Biosafety Levels

• 1 – standard teaching labs – Micrococcus, Lactobacillus,

Saccharomyces
• 2 – moderate potential to infect – Staph, enterics, Corynebacterium,
helminths, Hepa A, B, rabies , Cryptococcus, Blastomyces
• 3 – severe/lethal if inhaled – [Link], F. tularensis, Y. pestis, Brucella, C.
burnetti, C. immitis, yellow fever, WEE, AIDS
• 4 – highly virulent when inhaled – Flavi, Arena incl. Lassa, Filo incl.
Ebola and Marburg
Gram Negative Bacilli/Coccobacilli
(Aerobic)
• MacConkey Agar Growth
• Oxidase (-): Enterobacteriaceae, Acinetobacter, Stenotrophomonas,
• Oxidase (+): Pseudomonas, Burkholderia, Achromobacter, Rhizobium,
Ochrobactrum, Chyseobacterium, Sphingobacterium, Alcaligenes, Bordetella
(non pertussis), Comamonas, Vibrio, Aeromonas, Plesiomonas,
Chromobacterium
• McConkey Agar No Growth
• Growth Require Special Media
NLF on MAC; Nonfermentative but
oxidative
gram negative bacilli
• MAC: colorless, NLF
• Motile with peritrichous flagella, obligate aerobe, non fermentative
but oxidative
• Opportunistic, nosocomial
• Achromobacter denitrificans
• Nitrate reduction positive; in those w/ cystic fibrosis, small convex glistening
colonies
• Alcaligenes faecalis
• nitrate reduction negative; alpha hemolytic w/ feathery edge;
apple/strawberry-like odor
MAC (+), oxidase (+),
glucose fermenters,
aerobic / facultative anaerobic
• Family Vibrionaceae
• Vibrio cholerae, parahemolyticus, vulnificus, alginolyticus
• Plesiomonas shigelloides
• Family Aeromonadaceae
• Aeromonas caviae
• Aeromonas hydrophila
MAC (+) Nonsaccharolytic
(nonoxidative and nonfermentative)
• Oligella urethralis (Moraxella urethralis) – negative for urea hydrolysis
and negative for nitrae reduc
• Oligella ureolytica – urea hydrolysis; nitrate reducer
Pigmented on MAC; non fermentative
• Chryseobacterium meningosepticum
Gram (-) Rod, MAC (-), nonsaccharolytic
• Eikenella corrodens
• Normal flora of the mouth, upper respiratory tract, Git
• Corroding bacterium on BAP
• Bleach like odor
• Human bite infections, head and neck infections, SBE
Gram Negative Bacilli/Coccobacilli
(Aerobic)
• McConkey Agar Growth
• McConkey Agar No Growth
• Oxidase variable : Haemophilus
• Oxidase + : Sphingomonas paucimobilis, Moraxella, Neisseria elongata,
Eikenella corrodens, Weeksella virosa, Pasturella, Suttonella, Mannheimia
haemolytica, Actinobacillus, Kingella, Cardiobacterium, Capnocytophaga
• Growth Require Special Media
• Bartonella, Afipia, Campylobacter, Arcobacter, Helicobacter, Legionella,
Brucella, Bordetella pertussis, Bordetella parapertussis, Franciscella,
Streptobacillus moniliformis, Sprillum minus
Family Pasteurellaceae
• Genus Pasturella
• P. multocida
• Genus Haemophilus
• H. influenzae
• H. influenzae biogroup aegyptius
• H. ducreyi
• H. paraphrophilus
• H. parainfluenzae
• H. parahemolyticus
• Genus Actinobacillus
• A. actinomycetomcomitans
Fastidious Gram Negative Bacilli
• HACEK group
• Hemophilus esp. H. aphrophilus (A. aphrophilus) and H . paraphrophilus
• Aggregatibacter
• Cardiobacterium
• Eikenella corrodens
• Kingella sp.
• Capnocytophaga
• Pasturella
• Brucella
• Francisella
Gram Negative Bacilli
• Non Fastidious • Fastidious
• Fermenters 63 % • Hemophilic 13 %
• Enterobacteriaceae • Hemophilus
• Nonfermenters 20.3 % • Fermenters & Nonfermenters 1%
• Pseuomonas sp. • H. aphrophilus
• Acinetobacter sp. • E. corrodens
• Capnocytophaga
• Anaerobes 3%
• Bacteroides
• Fusobacterium
• Prevotella
Family Pasteurellaceae
• Genus Pasturella Respiratory tract of
• P. multocida humans and animals
• Genus Haemophilus
• H. influenza - pathogenic STD - chancre
• H. influenzae biogroup aegyptius - pathogenic
• H. ducreyi - pathogenic
• H. aphrophilus  Aggregatibacter
• H. paraphrophilus  Aggregatibacter
• H. segnis  Aggregativacter
• H. parainfluenzae
• H. hemolyticus
• H. parahemolyticus
• H. paraphrohemolyticus
• Genus Actinobacillus
• A. actinomycetomcomitans
HACEK group
• Hemophilus esp. H. aphrophilus (now Aggreatibacter aphrophilus)
and H . paraphrophilus
• Aggregatibacter
• Cardiobacterium
• Eikenella corrodens
• Kingella sp.
Pasteurellaceae
• Gram negative, pleomorphic, coccoid to rod shaped, non motile,
facultative anaerobes, nitrate reducers, oxidase and catalase positive
• Genus Pasturella
• Genus Hemophillus
Genus Haemophilus
• Most are opportunistic / non pathogenic
• X factor (unknown – hemin or hematin)
• V factor (vitamin – NAD)
• If with prefix “para” – needs V factor only
• Hemolytic on BAP
• CAP
• Satellitism – V factor production by
pyogenic cocci; X factor from BAP
 Hemophilus
• Hemophilic, pyogenic, capnophilic, pleomorphic, encapsulated
• Needs X and V factors
• Virulence factors:
- Capsular Ag - (major antigenic determinant – confers specificity & basis
for serovar grouping: a to f ; b has pentose – critical determinant of virulence,
essential for invasivenss)
- IgA protease (in H. influenzae only)
- Adherence mechanisms like fimbriae (none in H. influenzae systemic)
- LPS – paralyzes sweeping motion of ciliated respis
- outer membrane protein
Haemophilus
• All need X factor for growth except:
H. parainfluenzae, vaginalis
• All need V factor for growth except:
H. hemoglobinophilus, vaginalis, ducreyi
• All do not grow if with CO2 except:
H. vaginalis, ducreyi
• All are nonhemolytic except:
H. hemolyticus, vaginalis, ducreyi
* Except for H. ducreyi, all clinically significant species require V factor for growth
and display this unusual growth pattern.
Gardnerella vaginalis
• Bacterial vaginosis
• Clue cell
• Pap smear
• Absence of normal flora
• Lactobacillus acidophilus or
Doderlein’s bacillus
• Shift in normal flora
• Clinical significance
Hemophilus
• H. influenzae (Pfeiffer’s bacillus)
• H. aegyptius (Koch Weeks)
• H. ducreyi
• Opportunistic: H. aprophilus, parainfluenzae & parahemolyticus
• Nonpathogenic: H. hemolyticus
• Zoonotic: H. suis, hemoglobinophilus
H. influenzae
• Historically – “flu” due to the pandemic 1889 to 1890
• Coryza, myalgia, headache, bronchitis
• Frequent isolation & post mortem lung cultures
• Considered now as a secondary / opportunistic invader
H. influenzae – Clinical Manifestations
• Encapsulated strains – invasive disease – septicemia, meningitis,
arthritis, epiglottitis, tracheitis, pneumonia
• Nonencapsulated strains – localized – conjunctivitis, sinusitis, otitis
media with effusion; invasive in elderly – bronchitis & pneumonia
- may enter nervous system – meningitis in adults esp.
immunocompromised or debilitated
Haemophilus aegyptius
• Pink eye conjunctivitis - acute, contagious
H. influenzae Biogroup aegyptius
• Nonencapsulated
• Brazilian purpuric fever (BPF)
• Recurrent or concurrent conjunctivitis, high fever, vomiting, petechial /
purpuric rash, septicemia, shock and vascular collapse
H. ducreyi
• Chancroid or soft chancre / genital ulcer disease (GUD)
• 4 to 14 days incubation
• Suppurative (pus forming) enlarged, draining, inguinal lymph nodes
(buboes)
H. parainfluenzae
• Endocarditis
• Chronic insidious; a month after a dental procedure
• Mitral valve – primary site of infecton
H. parahemolyticus
• pharyngitis
Culture Media for Haemophilus
• Levinthal’s – large, opaque, flat
Filde’s – translucent, bluish
Rabbit blood agar – pinhead
CAP at 33C and 37C – colorless, transparent, dew drop, moist,
mousy odor ; 5 to 10 % CO2
CAP w/ Bacitracin 300mg/L: to reduce overgrowth of normal resp. flora
Avery’s Oleate Hb agar – inhibits Streptococcus
IsoVitaleX or Vitox
Nairobi biplate medium – GC agar base w/ 2% bovine Hb and 5% cholocalitized
horse blood agar; with vancomycin
Mousy/bleach like odor
• Capsule – halos
• School of fish
• Railroad tracks
• fingerprints
Laboratory Identification
• Growth on CAP, (-) sheep BAP & MAC
• X and V factor requirement
Porphyrin Test
H. influenzae
• Pappenheim’s stain
• Ito Reenstierna test – intradermal vaccine injection
(+) 7 mm or more within 48 hours
Treatment
• Cefotaxime
• Ceftriaxone
• TMP-SMX
• Imipenem
• Ciprofloxacin
• Chloramphenicol
• Increased resistance to: ampicillin & chloramphenicol/ampicillin
Bordetella
• Smooth, glistening, pearl – like pinpoint colonies
• Surface O Ag, Capsular Ag, Pertussis toxin, Hemagglutinins, Adenylate
cyclase, Dermonecrotic toxin, LPS endotoxin, Tracheal cytotoxin (TCT),
pertactin
• B. pertussis – catarrhal, paroxysmal, covalescent
• B. parapertussis – catalase (-)
• B. bronchiseptica – motile, oxidase (+)
HACEK
• Capnophilic
• Latter 4 – more dysgonic (slower or poorer growing)
• Predilection for attachment to heart valves
• Endocarditis
• Risk factors: tooth extraction, history or endocarditis, gingival surgery,
heart valve surgery, MVP
Aggregatibacter aphrophilus
• Aphros – foam loving or needing high CO2 concentration
• Most prevalent HACEK
• A. aphrophilus = H. aphrophilus & H. paraphrophilus
Aggregatibacter actinomycetecomitans
• Formerly under Actinobacillus– animal pathogens - but is urease
negative
• Associated with Actinomyces in a polymicrobic infection
• Periodontitis
• Virulence factors: Collagenase & leukotoxin
• Fermenter
• Star shape with 4 to 6 points
• Penicillin sensitive in vitro
Cardiobacterium
• C. hominis
• Pleomorphic, tear drop appearance
• BAP: small, slightly alpha hemolytic, smooth, round, glistening, opaque, +/-
pitting
• Habitat: normal flora of human upper respiratory tract
• Capnophilic
• Opportunistic endogenous infection
• Dx: endocarditis esp. in those with anatomic heart defects
• Rx: Penicillin
Cardiobacterium hominis
• Typical “rosettes”
• Cardiobacterium hominis and valvarum
• Aortic valve
• Vegetation
• Endocarditis
• Sticklike structures
• Oxidase positive, catalase negative, indole positive
Eikenella corrodens
• Opportunistic, immunocompromised
• Clenched fist wounds, human bites
• Pits or corrodes the agar
• Licking the needle – drug addicts
• Non motile, oxidase positive, asaccharolytic (like Moraxella sp.)
• Catalase negative, yellow pigment
• Adheres to sides of tubes – granules
• Chlorine, bleach – like odor
• Least common of the HACEK in endocarditis
Kingella
• Coccobacilli with short ends; in pairs or short chains
• Fastidious, oxidase positive, catalase negative
• Neisseria & Moraxella are catalase positive
• Tonsils
• May grow in Thayer Martin
• K. kingae - pedia
• K. denitrificans
• K. oralis
Pasturella
• Subspecies: P. multocida, septica, gallicida
• P. multocida – 5 serogrps defined by capsular Ags (A, B, D, E & F)
• Zoonosis
• Cutaneous infection – animal bites (birds and mammals)
• Ulceration – feline bites
• Capsular and somatic antigen; endotoxin
• Catalase weakly (+), usually oxidase (+), indole (+)
• Bipolar staining – safety pin appearance
Brucella
• Small, nonmotile, encapsulated, pleomorphic, obligate intracellular
• Potential for bioterrorism, with long term sequelae
• Outer membrane protein, cell wall
• Brucellosis, Undulant fever, malta fever, Gibraltar fever, Bang’s bacillus
• 3 clinical stages:
• Acute – 8 wks post exposure
• Subchronic/ undulant form
• Chronic – 1 year post exposure
Brucella
• B. melitensis
• B. abortus
• B. suis
• B. anis
• B. ovis
• B. neotomae
Brucella
Animal Thionine Bsc Fchs H2S Urease

[Link] goat + + - +

[Link] cow - + + +

B. canis dog + - - +

B. suis pig + v v +
Brucella
CO2 glucose maltose Mannitol

[Link] + + - -

[Link] + + - +

[Link] - + + +
Brucella
• Biphasic media, Abortus ban ringprobe test, Wisconsin medium, BCYE
Francisella
• F. tularensis (human pathogen – tularemia)& F. philomiragia
• Catalase weakly (+), oxidase (-), pleomorphic, non motile, urease negtive
• Tularemia – zoonotic, rabbit, deerfly and lemming fever, & water rat trapper’s disease
• Ingestion, inhalation, arthropod bite (e.g. ticks, biting flies) or contact with infected tissues
• Ulceroglandular, pneumonic, glandular, oropharyngeal, oculogalndular and typhoidal forms
- Polysaccharide Ag, cell wall, capsule
- 4 subspecies or biovars:
- Subsp. tularensis (type A) – most severe
- Subsp. holarctica (type B) – rarely fatal
- Subsp. mediasiatica – rarely fatal
- Subps. novicida – opportunistic, in immunocompromised
- F. tularensis var. tularemia – rabbit fever
- F. tularensis var. pallartica – rodents / mosquitoes
- Foshay’s antiserum test, BCYE agar
Family Legionellaceae
• 1976 American Legion Convention in Philadelphia
• L. pneumophila
• Motile, flattened rod with pili
• Flagellar antigen,
• Legionellosis:
• L. pneumophila, micdadei, longbeachae, dumoffi, bozemanii
• Dieterle’s silver impregnation test
• Catalase & oxidase (+)
• “ground glass appearance” – central portion of young colonies
• BCYE
• Urine antigen test
Legionella species based on colony
autofluorescence
• Yellow-green
• Blue-white
• Blue-white or yellow-green
• No color
Legionella Virulence Factors
• Ability to enter, survive, and multiply within the host’s cells, esp.
bronchoalveolar macrophages & the ability to produce proteolytic
enzymes
• Intracellular infections in human; survives in extracellular
environment
• Legionnaire’s disease – febrile – w/ pneumonia
• Pontiac fever – febrile w/o pulmonary involvement
• Asymptomatic infection
Legionnaire’s disease – 3 major patterns
• Sporadic cases – most common; occurs in communities
• Epidemic outbreaks – short duration; low attack rates
• Nosocomial clusters – compromised patient populations
• Pneumonia causes:
• S. pneumoniae
• M. pneumoniae
• C. pneumoniae
• L. pneumophila – atypical pneumonia
Pontiac fever
• Nonpneumonic form
• 2 days incubation period
• L. pneumophila
Bordetella
• Catalase positive
• Non fastidious:
• Oxidase negative: B. holmesii, B. parapertussis, B. trematum
• Oxidase positive: B. avium, B. bronchiseptica, B. hinzii
B. pertussis and B. parapertussis
• Virulence factors
• FHA – filamentous hemagglutinin & pertactin – facilitates attachment
to ciliated epithelial cells
• PT – pertussis toxin – interferes with signal transduction
• Adenylate cyclase toxin
• Tracheal cytotoxin - ciliostasis
Whooping Cough / Pertussis
• 1 to 3 weeks incubation
• Catarrhal stage – insidious / non specific; most communicable
• Paroxysmal stage – characteristic “whoop”
• Convalescent phase – 4 weeks of onset with a decrease in frequency
and severith
Bordetella
• Bordet Gengou – potato blood agar – glistening
• Regan Lowe – modified Bordet Gengou w/ horse blood
• Stainer Sholte – with cyclodextrin & cephalexin
Capnocytophaga
• C. gingivalis
• C. ochracea
• Fusiform bacilli with one rounded end and one tapered end ; occ.
Filamentous
• BAP: opaque, shiny nonhemolytic beige to yellow colonies and exhibit gliding
motility similar to swarming of Proteus
• Habitat: normal flora of human oral cavity
• Dx: periodontitis, bacteremia
Family Campylobacteraceae
• Campylobacter – formerly classified with the vibrios due to + oxidase
and microscopic appearance; darting motility
• Vibrios are fermentative; most Campylobacters are asaccharolytic.
• DNA homology
• Growth at 42C – Campylobacter, Pseudomonas, Aeromonas
• Campylobacter – single polar flagellum
• Helicobacter – multiple flagella at one pole
• Arcobacter
Campylobacter
• Sea gull shaped
• C. jejuni
• C. fetus
• C. pylori  H. pylori
• Campy BA, Butzler’s medium, Skirrow’s medium, CCDA
Campylobacter
Curved gram(-) rods, S-shaped arrangements
• “Seagull-wing”
• Oxidase(+)
• Catalase(+)
• Assacharolytic
• Darting motility
• Microaerophilic
Campylobacter
• Campylobacter jejuni
- most common cause of bacterial gastroenteritis
- associated with chickens and dogs
- grows at 42oC

• Campylobacter fetus
- bacteremia
- inhibited at 42oC; incubated at 37C
Guillain-Barre Syndrome
• Campylobacter related
• Autoimmune disorder; acute paralysis due to damage to PNS damage
• Abs produced during a Campylobacter infection bind to gangliosides
found on peripheral nerve cells
Helicobacter
• Helicobacter pylori
• Urea breath test
• Urea degraded by the urease activity of H. pyori releases CO2 which is
absorbed into the bloodstream and detected in the exhaled breath by a
scintillation counter
• Histopath – Giemsa, Brown and Brenn
• Fecal Ag detection test
• Christensen’s urea medium incubated at 37C x 2 hours
• Rapid color change
Chronic Gastritis
• Type A - autoimmune, fundic, with increase risk for vitamin
deficiencies, pernicious anemia and cancer
• Type B, the most common type, (environmental), antral is caused
by Helicobacter pylori, can cause stomach ulcers, intestinal ulcers,
and cancer.
• Type C - due to chemical irritants like NSAIDS, ROH or bile,  stomach
lining erosion and bleeding.
Helicobacter pylori
• Gastritis
• Peptic Ulcers
• Gastric carcinoma

Lab ID
- oxidase(+)
- catalase(+)
- strong urease activity

- urea breath test

Misc. Gram Negative Bacilli
• Bartonella bacilliformis – Carrion’ s disease:
Oroya fever (anemia), Veruga peruana (skin)
• Calymatobacterium granulomatis / Granuloma inguinale
Donovan bodies
• Gardnerella vaginalis
• Streptobacillus moniliformis – rat bite/haverhill fever; L
• Spirillum minor / minus - sodoku
• Bartonella / Rochalimea henselae – cat scratch
• Alfipia felis – cat scratch