Yersinia

GENERAL CHARACTERISTICS
 Small coccobacilli
 Pinpoint colonies on MAC
 All are non-motile @ 37deg C
 All are motile at RT except Yersinia pestis

Yersinia is suspected when TSI rxn is yellow over orange (weak acid productionn in the
slant w/ no change in butt)

3 important human pathogens:

 Yersinia pestis - BIOTERRORISM AGENT
 Yersinia enterocolitica
 Yersinia pseudotuberculosis

Spectrum of Disease

Yersinia Yersinia Yersinia enterocolitica

pestis pseudotuberculosis

Enterocolitis
Disease Bubonic plague Lymphadenitis
Appendicitis-like syndrome
Pneumonic plague
Septicemia

Mode of Ingestion of Ingestion of contaminated

Bite of rat flea contaminated food meat
transmission
and water

Bubonic Plague
– high fever and swelling of axilla and groin lymph nodes (buboes); rapidly
progresses to bacteremia that is often fatal

Pneumonic Plague
– malaise; resp infection results from bacteremia; rapidly fatal

Lab Dx of Yersinia pestis

Direct Detection Method

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Gram stain: coccobacilli or straight rods w/ rounded ends
Methylene blue or Wayson stain
- Y. pestis appears as a “closed safety pin”; rapid dx of plague

Wright’s stain: dark-stained, bipolar ends

Cultivation
on BAP, after 48-72 hours, grey-white to slightly yellow opaque raised, irregular “fried
egg” morphology; alternatively colonies may have a “hammered copper” shiny surface. Y.
pestis are best incubated at 25-30 deg C

Yersinia pestis
– rough, cauliflower appearance; in broth: “stalactite patterns” in w/c clumps of
cells adhere to one side

CIN Agar (Cefsulodin-Irgasan-Novobiocin)

Purpose:
 Selective for Yersinia spp esp. Yersinia enterocolitica; may be also useful for
the isolation of Aeromonas spp

Peptone base w/ yeast extract, mannitol and bile salts. Supplemented w/ the 3 antibiotics
(CIN); neutral red and crystal violet (indicators)

 CIN Agar – w/o mannitol

 YSA – CIN w/ mannitol
 CIN II Agar – modified CIN, can also isolate Aeromonas spp.

 “bull’s eye colonies” dark red or burgundy centers w/ translucent border (Yersinia
enterocolitica); seen after 48 hrs of incubation

Aeromonas will produce similar colonies to Y. enterolitica, to differentiate

perform oxidase test:
 Aeromonas (+) oxidase

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  Yersinia (-) oxidase

Speciation of Yersinia

REACTION Yersinia Yersinia Yersinia enterocolitica

pseudotuberculosis
pestis

ONPG + + +

Motility @ 37degC - - -

Motility @ 25deg C - + +

Fermentation of:
Sucrose - - +

Rhamnose - + -

Sorbitol - - +

Merthyl red + + +

Indole - - delayed

Christensen urea - + +

PAD - - -

ODC - - +

KLEBSIELLEAE
 Klebsiella
 Enterobacter
 Pantoea
 Serratia
 Cronobacter
 Hafnia

General Characteristics of Tribe

 The tribe Klebsielleae includes 6 genera; all members share similar biochemical
characteristics
 All members of this tribe:

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 1. Are Voges-Proskauer (VP) positive – all produce acetoin as end pdt in their
glucose metabolism
2. Produce large amounts of gas in their TSI deep portion

1.KLEBSIELLA

Important Characteristics:
1. TSI Rxn: AA+-
2. Most grow on Simmon’s citrate and in KCN broth
3. H2S negative
4. A few can hydrolyze urea
5. Negative MR, Positive VP
6. W / few exceptions, indole is not produced from tryptophan
7. Motility is variable

 90% of Klebsiella isolates belong to Klebsiella pneumoniae, the remaining

isolates are:
– K. pneumoniae subsp. ozaenae
– K. pneumoniae subsp. rhinoscleromatis
– K. oxytoca
– K. ornitholytica
– K. planticola
– K. terrigena

– Klebsiella pneumoniae

 aka “Friedlander’s bacillus” because it is encapsulated and appears as

mucoid colonies that tend to “string”.

 Carrier rate is high in health care settings

 Can cause:

 Severe pneumonia, neonatal meningitis

 Infections of the GIT, UTIs, wound infections

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  Most infections are nosocomial since org can colonize the hands, bowel and other
body sites of hospital personnel; isolated from contaminated medications, resp
care equipment and IV solns

 Pneumonia caused is very necrotic and hemorrhagic (“currant jelly-like”

sputum)

 Capsule enables the organism to resist phagocytosis; resistant to many

antibiotics including ampicillin and clindamycin

 At risk individuals are diabetics, alcoholics and infants

Other Klebsiella spp.

 Klebsiella oxytoca has been isolated from blood and stool cultures

 Indole positive- this differentiates it from other Klebsiella

 K. subsp. rhinoscleromatis causes granuloma (rhinoscleroma) of the nose

and oropharynx

2. K. ornitholytica – indole and ODC positive

3. K. planticola – isolated from urine, RT, and blood in humans

Biochemical reactions of Klebsiella

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2. ENTEROBACTER
– motile rods and ODC (ornithine decarboxylase) positive
– 12 spp. exist and are found widely in nature and dairy products
– Most infections caused by these genera are opportunistic: UTI, resp and wound
infections
– 2 most common isolates: E. cloacae and E. aerogenes

Enterobacter cloacae
- predominant isolate and associated w/ bacteremia, RT, UT and wound infections in
BURN PATIENTS

Enterobacter cancerogenus
- (formerly E. taylorae) unique in the genus since it is LACTOSE NEGATIVE BUT
ONPG POSITIVE

Enterobacter gergoviae
- resembles E. aerogenes but can be differentiated from it by its STRONG UREASE
ACTIVITY

Biochemical Reactions of Enterobacter

Cronobacter sakazakii - yellow pigment of colonies

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 - biochemically resembles E. cloacae but produces a YELLOW PIGMENT W/C
INTENSIFIES @ 25DEG C; assoc w/ neonatal sepsis and meningitis

SPECIATION OF ENTEROBACTER

REACTION E. aerogenes E. E. gergoviae E. C.

cloacae cancerogenus sakazakii

LDC + _ + - _
ADH - + + + +
ODC + + - + -
Fermentation of:
Lactose + + V - +
Sucrose + + + - +
Sorbitol + + - - -
Adonitol + V - - -
Urease - V + - -
(strong)
Yellow pigment - - - - +

3. SERRATIA
Opportunists that are unique in their ability to produce the enzymes:
 Deoxyribonuclease (Dnase)
 Lipase
 Gelatinase

Serratia marcescens
– most important species; associated w/ septicemia and pneumonia in
immunosuppressed patients especially those undergoing chemotherapy

 CA of COMMUNITY ACQUIRED ENDOCARDITIS among drug users

 Very destructive and invasive because of its ability to produce proteolytic

enzymes and resistance to antibiotics (cephalotin and colistin)

 Produces a RED pigment when incubated @ RT

Serratia liquefaciens

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 – differentiated from S. marcescens by its ability to ferment arabinose

Serratia rubidaea
– also produces a red pigment

Serratia odorifera
– produces a rancid, potato-like odor

Biochemical Reactions of Serratia

Serratia marcescens – red pigment of colonies

'Serratia marcescens‘ is positive for gelatinase production, as evidenced by the
liquidation of the media. 

4. HAFNIA
Hafnia alvei (formerly Enterobacter alvei) resembles Enterobacter but can be
distinguished by its inability to ferment:
 Lactose
 Sucrose
 Sorbitol
 Raffinose

Citrate negative; can be differentiated from Serratia because it is Dnase and lipase
negative

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 5. PANTOEA
Pantoea agglomerans (formerly Enterobacter agglomerans) has been isolated from
contaminated IV fluids in an outbreak of septicemia in the early 70’s

- Has a YELLOW PIGMENT

Can be identified by its negative reactions with:

 ADH
 LDC
 ODC

SUMMARY OF REACTIONS

PROTEEAE
 Proteus
 Providencia
 Morganella
1. PROTEUS

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 – Found in soil, water, and fecally contaminated objects

– This genus is characterized by its RAPID UREASE ACTIVITY and its typical
SWARMING MOTILITY on BAP.

Proteus mirabilis
– most frequently isolated human pathogen; associated w/ wound infections and
UTIs; cause of septicemia and pneumonia

 Indole negative
 Susceptible to both ampicillin and cephalosporin

Proteus vulgaris
– associated w/ similar infections as P. mirabilis but are often nosocomial and
affect immunosuppressed pxs and those in prolonged antibiotic therapy.

 Indole positive
 Resistant to ampicillin and cephalosporin

Proteus penneri
– resembles P. vulgaris but it is H2S, salicin and indole negative;
– it is the only indole negative Proteus spp
– resistant to chloramphenicol.

Biochemical Reactions of Proteus

DIFFERENTIATION OF PROTEUS Spp

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2. PROVIDENCIA
– Lactose negative, deaminase positive, H2S negative, motile members of Proteeae
– Ferment mannose; they do not swarm on BAP; most are citrate positive

Providencia rettgeri
- the only urease positive Providencia species;
- associated w/ nosocomial infections of UT and skin;
- UTIs caused often involve patients w/ predisposing urological problems;
- skin infections are usually seen in burn pateints

Providencia stuartii
– rare human pathogen, also associated w/ UTIs

3. MORGANELLA
Morganella morganii

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- is the only species w/in this genus; they are negative to the ff reactions:

 Lactose
 Citrate
 H2S
 LDC

- Deaminase positive
- Infections are nosocomial (UTIs and wound infections)
- Infections are also seen in patients in prolonged antibiotic therapy

 Citrobacteriaceae

 Resemble Salmonella but are ONPG positive and LDC negative

 Important spp: Citrobacter freundii and Citrobacter diversus
 Associated w/ septicemia, wound infections and gastroenteritis, meningitis

Diffferentiation of Citrobacter Species

 Edwardsiella

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 – Has been isolated from the environment; and from many cold- and warm-
blooded animals (reptiles, fish, frog and turtles)

Edwardsiella tarda
- most imptortant human spp in this genus; identified as the cause of GI infection
mainly in the tropics and sub-tropics; wound infections and abscesses, frequently
w/ trauma or accidents involving aquatic environment

 Identified by the abundance of H2S in TSI

 Biochemically resembles E. coli except that it is H2S (+) and lactose (-)

Biochemical Reactions of Edwardsiella

Klebsiella granulomatis
- Formerly known as Calymmatobacterium granulomatis

- CA of a sexually transmitted infection called DONOVANOSIS/ GRANULOMA

INGUINALE

- Scraping from genital lesions can be stained w/ Wright’s or Giemsa stain

DONOVAN bodies

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- groups of organism seen w/in mononuclear Abs; orgs stain as blue rods w/
prominent polar granules surrounded by a large, pink capsule

PENILE DONOVANOSIS
- painless “beefy red” ulcers on penis

- K. granulomatis will grow only in human monocytes from biopsy specimens of

genital ulcers w/ Donovanosis

Miscellaneous Gram Negative Bacilli

Vibrio, Aeromonas, Plesiomonas and Chromobacterium

General Characteristics
 Gram (-) bacilli, Oxidase (+), glucose fermenter
 Facultative anaerobes
 “curved” rods in initial Gram stain but are straight and can be highly pleomorphic
 All are halophilic except Vibrio cholerae and Vibrio mimicus

Epidemiology

Water
- brackish or marine water for Vibrio;
- fresh water for Aeromonas, Plesiomonas shigelloides and Chromobacterium
violaceum

 None are part of the normal human flora

 Ingestion of contaminated seafood and water; entry to open wounds and mucosal
surfaces; fecal-oral routes

 Most notable is Vibrio cholerae

VIBRIO

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 Clinically significant Vibrio species:
 Vibrio cholerae (subgroup 01)
 Vibrio parahaemolyticus
 Vibrio vulnifucus
 Vibrio mimicus
 Vibrio alginolyticus
 Vibrio fluvialis and Vibrio furnissi

Morphology and Epidemiology

- Vibrio are gram (-), straight or curved rods, motile with a single polar flagellum
- Facultative anaerobes or aerobic and some prefer increased CO2
- Natural habitants of sea water and all spp except V. cholerae and V. mimicus
require increased NaCl for growth (halophilic )

Antigenic Structure

3 Major Subgroups of Vibrio cholerae share a common O and H antigens

 Vibrio cholerae O1 – associated w/ epidemic cholera; serotypes:

 Ogawa (A, B)
 Inaba (A, C)
 Hikojima (A, B, C)

 Vibrio cholerae O139 – associated w/ epidemic cholera

 Vibrio cholerae non-O1 – strain that phenotypically resembles V. cholerae but

fails to agglutinate in O1 antisera

Epidemic Vibrio cholerae O1 strains occur in 2 biogroups:

 Classic
 El Tor – differs from Classic
- VP positive
- Hemolyzes rbcs
- Susceptible to polymyxin B (50ug)
- Agglutinates chicken rbcs
Pathogenesis of Vibrio cholerae

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  CHOLERA:
- profuse watery diarrhea (“rice water” stool: fluids and mucous flecks)
leading to dehydration, hypotension and often death; can be an epidemic
or pandemic in scope (7 pandemics)

 Non – epidemic diarrhea and, occasionally extraintestinal infections of wounds RT,

UT, and CNS

 Cholera toxin, Zot Toxin, Ace Toxin, O1 and O139 somatic Ags,
hemolysins/cytotoxins, motility, chemotaxis, mucinase and pili

Laboratory Diagnosis
Collection and Transport

 Stool specimens suspected of cholera should be collected in Cary-Blair medium

 Feces is preferable, but rectal swabs are acceptable in acute phase

Direct Detection
 V. cholerae in stool – ELISA or latex agglutination test
 V. cholerae are straight or slightly curved rods; when examined in dark-field, it
exhibits RAPID DARTING or SHOOTING STAR MOTILITY

Cultivation
 TCBS (thiosulfate citrate bile salts) – yellow or green colonies

 Alkaline peptone water – enrichment for stool samples; after inoculation, broth is
incubated for 5-8 hrs at 35 deg C and then subcultured to TCBS

 V. cholerae and V. mimicus are non-halophilic, so to separate them from

halophiles; halophiles are grown on NB with 6% salt

Vibrio Tests
STRING TEST – used to separate Vibrio from Aeromonas and P. shigelloides;
 orgs are emulsified in 0.5% Na desoxycholate w/c lyses the vibrio cells, but not
those of Aeromonas and Plesiomonas.

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  With cell lysis there is release of DNA, w/c can then be pulled up into a string
using an inoculating loop

VIBRIOSTATIC TEST
– using 0/129 (2,4-diamino-6,7-diisopropylpteridine) impreganted disks:
 Vibrios (susceptible)
 Other oxidase positive, glucose fermenters (resistant);

 Serotyping – serogroups 01 and 0139

 Chromobacterium violaceum
 Motile, facultative anaerobe, oxidase (+)
 It is found in soil and water
 Uniqueness: ability to produce violacein (purple pigment)

 Plesiomonas shigelloides
 An important causative agent of gastritis in Japan, in the tropics and
subtropics
 Organism is carried on various cold-blooded animals; found in water & soil
 Infection is acquired through ingestion of contaminated/unwashed foods
 Grows on BAP, MAC and EMB
 Differentiated from Enterobacteriaceae by a (+) oxidase rxn

 Aeromonas
 Aeromonas are oxidase (+), Gram (-) bacilli, most are motile (polar
monotrichous flagella)
 Naturally found in fresh and sea water and are known to cause infection in
cold-blooded animals
 Also found in drains, sink traps, distilled water and tap water
 Can grow in BAP, MaC, EMB, SSA and CIN
 Can be differentiated from Pseudomonas aeruginosa by their ability to
ferment glucose and produce indole

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  Aeromonas hydrophila
 Most common isolate; “water loving”
 Often associated w/ gastrointestinal disease
 Produces a heat-labile enterotoxin and heat stable cytotoxic enterotoxin
 Produces protease, lipase and nuclease

Differentiation of Aeromonas from Plesiomonas

Miscellaneous
Gram Negative Bacilli
 Campylobacter, Helicobacter,
 Legionella, Bordetella, Brucella,
 Pasteurella, Francisella, Streptobacillus

Campylobacter and Helicobacter

 CAMPYLOBACTER

General Characteristics
 Small, curved, motile, gram (-) rods

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  Slow growing and fastidious since it is microaerophilic and capnophilic
 Oxidase (+) and nonfermentative (asaccharolytic)
 Often associated w/ gastroenteritis and diarrhea

Campylobacter jejuni
- Single flagellum; w/ characteristic darting motility

Epidemiology and Pathogenesis

 Microaerobic inhabitants of the GIT of various animals (farm animals, domestic
pets); soil and water
 Campylobacter produce 3 syndromes in humans:
 Febrile systemic disease
 Periodontal disease
 Gastroenteritis
 C. jejuni and C. coli are most often assoc w/ human infections and
transmitted via contaminated food, milk or water (but they do not multiply in
food)
 Reservoir of infection – asymptomatic carriers
 Infection is seen more in temperate climates

Spectrum of Disease
 Gastrointestinal infections
 Self-limiting (no need for antimicrobial therapy)
 Stool characteristically contains segmented neutrophils and rbcs
 Severe form may lead to intestinal bleeding that mimics inflammatory bowel
disease
 Extraintestinal infections
 Meningitis, endocarditis and septic arthritis
 GUILLAIN-BARRE’ SYNDROME – acute demyelination (removal of myelin
sheath) of peripheral nerves

 Virulence factors:
 Cytotoxin, Cytotonic factor and Enterotoxin
 Campylobacter jejuni

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  C. jejuni subspecies jejuni
 C. jejuni subspecies doylei
 Campylobacter coli
 2nd most isolated Campylobacter species
 Campylobacter fetus
 causes infective abortion in cattle and sheep
 Can cause extraintestinal infections in immunocompromised pxs

Laboratory Diagnosis
 Specimens
 Feces, rectal swab, blood
 Shd be transported in Cary-Blair medium if there will be more than 2hrs
delay – processed immediately/or stored @ 4deg C
 Direct Detection/Microscopy
 Char morphology: S- shaped, spiral, seagull-winged, faintly staining gram (-)
 Wet mount (from colonies growing on selective media) – darting motility
 In Gram’s - safranin O is used as final stain

Stool
– Modified Skirrow’s: Columbia blood agar base, 7% horse lysed blood and
antibiotics (vancomycin, trimethroprim and polymyxin B) @ 42deg C under
microaerophilic condition for 72 hrs
– Campy-BAP: Brucella agar base w/ antibiotics (trimethoprim, polymyxin B,
cephalothin, vancomycin and amphotericin B) and 10% sheep blood @ 42deg C
under microaerophilic condition for 72 hrs

Blood
– Septicemia; 2 weeks for growth
– Chocolate blood agar incubated @ 37deg C in a CO2 enriched,
– microaerophilic environment

Incubation condition:
 37deg C; 42-43deg C,

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  microaerophilic, capnophilic environment of 85% nitrogen, 5% oxygen and
10% CO2
AST
 Not routinely performed but are susceptible to
 Macrolides
 Tetracyclines
 Aminoglycosides
 Quinolones
 Erythromycin – drug of choice for severe gastroenteritis

Helicobacter pylori

General Characteristics
 Previous name: Campylobacter pylori
 Differentiated fr C. jejuni by its 4-6 polar flagella and its strong UREASE activity
 Curved (helical), microaerophilic, capnophilic, prefers increased humidity
 Catalase (+), oxidase (+), gram(-) rods resembling C. jejuni
 Colonize human stomachs; can thrive the highly acidic environment of the
stomach
 Strongly associated w/ duodenal and peptic ulcers

Epidemiology
 Primary habitat – human gastric mucosa particularly in mucus secreting cells
 MOT: Oral – oral; Fecal – oral; Vertical
 Has been isolated from feces, dental plaque

Pathogenesis
 Colonizes the antrum & fundus of the stomach but does not invade the epithelium
 Motility allows H. pylori to escape stomach’s acidity and burrow and colonize the
gastric mucosa
 Urease plays a significant role in H. pylori’s growth and survival in the stomach by
creating an alkaline microenvironment

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  Other virulence factors:
– adhesins (colonization);
– mediators of inflammation;
– vacuolating cytotoxin (damages host cells)

Helicobacter pylori causes:

 Gastritis; Peptic ulcer; Gastric cancer

Laboratory Diagnosis

Specimen Collection, Transport and Processing

Specimens: stool, blood, tissue biopsy

Tissue biopsy shd be:
– placed directly in Stuart’s medium (to prevent drying)
– May be refrigerated up to 24hrs before processing
– Minced and gently homogenized

Direct Detection
– Biopsy specimens are stained w/ Warthin-Starry or other silver stain; Giemsa
– Squash preparations of biopsy specimen can be Gram stained
– 0.1% basic fuchsin enhances morphology

Presumptive test
– From biopsy: small portion of the processed tissue is placed directly into urease
broth or prepared kit (+) result.indicates presence of H. pylori

 UREA BREATH TEST

 Patient ingests radioactively labeled urea
 If infection is present – H. pylori breaks down urea to form ammonia and
labeled CO2
 Sensitive, specific, non-invasive test for H. pylori

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Cultivation

H. pylori from tissue biopsy is placed in:

– nonselective agar (Chocolate, Brucella)
– Selective (Skirrow’s, Modified Thayer Martin)

AST & Therapy

– Metronidazole
– H. pylori becomes resistant if metronidazole, clarithromycin, azithromycin, rifampin
or ciprofloxacin is given in a single event, so prescribed regimen is:
– Triple-drug therapy: metronidazole, bismuth-salt, amoxicillin/tetracycline

HAEMOPHILUS

Organisms to be Considered:
 Haemophilus influenzae
 Haemophilus influenzae biogroup aegyptius
 Haemophilus aegyptius
 Haemophilus ducreyi
 Haemophilus haemolyticus
 Haemophilus parainfluenzae

Others
 H. parahaemolyticus
 H. aphrophilus
 H. segnisH. Paraphrophilus

General Characteristics
 Tiny Gram (-) coccobacilli (can be pleomorphic) that are nonmotile and non-
spore forming

 Facultative anaerobes, reduce NO3 and utilize CHOs

 Most spp are oxidase and catalase (+)

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  Incubation requires 5-10% CO2 (capnophilic)

 Very prone to drying and chilling

 All species EXCEPT H. aphrophilus require these for in vitro growth:

 HEMIN or HEMATIN – X factor

 NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD) or COENZYME I – V factor

 Haemophilus means “blood loving”, hence, spp require particular iron containing

growth requirements: hemin and NAD

 Most species are non-pathogenic or opportunistic pathogens except for H.

influenzae and H. ducreyi.

 Many spp are considered as NF of the mucous membranes of the URT and oral cavity

Epidemiology

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Among H. influenzae strains, there are 2 broad categories:

TYPEABLE

– strains are typed based on capsule (w/c is made up of a sugar-alcohol PO4 like

polyribitol PO4)

– Differences in this complex serve as the bases for separating encapsulated strains

into 6 groups: type a, b, c, d, e, or f.

– Type b Haemophilus influenzae is most commonly encountered in serious

infections

NONTYPEABLE

– No capsule formation

– Part of normal flora of URT

Pathogenesis

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H. aphrophilus
- is an uncommon cause of endocarditis and is the H member of HACEK group of
bacteria associated with slowly progressive (subacute) bacterial endocarditis
HACEK

 Haemophilus aphrophilus
 Actinobacillus actinomycetemcomitans
 Cardiobacterium hominis
 Eikenella corrodens
 Kingella spp
New genus of H. aphrophilus and A. actinomycetemcomitans is Aggregatibacter

 Haemophilus influenzae

 Previous name: Pfeiffer’s bacillus

 Leading cause of meningitis among children less than 5y/o
 Has 6 serotypes: H. influenzae a, b, c, d, e and f
 Serotype b – most frequently encountered in infections
 Serotyping is based on capsular Ag through:

 Latex agglutination
 Capsular swelling
 Immunofluorescence
Carried as normal flora of the URT; nonencapsulated form, not virulent while
ENCAPSULATED STRAINS ARE PATHOGENIC AND PRODUCE RAPID, DEVASTATING
DISEASE IN CHILDREN LESS THAN 3Y/O (children do not develop Abs until 2-3yrs of
age)

Spectrum of disease

 Epiglottitis (2-4y/o)

o swollen, red, edematous epiglottitis; death may occur from suffocation

 Other RT infections – acute sinusitis, chronic bronchitis and pneumonia

 Systemic infections: bacteremia, septicemia and endocarditis
 Most of these cases are seen in children and immunosuppressed pxs!

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  Adults who develop H. influenzae meningitis may have a predisposing condition:
 Chronic sinus infection
 Alcoholism
 Head trauma
 Diabetes
 Ear infections
 Heart valve disease

Haemophilus influenzae Biogroup aegyptius

 Non-encapsulated

 CA of Brazilian purpuric fever - (severe infectionn ages 1-4); purulent meningitis,

bacteremia, high fever, vomiting, purpura (rash), & vascular collapse

 Mortality rate may reach as high as 70% within 48 hours after onset

Haemophilus aegyptius

 Previous name: Koch-Weeks bacillus

 Closely resembles H. influenzae biotype III (H. influenzae subspecies aegyptius)

 Causes “pink eye” – very contagious purulent conjunctivitis spread through

sharing of handkerchiefs and towels; occurs in seasonal epidemics esp in warmer
climates

Haemophilus ducreyi

 Infective agent of CHANCROID - Soft chancre venereal disease characterized by

the appearance of a ragged, painful ulcer on the genitalia

 Chancroid is found more frequently in Africa, Asia and the tropics

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  Appears as tiny gram (-) rods (intracellular to polymorphonuclear neutrophils) on
direct examination of the genital ulcer; resembles “school of fish”

 Very fastidious, require special media

H. haemolyticus
- Occasional normal flora of the URT
- Requires both X and V factors
- Shows a wide zone of beta-hemolysis on horse blood agar
- Maybe mistaken for Grp A Streptococcus on BAP; perform gram stain to
differentiate

H. parainfluenzae
- Normal flora of the URT
- Rarely infectious and requires only the V factor

 LABORATORY DIAGNOSIS

Specimen Collection and Transport

Points of Emphasis:
1. Haemophilus species are very susceptible to drying and temp extremes
 must be plated immediately esp if not submitted in a suitable transport
medium (Stuart’s or Amie’s charcoal)
 Recovery of H. ducreyi from genital ulcers require special measures (due
to its fastidious nature)
 Ulcer shd be cleaned w/ sterile gauze premoistened w/ sterile saline
 Cotton swab for collection shd be premoistened w/ PO4-buffered saline
(collection shd be at the base of the ulcer)
 Swab must be plated to special selective medium w/in 10 mins of collection

Specimen type for H. influenzae is dictated by the type of infection. Possible sites include:
o Blood o Infected joint fluid
o CSF o Nasopharyngeal swabs
o Middle ear aspirate o Eye swabs

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Isolation/Cultivation
 Sheep BAP IS NOT adequate to isolate Haemophilus spp because of the
requirement of NAD. Sheep rbcs release NADase w/c inactivates NAD.

 Horse or rabbit BAP can be used since neither contains NADase (but these
media are not usually maintained in the lab); preferred isolating medium then is
CHOCOLATE AGAR

 Chocolate agar

 Prepared by heating sheep rbcs to at least 8Odeg C or using enzyme treatment

 Either method will release hematin (X factor) and inactivate NADase
 Synthetic CA contains NAD, Fe, VitB12, thiamin, Mg, glucose, cysteine and glutamine

 Other methods for isolation

1. Staph streak
 culture suspected w/ Haemophilus is streaked heavily onto a sheep BAP
 A single streak of beta-hemolytic S. aureus is streaked through the inoculum
 Requirements are met since S. aureus synthesize NAD and hemolysis
releases hemin; X and V factors are present
 Haemophilus spp appear as tiny “satellite” colonies around Staphylococcus
aureus after incubation (satelliting phenomenon)

2. Horse blood-bacitracin agar

 Contains:
1. beef heart infusion, peptone, yeast, 5%
2. defibrinated horse blood (provides X and V) and
3. 300mg/L bacitracin (inhibits NF ; prevents overgrowth of H.
influenzae by mucoid P. aeruginosa)
 Hemolytic reaction can also be observed on media
 Especially used when specimen is resp secretions of px w/ cystic fibrosis.

3. Levinthal agar
 Clear medium containing X and V factors and growth factors

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  Encapsulated colonies of H. influenzae appear iridescent when viewed w/ an
oblique light
 Nonencapsulated colonies appear transparent, bluish and noniridescent

 H. ducreyi – needs special media:

1. Mueller-Hinton based chocolate agar supplemented w/ 1% IsoVitaleX and
3ug/ml vancomycin
2. Heart-infusion based agar supplemented w/ 10% fetal bovine serum and
3ug/ml vancomycin

 Identification
1. Staphylococcus streak
2. ALA
3. X and V Factors
4. Haemophilus ID Quad Plates

DELTA-AMINOLEVULINIC ACID TEST (ALA porphyrin test)

- Used to determine the requirement for X factor
- In this procedure, the synthesis of protoporphyrin from the substrate ALA is
determined.

RESULT:

 ALA positive : H. parainfluenzae since it does not require exogenous X factor; it

has enzymes to convert ALA to porphobilinogen and porphyrin to hemin

 ALA negative: H. influenzae since it requires exogenous X factor for growth

X AND V FACTORS
 Purpose: To speciate Haemophilus based on their specific growth requirements
for X and V factors
Media/Reagents: Mueller-Hinton Agar (MHA); BHI/TSB; X and V factors filter paper disks
 Filter paper w/ X factor and filter paper w/ V factor are placed on MHA

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  RESULT and INTERPRETATION:
 Growth about X factor and XV – X factor is required
 Growth about V factor and XV – V factor is required
 Growth about XV – both X and V factors are required

Haemophilus ID Quad Plates

 Purpose: determine the hemolytic properties and requirements for X and V
factors for particular Haemophilus species.

 Principle: the plate consists of 4 quadrants

 Quadrant I – BHI agar and X
 Quadrant II – BHI and Isovitale (V)
 Quadrant III – BHIA with X and V
 Quadrant IV – Horse blood agar (X) and NAD (V)

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 Interpretation:
 Interpret for visible growth and hemolysis
 Organism should grow in Quadrants III and IV
 Read Quadrant IV for hemolysis

Quadrant I – X
Quadrant II – V
Quadrant III – X and V
Quadrant IV – X and V and hemolysis

Direct Detection Methods

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Gram Stain
 Acridine orange stain (AO) is used to detect smaller numbers of organisms
 To increase sensitivity of GS esp of CSF, specimens are centrifuged (2000 rpm for
10 mins); fivefold to tenfold increased GS sensitivity; or cytocentrifugation
100 fold increased GS sensitivity

Appearance on Gram Stain

 H spp – pale pink
 H. influenzae – coccobacilli or small rods
 H. influenzae biotype aegyptius – long slender rods
 H. haemolyticus – small coccobacilli or short rods w/ occasional cells
appearing as tangled filaments
 H. parainfluenzae – small pleomorphic rods or as long filaments
 H. ducreyi – “school of fish” appearance

Incubation
 Can grow aerobically or anaerobically
 Growth is stimulated by 5% - 10% CO2 so that incubation in a candle extinction jar,
CO2 pouch, or CO2 incubator is recommended
 H. ducreyi may require up to 7 days for growth
 Optimal growth except for H. ducreyi is 35-37 deg C.H ducreyi at 33 deg C plus
high humidity (accomplished by placing a sterile gauze moistened w/ sterile water
inside candle jar or CO2 pouch)

AST
 H. influenzae – ampicillin (drug of choice); but if w/ resistance, give
chloramphenicol; ceftriaxone/cefotaxime
 H. ducreyi – erythromycin
 Other H spp - ceftriaxone/cefotaxime

Prevention
 Vaccine for H. influenzae type B – mutiple –dose CHON-polysaccharide
 Rifampicin as prophylaxis for H. influenzae type b meningitis
 PRP (purified polyribosyl ribitol PO4) – 1st vaccine for H. influenzae, 1985.

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