birth canal)

flu-like symptoms group B streptococci
headache E. coli
fever other GNR (Klebsiella)
nausea Listeria monocytogenes
vomiting young children (6 months - 5 years)
nuchal rigidity (stiff neck) Hib
mental status changes N. meningitidis
S. pneumoniae

Causative Agents Causative Agents

older children and adolescence neurosurgery and head trauma

meningococci and pneumococci pneumococci (most common)
Elderly S. aureus
pneumoncocci, GNR, Listeria monocytogenes Enterobacteriaceae
immunocompromised Pseudomonas
Listeria monocytogenes and encapsulated shunt: coagulase negative staph &
bacteria Corynebacterium

Specimen Collection Microscopic Examination

lumbar puncture gram stain smears sensitivity 75-90%

transported at RT within 15 minutes of thick smears
collection
STAT processing
stored at incubator or at RT
do not refrigerate - meningococci,
pneumococci, H. influenza
if >1 ml ==> centrifuge
sediment for smear and culture

2
Cultures Antigen Detection Tests

media available for group B streptococci, Hib,

BAP, CHOC meningococci, pneumococci
MAC or EMB if GNR seen in gram stained should supplement, not replace, smears
smears and cultures
THIO or BHI can be added
anaBAP if requested
⌧anaerobes rarely isolated
incubate at 35-37C in CO2

Chronic Meningitis Brain Abscesses

may or may not be immunocompromised caused by NF organisms

gradual symptoms anaerobic bacteria, staphylococci, viridans,
fever, headache, confusion other streptococci

causative agents aspirate and biopsy materials

Nocardia, Actinomyces, MTB, other transported in anaerobic conditions
mycobacteria examined microscopically
Cryptococcus neoformans in AIDS individuals cultured aerobically and anaerobically

Reporting Results

critical values
immediate notification
verbal communication followed by written
report

3
 Introduction
Genital Tract
male
urethra, prostate, epididymis
female
ovaries, fallopian tube, uterus, vulva, vagina,
cervix

Indigenous Microtobia Terms

NF of female varies with age non-gonococcal urethritis

affected with estrogen levels urethritis caused by C. trachomatis or U.
Lactobacillus (predominant in healthy vaginas) urealyticum
Staphyloccocci, Streptococci (group B) sexually transmitted disease - STD
Enterobacteriaceae venereal disease
Corynebacterium gonorrhea
Gardenerella vaginalis caused by N. gonorrhoeae
Mycoplasma & Ureaplasma
anaerobic organisms

Diseases Urethritis

some from endogenous microorganisms men or women

most traced to sexual activity N. gonorrhoeae or C. trachomatis (most
common)
GC, C. trachomatis, Enterobacteriaceae,
S. saprophyticus ==> acute urethral
syndrome in women
U. urealyticum, Haemophilus ==>
urethritis in men

1
Cervicitis Vulvovaginitis

N. gonorrhoeae or C. trachomatis Candida albicans*

Trichomonas vaginalis*
Enterobacteriaceae
N. gonorrhoeae
C. trachomatis
S. aureus
Actinomyces

Pelvic Inflammatory
Bacterial Vaginosis Disease

due to reduced numbers of lactobacilli include salpingitis, endometritis, tubo-

Gardnerella vaginalis ovarian abscesses, peritonitis
N. gonorrhoeae
C. trachomatis
NF (Bacteroides, streptococci,
Enterobacteriaceae
Actinomyces associated with use of
intrauterine devices

Genital Ulcers Prostatitis

Haemophilus ducreyi: chancroid Enterobacteriaceae

T. pallidum: syphilis N. gonorrhoeae
Calymmatobacterium granulomatis: C. trachomatis
granuloma inguinale P. aeruginosa
C. trachomatis (L1, L2, L3): enterococci
lymphogranuloma venereum S. aureus

2
Bartholinitis Postpartum Endometritis

Bartholin gland by vagina after childbirth

acute bartholinitis: N. gonorrhoeae, C. beta hemolytic streptococci
trachomatis Gardnerella vaginalis
gland ducts obstructed ==> abscesses by enterococci
aerobic and anaerobic NF Enterobacteriaceae
Mycoplasma hominis
anaerobic bacteria

Group B Streptococcal
Infections Epididymitis

NF N. gonorrhoeae
can cause meningitis in newborn C. trachomatis
screen pregnant women for group B Enterobacteriaceae
streptococci Pseudomonas aeruginosa

Orchitis Specimen Collection

Enterobacteriaceae (E. coli) general consideration

Pseudomonas aeruginosa calcium alginate & Dacron swabs
plastic or wire shafts
avoid cotton swabs or wooden shafts
routine culture specimens transported and
stored at RT

3
Specific Recommendations Specific Recommendations

urethra vagina
not urinated 1-2 hours before collection speculum inserted
catheter inserted in urethra sterile swab used to collect material deep
discharge collected by swab within vagina
cervix endometrium
use speculum special telescoping catheter
remove mucous and discharge material with
swab or cotton ball
sterile swab or cytobrush inserted, rotated,
carefully removed (avoid NF contamination)

Specific Recommendations Microscopic Examination

prostate gram stain

prostatic massage material diagnostic for BV, GC in men,
alternative: urine culture suggestive for other organisms
aspirate material ⌧school of fish ==> H. ducreyi

Bartholin gland, fallopian tubes, epididymis

buboes
biopsy & tissue

Cultures Media

can not test all organisms in genital BAP

specimen CHOC
physician request MTM
screening for GC or group B streptococci MAC or EMB
anaerobic cultures
phenylethyl alcohol (PEA) or colistin-
C. trachomatis, M. hominis, U. urealyticum
nalidixic acid (CNA)
require special culture conditions
anaerobic media

4
Incubation Conditions Workup

vary with culture medium and organism depends on

sought type of specimen
aerobic media: 35C in ambient air specimen quality
GC cultures in CO2 type of culture
Haemophilus in 35C in high humidity, CO2 GC, group B streptococci, Listeria
monocytogenes reported whenever
anaerobic organisms: anaerobically at 35C
present
S. aureus (other NF) worked up only
when predominant

5
 Introduction
Gastrointestinal Tract
esophagus
stomach
small intestines
duodenum, jejunum, ileum
large intestines
cecum, colon, rectum
anus

Terms Terms

gastritis diarrhea
inflammation of stomach abnormal increase in number of bowl
gastroenteritis movement
inflammation of stomach and intestines loose to liquid stool

enterocolitis dysentery
inflammation of small and large intestines diarrhea with cramping abdominal pain
proctitis
inflammation of rectal mucosa
N. gonorrhoeae, C. trachomatis, T. pallidum

Normal Flora Diseases

anaerobic bacilli Salmonella: gastroenteritis

Bacteroides, Clostridium Shigella: bacillary dysentery
GN enteric bacilli Campylobacter jejuni and C. coli: diarrhea
enterococci Y. enterocolitica: enterocolitis (can
S. aureus resemble acute appendicitis)
Yeast (Candida) Edwardsiella tarda: diarrhea (uncommon)
Vibrio cholrae: cholera (V.
parahaemolyticus: milder case)

1
Diseases Diseases

Aeromonas: unknown role but commonly S. aureus: food poisoning (enterotoxin)

isolated overgrowth due to antimicrobial therapy
Plesiomonas shigelloides: diarrhea Ps. Aeruginosa: diarrhea by overgrowing
H. pylori: chronic gastritis, peptic and Bacillus cereus: food poisoning (diarrheal
duodenal ulcers and emetic toxin)
C. difficile: antibiotic-associated diarrhea, Mycobacterium avium: GI diseases in
pseudomembranous colitis AIDS patients
C. perfringens: enterotoxin (food
poisoning)

Diseases Routes of Transmission

E. coli fecal-oral route

enterohemorrhagic organisms (Salmonella) or their toxins (S.
⌧E. coli O157:H7 aureus) ingested in contaminated water or
⌧cause of hemorrhagic colitis and hemolytic uremic food
syndrome (HUS)
enteroinvasive direct person-to-person contact (Shigella)
enterotoxigenic animal contact (Y. enterocolitica)
enteropathogenic need to survive pH of stomach
enteroadherent

Symptoms Inflammatory Diarrhea

nausea organisms invade intestinal mucosa

vomiting fever with loose, small-volume stool
abdominal discomfort fecal specimen contain PMN, blood,
diarrhea mucous
inflammatory Salmonella, Shigella, Y. enterocolitica, C,
non-inflammatory jejuni, enteroinvasive E. coli

2
Stool WBC Non-Inflammatory Diarrhea

bacterial toxin
afebrile with watery, large-volume stool
NO PMN, blood, mucous
V. cholerae and enterotoxigenic E. coli

Specimen Collection Specimen Collection

2-3 samples rectal swab specimens

fecal specimens preferred for isolation of Shigella
clean, non-sterile,
wide-mouth container young children
not contaminated with placed in transport medium
urine ⌧Cary-Blair or GN broth
process within 1-2
hours of collection other specimens
⌧transport medium and
refrigerate duodenal, colostomy, ileostomy material
⌧Cary-Blair medium diapers
food (public health labs)

Visual Examination Overgrowth of S. aureus

macroscopic examination
blood, mucous
consistency (watery, formed, loose)
color
microscopic examination
PMN (gram stain or iodine)
certain bacteria (darting motility of
Campylobacter, seagull wings)

3
C. difficle in stool Cultures

lots of NF
not practical to examine all isolates
look for Salmonella, Shigella,
Campylobacter, E. coli O157:H7
patient history and symptoms
rely on physician to request special
culture

Media Inoculation Routine Cultures

swab used to inoculate culture media panel grows Salmonella, Shigella,

agar plates inoculated by rotating swab Campylobacter, Aeromonas, Plesiomonas,
over small area S. auerus, P. aeruginosa, yeast, Y.
plates streaked for isolation enterocolitica, Vibrio
enrichment broth inoculated with swab
containing large amount of specimen

Media MAC

BAP
detects S. aureus and yeast overgrowth
MAC or EMB
differential and selective
LNF (Salmonella, Shigella, Y. enterocolitica,
E. tarda, Plesiomonas, Aeromonas, Vibrio)

4
Media Salmonella on HE

Xylose-lysine-desoxycholate (XLD) or
Hektoen enteric (HE)
differential and selective
Salmonella and Shigella
Campylobacter selective media
Campy-BAP
isolate C. jejuni and C. coli

Campy-BAP Media

enrichment broth
GN: Salmonella, Shigella
selenite F: Salmonella and some Shigella

Incubation Conditions Special Media

BAP, MAC, EMB, XLD, HE cefsulodin-irgasan-novobiocin (CIN) agar

ambient air at 35C Y. enterocolitica
Campy-BAP incubated aerobically at RT
microaerobic conditions at 42C Salmonella Shigella (SS) agar
enrichment media Shigella can be inhibited
few hours and then subcultured onto agar Sorbitol MAC
media E. coli O157:H7

5
E. coli vs. E. coli O157:H7
on sorbitol MAC Special Media

thiosulfate-citrate-bile salts-sucrose
(TCBS) agar
Vibrio

Workup Reporting Results

plates examined for suspicious organisms identification and susceptibility panel

tested to determine if enteric pathogen public health officials notified for
can use few biochemical tests for Salmonella and Shigella
screening before performing complete report overgrowth of S. aureus, yeast, Ps.
identification panel aeruginosa
negative results reported as: No
Salmonella, Shigella, or Campylobacter
isolated

6
 Introduction
Respiratory Tract
aURT
`nose, mouth, throat, epiglottis, larynx
`contains NF
`middle ear and paranasal sinuses connected
aLRT
`trachea, bronchi, bronchioles, lung alveoli
`below larynx is normally sterile

Normal Flora Pharyngitis

astaphylococci aNeisseria (N. asore throat (most common URT infection)

`S. epidermidis, S. meningitidis)
aureus
aagents
aGNR
astreptococci `group A streptococci & viruses
`enterics & NFGNR
`viridans and `less common: Corynebacterium diphtheriae,
aanaerobes
pneumococci N. gonorrhoeae, groups B, C, F streptococci,
aYeast
aenterococci `not S. pneumoniae, S. aureus, H. influenzae,
`Candida species
adiphtheroids N. meningitidis (can be NF)
a Haemophilus

Specimen Collection Microscopic Examination

aposterior pharynx and tonsils swabbed anot appropriate

aplaced in transport media `not diagnostic
amost labs seek only group A strep alots of NF
`antigen testing performed first, enough if
positive

1
Cultures Throat Culture

amainly for group A strep

aother organisms may be sought in certain
situations
`groups B, C, F, G
`N. gonorrhoeae
`Arcanobacterium haemolyticum
`C. diphtheriae

Epiglottitis Otitis Media

a2-6 years of age amiddle ear infection

aH. influenzae type b (almost exclusive) achildren <10
alife threatening disease - airways can atypically occurs after viral URTI
become obstructed aagents
abecoming rare due to Hib vaccine `S. pneumoniae, H. influenzae
adiagnosed clinically `others: group A strep, S. aureus, M.
catarrhalis, anaerobes, GNR
aepiglottal specimen collection is hazardous
ablood cultures performed (bacteremia)

Specimen Collection Otitis Media

atympanocentesis fluid (TF) collected

aaseptically puncturing tympanic
membrane and aspirating fluid
aplaced in anaerobic transport media
aswab can be used to collect fluid released
when eardrum rupture

2
Microscopic Examination Cultures

agram stain aaerobically and anaerobically

aBAP, CHOC, MAC, BHI, anaBAP
aidentification and susceptibility performed
aruptured ear drum fluid should be
cultured only aerobically

Sinusitis Specimen Collection

apreceded by a viral URTI adiagnosed clinically or radiographically

aacute sinusitis acollected when patient not responding to
`S. pneumoniae, H. influenzae therapy
`others: M. catarrhalis, group A strep, asinus aspirate or sinus opening (sinus
anaerobes, S. aureus, GNR ostium)
achronic sinusitis acollected in anaerobic transport medium
`anaerobes, S. aureus in adults
`S. pneumoniae, S. aureus, viridans
streptococci in children

Specimen Collection Microscopic Examination

agram stain

3
Cultures Sinusitis

aaerobically and anaerobically

aBAP, CHOC, MAC, BHI, anaBAP
aidentification and susceptibility performed

Lower Respiratory Tract

Infections Routes of Infection

abronchitis ainhalation of infectious aerosols

`mainly viral, sometimes bacterial ahematogenously (blood stream)
apneumonia aaspiration of oral secretions or gastric
`primary bacterial infections or secondary to contents
viral infection
aempyema
`pus in thorax
`associated with pneumonia

Causative Agents Causative Agents

aS. pneumoniae aEnterobacteriaceae

`pneumococcal pneumonia in adults `K. pneumoniae & S. marcescens
`most common aNFGNR
aS. aureus `P. aeruginosa & Burkholderia cepacia
`community acquired or nosocomial `CF and nosocomial
aH. influenzae aM. catarrhalis
`pneumonia in children and adults aanaerobic bacteria
aMycobacteria (MTB)

4
Causative Agents Specimen Collection

aLegionella alower RT specimens passing through URT

`immunocompromised individuals are usually contaminated
`community acquired or nosocomial alots of NF!!
aM. pneumoniae
`older children and young adults (<30)
`walking pneumonia
aChlamydia
`C. trachomatis in neonates
`C. pneumoniae in young adults

To Minimize Contamination To Minimize Contamination

aexpectorated sputum atracheostomy & endotracheal aspirates

`gargle and rinse mouth `suctioning secretions into tubes
`deep cough abronchoscopy specimens
`expectorate sputum into sterile container `fiberoptic scope through nose or mouth
ainduced sputum `bronchial lavage or washings collected
`under supervision of RT `bronchial brush
`patient inhales aerosolized saline until cough alung aspirates
is induced
aopen lung biopsies
`watery appearance (resembles saliva)

Specimen Transport Microscopic Examination

asterile screw-cap cup to tube agram stain

aanaerobic transport media appropriate for amay reveal etiologic agent
lung aspirates amainly to determine sputum quality -
abiopsies kept moist with saline suitability for culture
abronchial brush placed in sterile saline `sputum or saliva?
`At low power field, presence of many
epithelial cells indicates oropharyngeal
contamination
`at oil immersion, report gram stain,
morphology, quantity

5
Gram Stain Analysis of
Sputum Routine Cultures

ascan on low power afor sputum, tracheal aspirates, bronchial

anumber of epithelial and PMN (<10, >10, washings, bronchial brushings, bronchial
<25, >25) per LPF biopsies
`good specimen: <10 epithelial and >25 PMN aBAP, CHOC, MAC
per LPF aselective media for B. cepacia (OFPBL)
`reject if >25 epithelial/LPF when obtained from CF patient
aunder oil immersion, record quantity aincubated at 35C in CO2
(occasional, moderate, many) and type of
bacteria, intracellular or extracellular

Anaerobic Cultures Quantitative Cultures

alung aspirates and open-lung biopsies abronchial brushes and bronchoalveolar

lavage
avortex fluid in known amount of liquid
(saline)
ainoculate aliquot of suspension
aincubate
acalculate number of CFU/ml
a>106 CFU/ml is significant for brushes
a>105 CFU/ml significant for lavage

Work-Up Other Respiratory Cultures

adepends on type of specimen, specimen aDiphtheria

quality, type of culture `caused by C. diphtheriae
`cultured on BAP, Loeffler, tellurite media
aN. meningitidis
`carriage state
`BAP & MTM

6
Other Respiratory Cultures

aPertussis
`whopping cough
`caused by Bordetella pertussis
`cultured on Bordet-Gengou blood agar
anasal
`S. aureus carriage in anterior nares
anasopharyngeal
`B. pertussis, B. parapertussis, N.
meningitidis, C. diphtheriae

7
 Introduction
Urinary Tract
upper urinary tract
kidneys
ureters
lower urinary tract
bladder
urethra
(prostate)

Normal Flora Terms

sterile above urethra, NF in urethra bacteriuria

coag-neg staph bacteria in urine
Corynebacterium pyuria
Micrococcus WBC in urine
Streptococci dysuria
Enterobacteriaceae difficult urination
anaerobic bacteria
cystitis
yeast
bladder infection
Mycoplasma
dysuria with frequent urination

Terms Diseases - UTI

pyelonephritis caused by endogenous flora

kidney infection E. coli (most common)
fever, pain, dysuria, frequent urination other Enterobacteriaceae (Klebsiella)
acute urethral syndrome (AUS) Staphylococcus saprophyticus, S. aureus
in women enterococci
dysuria, pyuria, bacteriuria Pseudomonas
AUS: S. saprophyticus,
Enterobacteriaceae, N. gonorrhoeae, C.
trachomatis

1
Routes of Infection Epidemiology

ascending route predisposing factors

from urethra to bladder (to kidneys) urinary tract abnormalities
descending route ⌧enlarged prostate
⌧kidney stones
carried by bloodstream to kidneys
instrumentation
MTB and S. aureus
⌧catheterization
underlying medical conditions
⌧diabetes mellitus

Epidemiology Epidemiology

women children
most UTI occur in women neonates
short female urethra preschool children
hormonal changes school-aged children
sexual activity more common in girls
pregnancy
men
>60 years
associated with enlarged prostate

Specimen Collection &

Nosocomial Infections Transportation

UTI is most common infection in US general consideration: urine inside body is

hospitals sterile
associated with catheterization and contamination: urethral, vaginal, skin,
instrumentation fecal organisms during collection
periurethral area cleaned with mild soap
and rinsed
specimen collected in sterile container
bedpans and urinals should not be used

2
Timing Acceptable Specimens

urine should remain in bladder as long as clean-catch midstream

possible straight catheter
urine is good growth medium indwelling catheter
number of colony-forming unit (CFU)/ml suprapubic aspirates
increases with incubation
cystoscopy specimens
first morning specimen

Clean-Catch Midstream Straight Catheter

periurethral area cleaned in/out catheter urine

patient begins voiding periurethral urine cleaned
collects midstream specimen catheter inserted in bladder
first urine passed not collected midstream specimen collected
avoid NF from urethra

Indwelling Catheter Suprapubic Aspirates

clean catheter collection port with alcohol percutaneous aspiration

aspirate specimen with needle and suitable for anaerobic bacteria
syringe
should not be collected from catheter bag

3
Cystoscopy Specimens Transport

collected by cystosope transported at RT

urine from bladder and/or ureter cultured within 2 hours of collection
if not, urine should be refrigerated
preservatives prolong RT transport time to
24 hours
boric acid maintains original colony count

Unacceptable Specimens Urine Screens

urine catheter tips (Foley catheters used by some labs

pooled 24-hour urine rapid detection of bacteriuria, pyuria, or
unrefrigerated or unpreserved urine (if both
older than 2 hours) gram-stained smear
urine other than suprapubic aspirates for acridine orange stained smear
anaerobic culture urine sediment examination

Acridine Orange-Stained
Gram-Stained Smear Smears

one drop of well mixed, uncentrifuged urine same as gram stain

onto slide detects as few as 104 CFU/ml
drop NOT spread out
air dried, fixed, stained
examined for host cells and bacteria
>1 organism /oil immersion field ==> colony
count of >105 CFU/ml
>1 PMN/ oil immersion field ==> pyuria
contamination: many epithelial cells

4
Urine Sediment
Examination Chemical Methods

aliquot centrifuged leukocyte esterase test

wet mount of sediment nitrate test
examined for WBC and bacteria

Leukocyte Esterase Test Nitrite Test

enzyme present in WBC nitrate normal in urine (not nitrite)

dipstick some organisms reduce nitrate ==> nitrite
dipstick detects nitrite in urine
positive ==> pyuria
positive: significant bacteriuria
false negative: organism can not reduce nitrate
(enterococci)
false positive: speciemen nor properly
preserved, contamination from nitrate reducing
organism

Instrumentation Media

photometry BAP
urine aliquot in broth medium enteric agar (MAC, EMB)
incubated and turbidity changes monitored other in special situations
bioluminescence CHOC if Haemophilus suspected
uses light to measure bacterial ATP in urine
colorimetric filtration
urine aliquot filtered
filter stained with safranin
amount color correlates with CFU/ml

5
Inoculation Loops

cultured quantitatively
CFU/ml is important diagnostic tool
calibrated loops that delivers 0.001 or
0.01 ml
loops dipped into well-mixed urine
loop completely covered
streaked down center of plate and then
spread over surface of agar

Streaking Incubation

overnight at 35C

Colony Count Workup

CFU/ml calculated factors

number of colonies on plate x 100 = type of specimen
CFU/ml colony count
if 0.01 ml loop used number of colony types present
number of colonies on plate x 1000 = patient history and symptoms
CFU/ml presence or absence of pyuria
if 0.001 ml loop used

6
General Guidelines General Guidelines

any urine, one organism, >105 CFU/ml suprapubic aspirates

ID and sensitivity to be performed ID and sensitivity as appropriate
voided urine, >3 organisms, each >105 special request - non-routine
CFU/ml catheterized,
catheterized any count
contaminated specimen, reject ID and sensitivity
request new specimen
voided urine, one pathogen, symptomatic
patient, >102 CFU/ml
ID and sensitivity to be performed

One Type of Possible

NO Growth Pathogen

0-999 organisms present <10,000 CFU/ml

report No growth at xx hours possible contamination
no work-up
state colony count and description of
isolate

One Type of Possible One Type of Possible

Pathogen Pathogen

10,000 - 100,000 CFU/ml >100,000 CFU/ml

possible infection probable infection
ID and sensitivity ID and sensitivity

7
Two Types of Possible Two Types of Possible
Pathogens Pathogens

each >10,000 CFU/ml one >10,000 CFU/ml, other <10,000

possible infection CFU/ml
ID and sensitivity on both possible infection by predominant
organism
ID and sensitivity on first one, count and
description of other

Two Types of Possible

Pathogens Three or More Pathogens

both <10,000 CFU/ml if one predominant (>100,000) - ID and

probable contamination sensitivity on that one
state colony count and description describe the others
no work-up all similar counts ==> contamination

8
 Introduction
Wound Infections
skin
epidermis
dermis
hair follicles, sebaceous glands, sweat glands
Skin & Soft Tissue subcutaneous layer (fat)
fascia (fibrous tissue)
muscles

Normal Flora Diseases

staphylococci (S. aureus & S. epidermidis) result of trauma (minor or severe)

micrococci surgical incision sites
streptococci (non-hemolytic) infecting organisms may be endogenous
enterococci or exogenous
diphtheroids single or polymicrobial infections
GNR (enterics & non-fermenters)
anaerobes
yeast and fungi

Abscesses Pyoderma

collection of pus skin infection with pus

skin and subcutaneous tissue

1
Folliculitis Furuncles

infected hair follicle boils

S. aureus small abscesses located deep in hair
P. aeruginosa (contaminated hot tubs) follicles
S. aureus

Carbuncles Cellulitis

subcutaneous abscesses spreading inflammation of connective

involves several hair follicles tissue in dermis
S. aureus group A streptococci
S. aureus
Hib
Vibrio
Clostridium

Impetigo Erysipelas

blister-like superficial skin infection dermis and superficial lymphatics

group A streptococci painful lesion
S. aureus very red appearance
group A streptococci

2
Wound Infections Wound Infections

infections in injured tissue burn wounds

caused by many organisms S. aureus
surgical wound infections P. aeruginosa
S. aureus GNR
GNR
streptococci
anaerobes

Animal Bite Sinus Tracts

Pasteurella maltocida channels connecting deep infected sites

S. aureus with skin surface
Capnocytophaga canimorsus
anaerobes
human bite
S. aureus, group A strep, viridans, Eikenella
corrodens, Prevotella, Fusobacterium

Myonecrosis Necrotizing Fasciitis

gas gangrene infection of fascia

severe muscle infection very severe
C. perfringens group A sterp
S. aureus
anaerobic bacteria

3
Decubitus Ulcers Diabetic Foot Ulcers

bed sores or pressure sores injuries heal slowly

Enterobacteriaceae S. aureus
Pseudomonas streptococci
enterococci enterococci
S. aureus Enterobacteriaceae
Bacteroides Pseudomonas aeruginosa
Clostridium anaerobes

Specimen Collection and

Transport Microscopic Examination

avoid surface contamination gram stain

skin or mucous membrane some clinically significant organisms can be
decontaminated before collection detected (Clostridium)
determine specimen quality
tissue and pus aspirates preferred
⌧reject if many epithelial cells seen
specimens
swabs are least desirable
tissue should be kept moist
swabs placed in transport medium
anaerobic

Cultures Anaerobic

routine media vary with setting, site, recommended for closed wounds and
organisms suspected abscesses
BAP, CHOC, MAC should be cultured aerobically also
35C in CO2

4
Quantitative Cultures Formula

performed on tissue specimens from burn number of colonies X dilution factor ÷

or trauma patients weight of tissue (grams) = CFU/gram
tissue is weighed >105 organisms per gram: clinically
homogenized in known amount of saline significant
serial dilution made
aliquot from each dilution inoculated onto
BAP, incubated
number of organisms per gram calculated

5
 Case Study
Bacterial Morphology And Structure
‹ A 4 year-old presented with symptoms of redness,
burning, and light-sensitivity in both eyes
‹ Exudative discharge made eyelids stick together
‹ Gram stain of conjunctival discharge showed Gram-
positive intracellular diplococci
‹ Quality control slides showed gram positive reactions for
both staphylococci and E.coli
‹ Procedure was repeated on the exudate and the quality
control organisms

Points to Consider Bacterial Structure: Introduction

‹ Why did the technologist repeat the procedure? ‹ Animal
‹ What may have caused the discrepancy? ‹ Plant
‹ What cell structure determines the Gram stain reaction ‹ Protista
of the bacteria?  Eukaryotes
‹ Why are older cells more easily decolorized than  Prokaryotes
younger colonies?  Archaebacteria
‹ Other points to consider

Bacterial Structure: Introduction Bacterial Structure: Introduction

‹ Prokaryotes
 Primitive nucleus ‹ Eukaryotes
)True bacteria.  True nucleus bound by a
"eubacteria” nuclear membrane
)Fungi, protozoan

Prokaryotic cell

Eukaryotic cell

1
The Bacterial Cell: The Cell Wall The Bacterial Cell: The Cell Wall

‹ Functions
‹ Peptidoglycan:A network of N-
 Provides strength acetyl Glucosamine (NAG) and
 Protects the internal contents N-acetylmuramic acid (NAM)
 Determines the shape connected by peptide bonds

The Gram-Positive vs. The Gram-Positive vs.

The Gram-Negative Cell Wall The Gram-Negative Cell Wall

Gram-positive bacteria Gram-negative bacteria

Morphology of Bacterial Species

Other Optional Parts
‹ Capsule
‹ Flagella
‹ Pili
‹ Endospores

Cocci in clusters

Cocci in chains

Streptococcus pneumoniae with capsules

Large spore-forming bacilli

Small non–spore-
Clostridium sp. with endospores
forming bacilli

2
Special Stains Special Stains

Fluorescent India Ink

Acridine orange
Lactophenol cotton blue

Löffler’s alkaline methylene blue

Acid-fast

Microbial Growth and Nutrition Bacterial Biochemistry and Metabolism

Bacterial Biochemistry and Metabolism Bacterial Biochemistry and Metabolism

3
Bacterial Biochemistry and Metabolism Bacterial Genetics

Points to Remember
‹ Sources of error when performing the Gram stain
procedure
‹ Other reasons for inaccurate interpretation of Grams
stain reactions shown in the stained smear
‹ Bacterial structures such as capsule, fimbrae, and spore
as virulence factors
‹ Significance of genetic mutations and alterations
clinically and in laboratory diagnoses of infectious agents

4
 Infections of the Case Study
Central Nervous Systems
‹ A 3-year-old male with a recent history of otitis
media was brought to the emergency room because
of fever and lethargy
‹ He had no rash and his vaccination history was up-
to-date
‹ His complete blood count showed leukocytosis with
shift-left and toxicity
‹ Blood and cerebrospinal fluid (CSF) cultures were
drawn. Cell count; CSF protein, and glucose levels
were also requested

Points to Consider Points to Consider

‹ Why is it important to determine the age group, ‹ How would you correlate the laboratory findings with
population, and immunization history of the patient? the possible etiology of the infection?
‹ What is the significance of the presence of rash in ‹ Why are the direct smear findings critical in the
this type of infection? presumptive diagnosis of this infection?
‹ What predisposing risk factors contribute to the ‹ Other points to consider
pathogenesis of this infection?

Components of the Central Nervous System Portals of Entry that may Result in Meningitis
and the Flow Pattern of CSF or Other CNS Infections

1
Bacteria Involving the CNS: Bacteria Involving the CNS:
Acute Meningitis Related to Age Chronic Meningitis
‹ Premature neonate Gram-negative bacilli
‹ Infants Listeria monocytogenes ‹ Mycobacterium tuberculosis and other nontuberculous
Group B Streptococci mycobacteria
S. pneumoniae
H. influenzae ‹ Treponema pallidum
‹ Children N. meningitidis ‹ Borrelia burgdorferi
S.pneumoniae ‹ Borrelia recurrentis
H. influenzae
‹ Leptospira sp.
‹ Adolescents N. meningitidis
‹ Adults S.pneumoniae
‹ Elderly Gram-negative bacilli

Infections of the CNS: Infections of the CNS:

Laboratory Diagnosis Laboratory Diagnosis
Characteristic Findings in Meningitis
Lumbar puncture: CSF Test Normal CSF Acute Bacterial
is obtained by inserting Meningitis
a long, sterile, hollow Appearance Clear/colorless clear/turbid
needle into the spinal Total protein Adults greatly increased
subarachnoid space in (mg/dL) 15 to 45 (usually 8 to 500)
the lower back.
Glucose 60% to 80% Usually greatly
of blood level reduced

Cell count 0 to 10 Greatly increased

>90% PMNs

Grams stain No bacteria + in 60 to 70% cases

CSF Direct Microscopic Exam: Bacterial CSF Direct Microscopic Exam: Bacterial
Infections Infections

Direct smear of CSF from a child showing

Direct smear of CSF from an infant
gram-negative pleomorphic coccobacilli
showing gram-positive cocci in chains
characteristic of Haemophilus influenzae
characteristic of Group B streptococci

2
CSF Direct Microscopic Exam: Bacterial CSF Direct Microscopic Exam: Bacterial
Infections Infections

Direct smear of CSF from a high-school student

showing clusters of gram-negative intracellular Lancet-shaped gram-positive diplococci
diplococci consistent of Neisseria meningitidis characteristic of Streptococcus pneumoniae

CSF Direct Microscopic Exam: Bacterial CSF Direct Microscopic Exam: Bacterial
Infections Infections
CSF direct smear from a
newborn delivered from a woman
with amnionitis secondary to
premature rupture of the
membranes; Bacteroides species
were suspected

Direct smear of posttraumatic acute bacterial

Direct smear of aspirated brain
meningitis; encapsulated gram-negative bacilli
abscess contents; Intracellular
typical of Klebsiella pneumoniae are seen
gram-positive cocci are seen

CSF Direct Microscopic Exam: Bacterial

Infections Case Study

‹ A 52-year-old white male arrived at the Emergency

Room in a disoriented and poorly responsive state
‹ He was febrile, lethargic, and in respiratory failure
‹ He had poorly controlled diabetes and chronic
obstructive pulmonary disease

Congenital listeriosis suspected. This is a

Gram-stained smear of the amniotic fluid
showing purulence and gram-positive small
bacilli typical of Listeria monocytogenes

3
 CSF Direct Microscopic Exam:
Case Study Fungal Infections
Cryptococcal meningitis in
‹ Current medications included steroids partially immunocompetent
patient showing inflammatory
‹ CSF sample showed encapsulated budding yeasts cells and budding yeast
‹ The patient died on the third day of hospitalization

Cryptococcal meningitis in an
immunosuppressed host
showing numerous yeasts with
scarce inflammatory cells

CSF Direct Microscopic Exam: CSF Direct Microscopic Exam:

Fungal Infections Viral Infections

Brain abscess smear, toluidine blue stain showing In a case of “aseptic” meningitis,
fungal hyphae septate suggesting Aspergillus spp; lymphocytes are predominantly
suspected cerebral aspergillosis present; no organisms seen.

CSF Direct Microscopic Exam: CSF Direct Microscopic Exam: Parasitic

Viral Infections Infections

Reactive lymphocytes with “monocytoid” features

are the only clue that this is not a viral infection; Touch preparation of brain tissue showing typical
CSF in meningitis resulting from tularemia “floret” of Toxoplasma gondii trophozoites

4
Points to Remember

‹ Host-related risk factors and virulence factors

associated with specific pathogens
‹ Age and clinical history of the affected population
‹ Characteristics of the CSF
‹ Characteristics of the microbial agents on direct
smear preparations

5
 Gastrointestinal Infections Gastrointestinal Infections: Introduction
and Food Poisoning
‹ Anatomical
Considerations
 Gastric acidity
 Motility
 IgA
 Gut flora
‹ The role of the
usual flora

Case Study Points to Consider

‹ What clinical findings are significant?
‹ A 32 year old man traveled to a small village in Mexico
and experienced a sudden onset of diarrhea 4 days ‹ What are the travel and food intake histories?
after his arrival ‹ What pathogenic mechanism is employed by the
‹ He complained of nausea, and the stool was watery, suspected agent?
without gross blood, pus, or mucous ‹ How would you differentiate food poisoning from
‹ He had no fever gastrointestinal infection?
‹ What methods of recovery would be appropriate?
‹ Other points to consider

Gastrointestinal Infections and

Food Poisoning:A Practical Approach Food Vehicle Pathogen or Toxin
Undercooked chicken Salmonella sp. Campylobacter sp.
‹ Clinical history Eggs
Unpasteurized milk Salmonella, Campylobacter,
 Travel Yersinia sp
Water G. lamblia, Norwalk virus,
 Food ingestion
Campylobacter, Cyclospora,
‹ Physical examination Cryptosporidium
Fried rice Bacillus cereus
 Clinical presentation
Fish-shellfish Vibrio sp. neurotoxic shellfish
‹ Laboratory diagnosis poisoning, Norwalk virus

Tuna, mackerel, Scombroid Ciguatera Anisakis

grouper, amberjack-
sushi
Beef gravy Salmonella, Campylobacter,
C. perfringens

1
Practical Approach to the Diagnosis of GI Practical Approach to the Diagnosis of GI
Infections:Pathogenic Mechanisms Infections:Pathogenic Mechanisms

‹ Enterotoxin-mediated ‹ Preformed toxin ‹ Invasive organisms ‹ Localized invasion

diarrhea  S. aureus  Longer incubation  Nontyphoidal Salmonella
 C. perfringens period  Shigella spp.
 Rapid onset  C. botulinum  Fever present  Campylobacter jejuni
 No fever ‹ Colonization and Toxin  WBCs and RBCs  Vibrio parahaemolyticus
Production present
 No leukocytes  Yersinia enterocolitica
 Clostridium difficile ‹ Systemic invasion  Enteroinvasive E. coli
 Vibrio cholerae
 Salmonella typhi
 E coli O157:H7
 Aeromonas
 Enterotoxigenic E. coli

Gastrointestinal Infections:
Laboratory Diagnosis A Practical Approach

‹ Culture of appropriate ‹ Other special/selective

body fluids media
‹ Routine culture media  TCBS
 BAP—blood agar  CCFA
 MAC—MacConkey  SMAC
 HE—Hektoen
 XLD—Xylose lysine
desoxycholate Direct fecal smear gram-stained to show
the presence of white blood cells,
 CAMPY agar
indicative of an invasive process and
not due to an enterotoxin

Common Gastrointestinal Common Gastrointestinal

Infections and Their Agents Infections and Their Agents
Campylobacteriosis Salmonellosis

Grams stain of Campylobacter colony showing

the typical microscopic morphology described Culture and serologic
as “seagull wings”; Campylobacter jejuni is the diagnosis of typhoid fever
most common cause of bacterial diarrhea in
the United States.

2
Common Gastrointestinal Common Gastrointestinal
Infections and Their Agents Infections and Their Agents
Salmonellosis Shigellosis (Left) Lactose-negative
Salmonellae on agar showing appearance of Shigella sonnei
black centers resulting from growing on MAC agar at 18 to
hydrogen sulfide production 24 hours of incubation.(Left)
(Right) Lactose-positive
Shigella sonnei on MAC agar
after 48 hours of
incubation(Right)

Hydrogen-sulfide producing
Shigellae colonies growing on
colonies of salmonellae
HE agar showing clear green
growing on XLD
colonies

Common Gastrointestinal Common Gastrointestinal

Infections and Their Agents Infections and Their Agents
Diarrheogenic Escherichia coli Helicobacter pylori
Microscopic morphology of
Helicobacter pylori gram-
stained from a colony

Urea breath test for

diagnosis of Helicobacter
Escherichia coli O157:H7 growing on MAC agar(Left); pylori infection
Escherichia coli O157:H7 on sorbitol MAC(right)

Common Gastrointestinal Common Gastrointestinal

Infections and Their Agents Infections and Their Agents
Parasitic agents
Watery, frothy diarrheic
stool smear; acid-fast
stain showing oocysts of
Cyclospora spp.

• Diarrheic stool smear, modified

Watery, frothy diarrheic stool Weber stain
smear; acid-fast stain showing
oocysts of Cryptosporidium • Small spore morphology consistent
parvum with enterocytozoon

3
Common Gastrointestinal
Infections and Their Agents Points to Remember
Amoebiasis ‹ Significant clinical findings
Entamoeba histolytica trophozoite ‹ Travel history and food intake
‹ Incubation period and initial laboratory findings
‹ Methods of recovery
Giardiasis ‹ Characteristics of intestinal pathogens on
screening media
‹ Key identification features of each pathogen

Giardia lamblia trophozoite

4
 Case Study
Respiratory Tract Infections
‹ An 8-year-old girl was brought to the emergency room
because of complaints of fever and sore throat
‹ The child had had a runny nose and cough for the last few
days
‹ Her pharynx was red and her tonsils were slightly swollen
‹ No exudates were present and there were no swollen
lymph nodes

Points to Consider Anatomy of the Respiratory Tract

‹ Etiology and pathogenesis of respiratory tract

infections
‹ Presenting symptoms and predisposing factors
‹ Age and immune status of the affected population

Usual Flora in the Respiratory Tract Respiratory Tract: Definitive Pathogens

‹ Rarely Pathogenic ‹ Possible Pathogens ‹ Corynebacterium diphtheriae ‹ Pneumocystis carinii
 Nonhemolytic streptococci  Beta hemolytic streptococci (toxin+) ‹ Viruses
 Staphylococci  S.pneumoniae ‹ Neisseria gonorrhoeae  RSV, HSV
‹ Mycobacterium tuberculosis  influenza
 Micrococci  S. aureus
‹ Chlamydia trachomatis ‹ Fungi
 CNS  H. Influenzae
‹ Mycoplasma pneumoniae  Coccidioides immitis
 Nonpathogenic Neisseria  N. meningitidis ‹ Bordetella pertussis  Histoplasma capsulatum

 Stomatococci  Moraxella catarrhalis ‹ Legionella pneumophila

 Veillonella  Enterobacteriaceae

 Spirochetes  Fungal agents

 Anaerobes

1
 Upper Respiratory Tract Upper Respiratory
Infections: Pharyngitis/Tonsillitis Tract Infections: Diagnosis
‹ Etiology
 Streptococcus pyogenes (most common)
 Neisseria gonorrhoeae
 Corynebacterium diphtheriae Specimen collection from the
 Viruses throat to differentiate bacterial
‹ Clinical signs pharyngitis due to streptococci
from viral origin
 Abrupt onset
 Fever
 Headache
 Sore throat
 Exudates

Upper Respiratory Other Upper Respiratory

Tract Infections: Other Infections Tract Infections: Whooping Cough
‹ Bordetella pertussis and ‹ Clinical manifestations
B. parapertussis  Prodromal: 5 to 10 days after
‹ Highly communicable exposure
 Catarrhal
‹ Usually found in children )Cold-like symptoms
‹ Increased incidence )Cough/runny nose for
among teenagers several weeks
 Paroxysmal:“whooping”
‹ Adults serve as reservoir
stage
and source of infection )Severe, violent coughing
Maxillary sinus aspirate; gram-stained smear showing a polymicrobial stage—can occur in 24
infection; gram-positive cocci in chains and gram-positive coccobacilli hours of exposure
with heavy purulence are noted  Convalescent: symptoms
slowly decrease 6 months

Other Upper Respiratory Tract Infections: Other Upper Respiratory Tract Infections:
Whooping Cough Case Study

‹ A 4-year-old boy was brought to the physician’s clinic

because of fever and trouble swallowing
‹ Inspiratory stridor was noted
‹ He was taken to the emergency room immediately,
where his epiglottis was examined
‹ Epiglottitis was suspected

Bronchoalveolar lavage; Wright-Giemsa stained showing lymphocytes

typically seen in pertussis; ciliated columnar epithelial cells with
numerous small bacilli adherent to the cilia; morphology consistent
with Bordetella pertussis

2
Other Upper Respiratory Tract Infections: Lower Respiratory
Epiglottitis Tract Infections: Epidemiology
Age Etiology
Haemophilus influenzae type b Neonates/infants RSV, influenza, parainfluenza, adenovirus,
causes acute epiglottitis, a life- Chlamydia trachomatis, or P. carinii
threatening infection of the
epiglottis Children
5 to 18 months S. pneumoniae, H. influenzae
3 months to teens Viruses, Mycoplasma pneumoniae,
S. aureus

Young adults Chlamydia pneumoniae, Mycoplasma

Older adults S. pneumoniae, Legionella sp

Gram-stained smear of
sputum/exudate with H. influenzae Institutionalized adults Gram-negative rods, S. aureus,
S. pneumoniae

Community-Acquired Pneumonia Community-Acquired Pneumonia

A B
Chest radiographs before (A) and after (B) development of an acute,
community-acquired pneumococcal pneumonia; in B, consolidation of
the right upper lobe of the lung is evidenced by the dense, whitish Gram-stained smear of Streptococcus pneumoniae isolated from the
opacity in the area blood culture of a patient with pneumococcal pneumonia

Community-Acquired Pneumonia Community-Acquired Pneumonia

Bronchoalveolar lavage; Gram’s
stain showing purulence and
gram-negative bacilli

Bronchoalveolar lavage, same

Expectorated sputum smear, Gram’s stain; typical presentation of early sample, using direct fluorescent
stages of pneumococcal pneumonia with migration of neutrophils; culture antibody (DFA) and Legionella
yielded S. pneumoniae pneumophila antiserum

3
Community-Acquired Pneumonia Community-Acquired Pneumonia

Chlamydia pneumoniae detected from direct sputum smear using

fluorescent-labeled monoclonal antibody; shows cytoplasmic
Typical chest radiograph of a patient with a 3-week course of
inclusions
atypical pneumonia

Community-Acquired Pneumonia Community-Acquired Pneumonia

Mycoplasma pneumoniae on ciliated Typical large Mycoplasma

tracheal cells; infected animal model colony showing “fried egg”
appearance

Diene’s stain of Mycoplasma colony

showing “fried egg” appearance
Uninfected animal model

Hospital-Acquired Pneumonia Hospital-Acquired Pneumonia

‹ Risk factors ‹ Etiology

 Increased colonization of the  Klebsiella pneumoniae
upper respiratory tract with  Other Enterobacteriaciae
bacterial pathogens  P. aeruginosa
 Compromise of the barriers that  Other organisms, such
normally protect the lower as anaerobes
respiratory tract

Expectorated sputum with light purulence; gram-negative bacilli

suggest an enteric bacillary infection

4
Hospital-Acquired Pneumonia Hospital-Acquired Pneumonia

Expectorated sputum smear, Gram’s

stain; Pseudomonas aeruginosa
suspected

Expectorated sputum smear, Gram’s

stain; mucus present with gram-negative
bacilli enveloped in slime; morphotype
Expectorated sputum smear; Gram’s stain showing moderate mucus
suggested Pseudomonas aeruginosa
and purulence; encapsulated gram-negative bacilli; morphology
suggests antibiotic-affected Klebsiella pneumoniae

Lower Respiratory Infections:

Aspiration Pneumonia Sputum Evaluation for Culture

Aspirated sputum smear, Gram’s stain; light purulence with gram- Expectorated sputum smear, Gram’s stain; numerous
positive bacilli, gram-negative bacilli, and gram-positive cocci; white blood cells, and gram-positive diplococci acceptable
Polymicrobial infection with fecal flora suspected for culture

Lower Respiratory Infections: Lower Respiratory Infections:

Sputum Evaluation for Culture Sputum Evaluation for Culture

Expectorated sputum smear, Gram’s stain; no visible purulence; Expectorated sputum smear, Gram’s stain; heavy purulence;
contaminating bacteria and squamous epithelial cells heavy; sample is Curschmann’s spirals present; this is local to the tracheobronchial tree
saliva, not sputum, making it unacceptable for culture. but is not normal

5
Lower Respiratory Infections: Other Types of Respiratory
Sputum Evaluation for Culture Tract Infections: Tuberculosis

Acid-fast bacillus stain of sputum

containing mycobacteria

Expectorated sputum; concentrated,

Aspirated sputum smear, Gram’s stain; alveolar macrophages and
fluorochrome stain for acid-fast bacilli;
mucus present; no purulence; no organisms seen; sputum was sampled
Mycobacterium tuberculosis was grown
but no evidence of infection
in culture

Other Types of Respiratory Other Types of Respiratory

Tract Infections: Fungal Agents Tract Infections: Fungal Agents

Expectorated sputum smear, Gram’s

stain; shows pseudohyphae consistent
with Candida sp.

GMS stain H & E stain

Expectorated sputum smear,
calcofluor white stain; shows broad-
Lung exudate from a patient with hematologic disorder; shows
based yeast, suggesting Blastomyces
alveoli that contains branching fungal elements
dermatitidis

Other Types of Respiratory Tract Infections: Other Types of Respiratory Tract Infections:
Miscellaneous Agents Miscellaneous Agents

Bronchoalveolar lavage, GMS stain; Bronchoalveolar lavage, Wright-

shows cysts of Pneumocystis carinii Giemsa stain; shows crescent-
shaped cells with central nucleus

Bronchoalveolar lavage, Acridine

Bronchoalveolar lavage, calcofluor orange stain; shows crescent-shaped
white stain; shows coccoid bodies cells with central nucleus consistent
consistent with Pneumocystis carinii with Toxoplasma gondii

6
Other Types of Respiratory Tract Infections:
Miscellaneous Agents Points to Remember
‹ Clinical history of the patient including
Expectorated sputum growing on 5%
sheep blood agar; heavy bacterial  Age group and population
growth with thin trails of colonies lacing  Predisposing risk factors
the surface of the agar  Clinical sites of infection
 Clinical presentation and manifestations
‹ General characteristics of the isolate
‹ Appropriate samples for maximum recovery of the
etiologic agent

Aspirated sputum showing coiled

larvae; morphology consistent with
Strongyloides stercoralis

7
 Case Study
Skin and Soft Tissue Infections
‹ A 37-year-old Haitian woman presented with
complaints of a swollen, painful, right breast
‹ She had low-grade fever, and a mass was detected
on her right breast
‹ Pus was aspirated from the lesion

Points to Consider Anatomy of the Skin

‹ Why is it important to determine how the patient
acquired the infection?
‹ What are the possible sources of infection for this
type of wound?
‹ How do the causative organisms initiate the
infectious process?
‹ What clues would indicate the presence of infection?
‹ Other points to consider

Usual Skin Colonizers Most Common Primary Pyodermas

‹ Coagulase-negative staphylococci Infection Organism

‹ Diphtheroids Impetigo S. pyogenes
‹ Propionibacteria S. aureus
Erysipelas S. pyogenes
‹ Streptococci Other β-hemolytic streptococci
Cellulitis S. pyogenes
‹ Yeasts S. aureus; H. influenzae
Carbuncle S.aureus

Folliculitis S. aureus, Candida; gram-negative

bacilli
Paronychia S. aureus, Candida; gram-negative
bacilli
Furuncle S. aureus

1
Clinical Infections: Clinical Infections:
Bacterial Skin Infections Bacterial Skin Infections

Staphylococcus aureus furuncle of the breast

Bullous impetigo caused by S. aureus

Clinical Infections:
Bacterial Skin Infections Case Study
‹ An 18-year-old man suffered a severe crush injury to
his right forearm
‹ Within 18 hours of his admission to the hospital, he
developed a high fever and systemic toxicity
‹ The site of injury showed progressive necrosis and
hemorrhagic bullae
‹ He was transferred immediately to another facility for
hyperbaric oxygen therapy
‹ Broad-spectrum antibiotics were started

Erysipelas due to Streptococcus pyogenes

Clinical Infections: Cutaneous Manifestations of Systemic

Bacterial Skin Infections Bacterial Infections

A severe soft tissue infection

with sepsis syndrome in a 55-
year-old man who had history
of alcohol abuse and hepatitis;
an ulcerative cellulitis with
overlying exudate developed.

Diabetic foot infection with soft-

tissue gas formation
Clostridial myonecrosis or gas gangrene, typically caused by
Clostridium species

2
Cutaneous Manifestations of Systemic Other Types and
Bacterial Infections Sources of Skin Infections

Petechial lesion in meningococcemia Subcutaneous nodules and cellulitis due to

disseminated Mycobacterium chelonei

Penile syphilitic chancre caused by

Hemorrhagic vasculitis lesion of Treponema pallidum
Staphylococcus aureus

Other Types and

Sources of Skin Infections Fungal Skin Infections

Eczema herpeticum due

to herpes simplex Giant molluscum contagiosum Tinea corporis Tinea capitis

Fungal Skin Infections Fungal Skin Infections

Tissue cross-section of a nodule containing Onchocerca

Hypopigmented Hyperpigmented
volvulus microfilaria
Tinea versicolor

3
 Skin and Soft Tissue Infections: Laboratory
Parasitic Skin Infections Diagnosis

Sarcoptes scabiei in an adult showing ‹ Specimen collection/processing

short legs and conical spines ‹ Smear examination
 Grams Stain
 KOH preparation
 Calcofluor white
 Acid-fast stain
‹ Primary isolation
‹ Identification/susceptibility testing

Tissue cross-section of scabies

lesion showing larvae burrowed into
the epidermal layer of the skin

Skin and Soft Tissue Infections: Skin and Soft Tissue Infections: Direct Smear
Direct Smear Examination Examination

Abscess aspirate smear, Grams stain; Heavy purulence with gram-

Wound smear, Grams stain; moderate purulence with Gram-positive
positive coccic in lusters suggestive of staphylococcal disease
cocci in chains suggestive of streptococcal disease

Skin and Soft Tissue Infections: Direct Smear Skin and Soft Tissue Infections: Direct Smear
Examination Examination

Abscess aspirate smear, Grams stain

with purulence; gram positive bacilli,
beaded; Suspected mycobacteria

Abscess aspirate smear, Ziehl-

Wound cellulitis smear, Grams stain; gram-positive large bacilli
Neelsen (AFB stain); acid-fast bacilli
morphology consistent with Clostridium perfringens
seen; Culture grew M. kansasii

4
 Skin and Soft Tissue Infections: Direct Smear Skin and Soft Tissue Infections: Direct Smear
Examination Examination

Cutaneous sinus tract aspirate, Grams

stain; heavy purulence and Gram-
positive filamentous, beaded;
branched bacilli; AFB stain negative,
suggesting Actinomycosis present

Cutaneous sinus tract aspirate

colonies on anaerobic blood agar Wound smear, Grams stain; heavy purulence with gram-negative
plate; molar tooth colony-type seen bacilli and gram-positive cocci
consistent with Actinomyces israelii

Skin and Soft Tissue Infections: Direct Smear Skin and Soft Tissue Infections: Direct Smear
Examination Examination

Skin scales, KOH wet preparation; Hyphae present, septate, Skin scales from scrapings, calcofluor white stain; hyphae present,
suggest dermatophyte suggesting dermatophytosis

Skin and Soft Tissue Infections: Direct Smear Skin and Soft Tissue Infections: Direct Smear
Examination Examination

Skin, vesicle fluid, Tzanck preparation, H &

E stain; multinucleated epithelial cells
present; intranuclear inclusions consistent
with herpes viral inclusions

Skin, vesicle fluid, Tzanck

preparation; antibody stain for herpes
Soft tissue abscess smear, calcofluor white stain; Fungal hyphae,
simplex; Herpes simplex infection
septate and branched; dermatophyte was isolated in culture.
confirmed

5
Points to Remember
‹ Types of skin and soft tissue infections
‹ Sources and agents of infections
‹ Clinical and bacteriological clues to skin and soft
tissue infections
‹ Characteristic morphology of representative species
‹ Tests to presumptively identify isolated agent

6
 Sexually Transmitted Diseases and Case Study
Urinary Tract Infections
‹ A 24-year-old female presented with fever, pain, and
tenderness around her left elbow joint
‹ The patient had noticed a slight rash around her left
hand 10 days earlier
‹ On examination, the patient showed rash on her
extremities, swollen joints, and petechiae with
vesicular eruptions
‹ Based on the physical findings, the physician obtained
an aspirate of the joint fluid and ordered routine and
anaerobic cultures, along with appropriate cultures for
Neisseria gonorrhoeae

Common Exudative Sexually Transmitted

Points to consider Infections: Gonorrhea
‹ Why did the physician request a culture for N.
gonorrhoeae ?
‹ Which of the clinical findings indicate a gonococcal
infection?
‹ How is the infection acquired?
‹ What complications may develop at the initial site of
infection?
‹ Other points to consider

Reported cases of gonorrhea since 1978

Common Exudative Sexually Transmitted Common Exudative Sexually Transmitted

Infections: Gonorrhea Infections: Gonorrhea
‹ Clinical Manifestations
‹ Gonorrhea
 In women
 Caused by gram-negative diplococcus Neisseria gonorrhoeae )Endocervix is the primary site of infection
 One of the most commonly diagnosed, reportable, sexually )Only about 20% show overt symptoms
transmitted diseases )If it remains undiagnosed, may develop salpingitis and pelvic
 One third of the infections occur in adolescents inflammatory disease
 In men
)Symptoms include urethral inflammation with dysuria and pyuria
)Sequelae include prostatitis, epididymitis, and urethral stricture

1
Common Exudative Sexually Transmitted Common Exudative Sexually Transmitted
Infections: Laboratory Diagnosis Infections: Laboratory Diagnosis

JEMBEC plate streaked in a Z

Direct gram-stained smear of pattern showing growth of N.
male urethral discharge gonorrhoeae
showing intracellular gram-
negative diplococci

Direct smear showing

numerous PMNs but no
bacteria, suggesting N. gonorrhoeae after 24 hours
nongonococcal urethritis of growth on modified Thayer-
Martin agar

Common Exudative Sexually Transmitted Common Exudative Sexually Transmitted

Infections: Laboratory Diagnosis Infections: Laboratory Diagnosis

Candle extinction jar with

inoculated Thayer-Martin agar
plates; N. gonorrhoeae requires a
3% to 5% carbon dioxide
atmosphere
Conventional CTA sugars showing acid production in the glucose
tube only, identifying the organism as N. gonorrhoeae

Common Exudative Sexually Transmitted

Case Study Infections: Genital Chlamydiosis
‹ A 24-year-old female went to her gynecologist for a ‹ Clinical Manifestations
routine pelvic examination; It had been several years  Caused by Chlamydia trachomatis
since her last examination  Most common sexually transmitted bacterial infection
‹ She was currently in a monogamous relationship, but  Up to 85% of women with this condition are asymptomatic
her sexual history included other sex partners  Highest rate of infection is among adolescent girls
 In men, infection appears as urethritis
‹ The patient had no symptoms, but the physician
collected routine screening tests for gonorrhea,
chlamydiosis, HIV, and syphilis
‹ All laboratory test results came back negative except
for the Chlamydia test, which was positive

2
Common Exudative Sexually Transmitted Common Exudative Sexually Transmitted Infections:
Infections: Genital Chlamydiosis Genital Chlamydiosis

Chlamydia spp. growth cycle

showing initial bodies

Chlamydia spp. growth cycle showing

Life cycle of Chlamydia infective elementary bodies

Common Exudative Sexually Transmitted Common Exudative Sexually Transmitted

Infections: Laboratory Diagnosis Infections: Laboratory Diagnosis

Iodine-stained inclusion
Endocervical specimens bodies from Chlamydia-
stained with Papanicolaou trachomatis–infected
stain showing inclusion McCoy cells
bodies consistent with
Chlamydia trachomatis

Case Study Case study, cont’d

‹ A black male infant was born at 33 weeks of gestation ‹ Laboratory findings also showed pancytopenia,
to a 17-year-old mother hypoglycemia, coagulopathy, oliguria, and acidosis in
‹ The mother had no prenatal care and had a reactive the infant
rapid plasma reagin (RPR) test result (1:128) on ‹ The infant received multiple transfusions of washed,
admission to an obstetric service packed red blood cells, platelets, and fresh-frozen
‹ The infant was delivered by Cesarean section plasma
because of fetal distress ‹ He was treated with penicillin and gentamicin but died
‹ On examination, physical findings revealed that the 3 days after birth
infant was hydropic and showed jaundice and ‹ Examination of the placenta showed spirochetes in the
hepatosplenomegaly umbilical cord

3
Common Ulcerative Sexually Transmitted Common Ulcerative Sexually Transmitted
Infections: Syphilis Infections: Syphilis
‹ Clinical Manifestations
 Caused by Treponema pallidum subsp. Pallidum
 The second most common ulcerative sexually transmitted
infection
 Primary syphilis
)Appearance of chancre
 Secondary syphilis
)Spirochetemia
)Fever, lymphadenopathy, macular skin lesions
 Latent—no symptoms
 Tertiary
)Immune sequelae
Reported cases of syphilis since 1978

Common Ulcerative Sexually Transmitted Common Ulcerative Sexually Transmitted

Infections: Syphilis Infections: Laboratory Diagnosis
‹ Nontreponemal (nonspecific) antibody tests
 RPR (Rapid plasma reagin)
 VDRL (Venereal Disease Research) Laboratory
‹ Treponemal antibody tests
 FTA-ABS(Fluorescent treponemal antibody
absorption)
 MHA-TP ( microhemagglutination for
T. pallidum)

Penile syphilitic chancre caused by Treponema pallidum

Common Ulcerative Sexually Transmitted Common Ulcerative Sexually Transmitted

Infections: Chancroid (Soft Chancre) Infections: Laboratory Diagnosis
‹ Clinical Manifestations ‹ Haemophilus ducreyi
 Caused by Haemophilus ducreyi
 Pleomorphic gram-negative coccobacilli that may occur in
 Endemic in southeast Asia, Africa, and India
chains resembling a “school of fish” arrangement
 Of the ulcerative sexually transmitted infections, the most difficult
 Very fastidious pathogen that requires special growth and
infection to diagnose
environmental supplements
 Chancroid develops on the external genitalia of both men and
women
 Inguinal lympadenopathy resembling buboes may be present

4
Common Ulcerative Sexually Transmitted Common Ulcerative Sexually Transmitted
Infections: Chancroid Infections: Chancroid

Reported cases of chancroid since 1978 Lesions of chancroid on the penis, showing draining buboes
in the adjacent groin area

Common Ulcerative Sexually Transmitted

Infections: Genital Herpes Points to Remember
‹ Herpes simplex virus (HSV) infection ‹ Clinical presentation or manifestation
 The most common ulcerative sexually transmitted infection
‹ Complications or sequelae that may develop
 Two serotypes of HSV
)HSV1 causes 80% of oral infections and 20% of genital infections
‹ Diagnostic testing for the suspected agent
)The reverse is true for HSV2 ‹ Characteristic features of each of the agents
‹ Clinical manifestations
 Painful erythematous lesions appear at the site of infection
 May be accompanied with fever, myalgia, and malaise
 Recurrence is not uncommon

Case Study
Urinary Tract Infections
‹ A 77-year-old surgical patient developed dementia with
concurrent fever and elevated peripheral white blood cell
count
‹ Blood and urine samples were taken for culture
‹ The patient has been recently discharged to a long-term
facility
‹ Urine analysis showed positive leukocyte esterase and
the presence of yeasts and gram-negative rods
‹ Culture grew >100,000 cfu/mL of urine identified as
Escherichia coli, Klebsiella pneumoniae, and other
organisms

5
 Urinary Tract Infections: Epidemiology and
Urinary Tract Infections Risk Factors

Anatomy of the urinary tract Frequency of urinary tract infection over time(X, female; O, male.

Schema that may be used to

evaluate women with dysuria Urinary Tract Infections: Etiology

Urine; direct smear; gram-stained showing heavy purulence, gram-

negative bacilli, and gram-positive cocci; urine culture grew
Escherichia coli and Enterococcus faecalis with a bacterial density of
>100,000 cfu/mL of urine

Points to Consider
‹ Clinical findings or presence or absence of symptoms
‹ Age and population
‹ Predisposing risk factors present
‹ Presence or absence of pyuria
‹ Significant colony count of a pure or predominant
organism
‹ Characteristic features of the associated agents
associated

6
 Case Study
Zoonotic Infections
‹ A 16-year-old female went to the emergency
room(ER) because of pain and numbness in her left
axilla and arm
‹ She reported falling from a trampoline 4 days prior
and was diagnosed with possible brachial plexus
injury
‹ Two days after she was seen in the emergency room,
she was found semiconscious at home

Case Study, cont’d Case Study, cont’d

‹ Within hours after she was taken to the hospital, she ‹ Her condition deteriorated rapidly, and she later died.
experienced respiratory arrest and was intubated ‹ Sputum, blood, and spinal fluid drawn from her
‹ Numerous gram-positive diplococci were found in her revealed the following organisms:
blood, and a chest x-Ray revealed bilateral  CSF: Streptococcus pneumoniae
pulmonary edema  Respiratory aspirate: Yersinia pseudotuberculosis
‹ She was treated with antibiotics and was transferred  Blood: Yersinia pestis
to a referral hospital where she was diagnosed with
septicemia, DIC, adult respiratory distress syndrome,
and meningitis

Points to Consider Zoonotic Infections Transmitted by Scratches

and Bites
‹ What are the significant clinical findings?
‹ How would the clinical, social, and occupational
history of the patient help in determining the
possible source of infection?
‹ What are the possible sources of infection?
‹ Are there risk factors associated with these
infections?
‹ How are the infections acquired?
‹ Other points to consider

1
Plague and Yersinia pestis Plague and Yersinia pestis: Life Cycle

‹ Epidemiology: Three Pandemics Principal cause of massive

 Egypt in 542 AD epidemics domestic rat is
 The Black Death in the Middle Ages beaten by infective flea. Rat
 1890s to present dies, flea looks for new host.
)Introduced in the United States
)Vietnam
)Exists in all continents except Australia

Urban Cycle

Plague and Yersinia pestis Plague and Yersinia pestis

‹ Wild rodents as
‹ Bubonic: initiated by bite of infected flea
reservoirs
 Organisms reach the regional lymph nodes, where they
 Transmission among then multiply
these animals is
 Produce buboes
accomplished by fleas or
ingestion of infected  Leads to bacteremia and seeding of the lungs, liver, and
rodents spleen
 Humans infections are ‹ Pneumonic: fatal hemorrhagic pneumonia that
accidental leads to cyanosis (Black Death)
 Highly contagious
 Spread by droplets

Sylvatic cycle

Plague and Yersinia pestis: Plague and Yersinia pestis:

Laboratory Diagnosis Laboratory Diagnosis

‹ Specimen collection ‹ Culture

 Aspirates of buboes  Grows slowly on sheep’s
 Sputum blood agar
‹ Direct microscopic  Colonies are
nonhemolytic, smooth
examination and slightly opaque
 Gram-negative bacilli with
bipolar or “safety pin”
appearance
Smear from lymph gland of
patient with plague Small, translucent gray colonies
of Y. pestis

2
 Lyme Borreliosis (Lyme Disease) and Borrelia
Case Study burgdorferi

‹ A 28-year-old woman presented at the emergency ‹ Epidemiology

room with shaking chills and perspiration  1908: First noted in Sweden
‹ She had swollen ankles, knees, wrists, and elbows;  1970: First reported in the United States
synovitis was noted on these areas  1975: Outbreak in Lyme and Old Lyme, Connecticut
‹ No rash or lymphadenopathy  Dr. Willy Burgdorferi recovered spirochetes in the blood of ticks
(Ixodes dammini, deer tick) recovered from Old Lyme
‹ She lived in a rural area and had evidence of multiple
insect bites

Lyme Borreliosis: Lyme Borreliosis (Lyme Disease) and Borrelia

Transmission to Humans burgdorferi

‹ Ticks feed twice in life cycle

 First feed is on small rodents
 Ticks grow from nymphs to adults
Usual host is white-tail deer; humans are accidental hosts
 As tick feeds, it regurgitates into the tissue; Borrelia from the
midgut are then directly placed into the host tissue

Transmission electron micrograph of Borrelia

burgdorferi between midgut epithelial cells of
Ixodes dammini

Lyme Borreliosis (Lyme Disease) and Borrelia Lyme Borreliosis (Lyme Disease) and Borrelia
burgdorferi burgdorferi
‹ Early stage Annular lesion
 Red papule at site of tick bite associated with Lyme
borreliosis
 Papule extends to form concentric rings (annular rings)
called erythema migrans
 Patient may have flu-like symptoms due to spirochetema
‹ Late stage
 Relapsing (migratory) arthritis
 Symptoms most likely caused by overactive immune
response

Patient with multiple

erythema migrans lesions

3
Lyme Borreliosis:
Laboratory Diagnosis Case Study

‹ A 6-month-old girl was taken by her parents to an

‹ Direct examination: spirochete is difficult to detect from
emergency room because of irritability and lethargy
peripheral smear; silver-stain preparations of tissues
may show the spirochetes ‹ She had a low-grade fever and a nonerythematous
nodule on the right upper arm
‹ Culture:specimens such as blood, skin, CSF
‹ There was no sign of rash or lymphadenopathy
‹ Media: modified Kelly
‹ Serodiagnosis: IFA, ELISA

Pasteurella multocida:
Case Study, cont’d Pasteurellosis in Humans

‹ There were two shallow abrasions, possibly from the

pet cat, near the nodule ‹ Clinical manifestations
‹ Laboratory findings showed leukocytosis with left-shift  Local infection after bite or scratch
‹ CSF WBC was 327/µL, glucose was 48 mg/dL, and  Respiratory tract infection
protein was 70 mg/dL  Life-threatening systemic diseases of meningitis or
bacteremia

Pasteurellosis in Humans: Pasteurellosis in Humans:

Laboratory Diagnosis Laboratory Diagnosis

Microscopic examination:
‹ Material from wounds very small gram-negative
‹ Blood rods; bipolar staining with
Giemsa or methylene blue;
‹ Respiratory tract specimens “safety-pin” appearance
‹ Pleural or cerebrospinal fluid

Grams stain of Pasteurella multocida

culture (left) and P. multocida from
mouse heart blood showing
encapsulated gram-negative bacilli

4
Pasteurellosis in Humans: Laboratory
Diagnosis Zoonotic Infections Transmitted by Direct
Contact or Inhalation
‹ Culture characteristics
 Growth on 5% blood
or chocolate shows
small, smooth, convex
colonies
 “Musty” odor
 No growth on
MacConkey agar;
oxidase-positive
Pasteurella multocida colonies
on sheep’s blood agar

Case Study Case Study, cont’d

‹ A 57-year-old male patient, an electrician, felt ‹ He was diagnosed as having streptococcal cellulitis
feverish and had collapsed at home when he tried to ‹ In spite of antimicrobial therapy, the patient worsened
stand ‹ It was later revealed that he had been working for a
‹ He reported being bitten by an insect on the upper leather firm and had not worn his shirt while at work
left chest while at work the previous day
‹ The patient was hypotensive, febrile, and showed a
necrotic lesion with edema at the site of the insect
bite

Anthrax: Epidemiology Anthrax: Clinical Manifestations

‹ Cutaneous anthrax begins 2 to 5 days after
‹ Primarily a disease of herbivorous animals such inoculation of spores (95%)
as sheep, cattle, goats, and horses  Lesion starts as an erythematous papule that progresses
‹ Humans acquire the infection accidentally in into an ulcerative black eschar or “malignant pustule”
agricultural or industrial setting ‹ Pulmonary anthrax (rare) is acquired by inhalation
 During processing of hides or animal hair. of spores
 Gains access through cuts or inhalation
‹ Malaise, mild fever, nonproductive cough
follows
‹ Gastrointestinal (very rare)

5
Anthrax: Laboratory Diagnosis Anthrax: Laboratory Diagnosis
‹ Colony morphology
‹ Microscopic  Nonhemolytic, white to
morphology gray on sheep’s blood
 Large gram-positive agar
bacilli in short chains  "Medusa head”
appearance

Methylene-blue–stained Impression colony of Bacillus

preparation from peripheral blood anthracis stained with methylene
collected from a cow that was dying blue showing the “medusa-head”
of anthrax appearance of the colony

Case Study Case Study, cont’d

‹ A 63-year-old male noticed swelling and localized ‹ Five days after admission, the patient developed
pain on his left thumb 5 days after a cat bite shortness of breath and pneumonic infiltrates, and
‹ He continued to experience pain, general malaise, later axillary lymphadenopathies were detected
fever, and vomiting in spite of antimicrobial intake ‹ Wound culture grew what was later identified as
and was admitted to the hospital Francisella tularensis
‹ The physician incised and drained what he suspected
as an abscess on his thumb and hand but no
abscess was found

Tularemia: Disease of Wild Animals Tularemia:Clinical Manifestations

‹ Epizootic outbreak of plaque-like ground squirrel ‹ Infection follows after contact with the skin or mucous
disease is 1911 membrane.
‹ Organism named Bacillus tularense after Tulare ‹ Incubation period: 3 to 10 days; symptoms are fever,
County, CA, where it was discovered chills, and malaise
‹ Humans become infected by direct contact with an ‹ Ulceroglandular
infected animal (rabbit) or through the bite of a ‹ Oropharyngeal
vector (tick or deer fly). ‹ Oculoglandular
‹ In 1959, the genus name was changed to ‹ Pleuropulmonary
Francisella in honor of Dr. Francis, who first isolated
the organism ‹ Typhoidal

6
Tularemia: Laboratory Diagnosis Tularemia: Laboratory Diagnosis
‹ Culture and biochemical
testing is DANGEROUS ‹ Culture requirements
‹ Microscopic morphology  Blood-cysteine-glucose (BCG) agar
 Save portion (-30° C to -70° C) for direct FA
 Gram-negative,
pleomorphic, cocco-bacilli  Colony formation may take up to 10 days; Slants retained
for 3 weeks, examined daily
 Shows bipolar staining

Francisella tularensis seen in

tissue specimens (top), infected
liver (bottom); Grams stain from
broth culture

Tularemia: Laboratory Diagnosis Brucellosis: Infections in Humans

‹ Synonymous terms
 Undulant fever, Malta fever, Gibraltar fever,
‹ Culture Mediterranean fever
 Growth in 2 to 5 days on chocolate agar; minute,
 In animals: contagious abortion, abortus fever, infectious
transparent, drop-like colonies.
abortion, epizootic abortion
 Chocolate: lush, gray to white, butyrous growth
 Bang’s disease
 No growth on heart infusion agar or TSA
 Ram epididymitis
 Slight growth/no growth on BAP
 Greening under the colonies on BCG media

Brucellosis:
Brucellosis: Infections in Humans Pathogenesis of Infections
‹ Four species that are pathogenic in humans
‹ Ingestion of animal products (cheese and raw milk of
 Brucella melitensis (goats)
goats most common); Raw vegetables contaminated
 Brucella abortus (cattle)
with animal excreta also a possible route
 Brucella canis (canines)
 Brucella suis (swine) ‹ Direct contact with contaminated tissues
‹ Inhalation of airborne agents; especially likely in
laboratories from centrifuge use.

7
Brucellosis: Pathogenesis of Infection Brucellosis: Laboratory Diagnosis
‹ Incubation
 1 week to several months; septicemic, insidious onset with ‹ Specimens may include:
irregular (undulant) fever  Blood cultures
‹ Symptoms  Bone marrow biopsies
 Lymph node aspirates
 Malaise, chills, fever 7 to 12 days after infection
 Cerebrospinal fluid
 Chills, sweats, insomnia, anorexia, headache, arthralgia,
marked effect on nervous system..  Abscess apirates
 Organisms are carried within the polymorphonuclear cells
 Multiply in macrophages in the RES
 Infection progresses with formation of granulomas and organ
abscesses

Brucellosis: Laboratory Diagnosis Brucellosis: Laboratory Diagnosis

‹ Microscopic
examination Dye Inhibition Test to Identify Brucella species
 Minute gram-negative
coccobacilli
‹ Colonial morphology
 Non hemolytic,
translucent, and
opalescent

Culture of B. melitensis on sheep’s

blood agar after 48 hours
Basic Fuchsin Thionin

B. melitensis (top), B. abortus (middle); B. suis (bottom)

Relapsing Fever: Borrelia recurrentis

Borreliosis and Leptospirosis
‹ Endemic relapsing fever;
worldwide distribution
 Transmitted to humans by
ticks during recreational
activities
‹ Epidemic relapsing fever
 Human: - spread by contact
with body louse
‹ Large spirochete with irregular
spirals Borrelia recurrentis in
 Mutability is characteristic blood smear stained with
antigenic property Giemsa/Wright

8
Relapsing Fever: Pathogenesis Leptospirosis: Weil’s Disease
‹ Usually transmitted to
‹ Incubation period is 2 to 15 days (mean: 7 days) humans through water
‹ Symptoms include high fever, severe headache, contaminated with
muscle pains and weakness animal urine
‹ Febrile period lasts for a week and relapses days or ‹ Morphology: slim
weeks later spirochete with fine,
closely wound spirals
and hooked ends Scanning electron micrograph
of Leptospira interrogans
isolated from the blood of a
patient

Leptospirosis in Humans Leptospirosis: Laboratory Diagnosis

‹ Infection results from contact with water
‹ Detection is accomplished by dark-field or
contaminated with the urine of zoonotic reservoirs
immunofluorescent microscopy
(cattle, dogs, rats)
‹ Blood or CSF maybe cultured during the first 10 days
‹ Subclinical disease develops after the organism
of illness (acute phase)
gains entrance through broken skin, conjunctiva, or
ingestion ‹ Urine must be cultured after the first week
‹ Incubation period: 7 to 13 days, febrile and muscle ‹ Fletcher semisolid medium is used
pain
‹ The most severe form is Weil’s disease; which has a
10% mortality rate

Points to Remember
‹ Clinical presentations of the infection
‹ Epidemiology of the infection
‹ Modes of transmission of the organisms involved
and sources of infection
‹ Clinical, social, and occupational history of the
patient
‹ Methods to recover and identify the possible agents
of infection
‹ Characteristic features of the isolated agent