Haemophilus Species

Overview
Haemophilus
influenzau
Epidemiology
Clinical manifestation
Diagnosis and treatment
Case study

Haemophilus ducreyi
Epidemiology
Clinical manifestation
Diagnosis and treatment
Case study

Haemophilus

aegypti
Epidemiology
Clinical

manifestation
Diagnosis and
treatment
Case study

Haemophilus species
Haemophilus = blood loving
Require either heme (X factor) or NAD (V
factor)
Haemophilus is facultative and can grow
anaerobically
Organism is sensitive to drying and extremes
in temperature
Distinctive mousy or bleach-like odor

HAEMOPHILUS
SPECIES:
IDENTIFICATION

Quad plates
Contain X and V
factors & sheep
blood
agar

HAEMOPHILUS SPECIES: IDENTIFICATION

V=variable

Haemophilus influenzae

Haemophilus influenzae
Bacteria, mainly causes illness in babies and young
children; can cause infections in people of all ages ranging
from mild, such as an ear infection, to severe, such as a
bloodstream infection.
H. influenzaedo not cause influenza (the "flu").
Six identifiable types ofH. influenzaebacteria (a through f)
and other non-identifiable types (called nontypeable).
People are most familiar with isH. influenzaetype b, or Hib,
that can be prevented with a vaccine. However, the vaccine
does not protect against other types of the bacteria.

When the bacteria invade parts of the body that are normally free
from germs, like spinal fluid or blood - "invasive disease." Invasive
disease - usually severe and can sometimes result in death.
Most common types of invasive disease caused byH. influenzaeare:
Pneumonia* (lung infection)
Bacteremia (blood infection)
Meningitis (infection of the covering of the brain and spinal cord)
Epiglotittis (swelling of the windpipe that can cause breathing
trouble)
Cellulitis (skin infection)
Infectious arthritis (inflammation of the joint)
H. influenzaecan also be a common cause of ear infections in children
and bronchitis in adults

 H. influenzae(a through f) and other nonidentifiable types (called nontypeable)

Usually asymptomatic in areas like nose and
throat
Infectious if moved to other parts of the body and
possibly be invasive
Incubation period ofH. influenzaedisease is not
certain, but could be as short as a few days.

Most of the time,H. influenzae are asymptomatic and are

spread via:
Respiratory droplets
coughing and sneezing

How It Spreads

Lengthy contact with an infected person can transmit the

disease; it
can be prevented via:
Antibiotic prophylaxis
the prevention of infection complications using
antimicrobialtherapy

 Babies and children younger than five years old.

Adults 65 years or older
American Indians, and Alaska Natives are also at increased risk for
getting sick with invasiveH. influenzaedisease.

People at Increased Risk

People with certain medical conditions are also at increased risk for developing
H. influenzaedisease. Those medical conditions include:

Sickle cell disease

Asplenia (no spleen)
HIV (human immunodeficiency virus) infection
Antibody and complement deficiency syndromes
Receipt of chemotherapy or radiation therapy for malignant neoplasms
Receipt of hematopoietic stem cell

Signs and Symptoms

Haemophilus influenzae, including Hib, disease
causes different symptoms depending on which part
of the body is affected. The most common severe
types of
H. influenzaedisease are:
Pneumonia (lung infection)
Bacteremia (bloodstream infection)
Meningitis (infection of the covering of the brain and
spinal cord)

 Diagnosis
Haemophilus influenzae, including Hib, disease is
usually diagnosed with one or more laboratory tests using
a sample of body fluid, such as blood or spinal fluid.
Treatment
Haemophilus influenzae, including Hib, disease is
treated with antibiotics, usually for 10 days.
Most cases of invasive disease require care in a
hospital.
WhenH. influenzaecause a non-invasive infection, like
bronchitis or an ear infection, antibiotics may be given to
prevent complications.

Complications
Meningitis(infection of the covering of the brain and spinal
cord),
a person can suffer from brain damage or hearing loss.
Bacteremia (blood infection)
can result in loss of limb(s). InvasiveH.
influenzaeinfections can sometimes result in death.
Even with antibiotic treatment, about 3 to 6 out of every 100
children with meningitis caused by Hib die from the disease.

Colored Plates

Haemophilus
influenzae using
immunoflourescence

Photomicrograph of Haemophilus
influenzae as seen using a Gramstain techniques

Blood Agar Plate culture of

Haemophilus influenzae

Haemophilus
Influenzae
satelliting around
Staphyloccocus
aureus

Blood Agar Plate culture of

Haemophilus influenzae

CSF culture positive for

Haemophilus influenza,
type b(Gram stain)

Case Study
Haemophilus influenzaeType b Meningitis in the Short Period after
Vaccination: A Reminder of the Phenomenon of Apparent Vaccine Failure
Abstract: We present two cases of bacterial meningitis caused
byHaemophilus influenzaetype b (Hib) which developed a few days after
conjugate Hib vaccination. This phenomenon of postimmunization
provocative time period is reviewed and discussed. These cases serve as
a reminder to clinicians of the risk, albeit rare, of invasive Hib disease in
the short period after successful immunization.
Introduction: Haemophilus influenzaetype b (Hib) was the leading
cause of bacterial meningitis in children worldwide until the introduction
of the Hib conjugate vaccine in the early 1990s. Since then, the incidence
of Hib disease has declined dramatically in high-income countries and
virtually eliminated in parts of the United States and Europe.
Over the past 20 years, there have been some reports of invasive Hib
disease within a short period after administration of the vaccine. Report
describes two children in whom Hib meningitis developed a few days
after vaccination. These cases serve as a reminder for clinicians of a

 Discussion: The Hib vaccine targets the organisms capsular polysaccharide,

polyribosylribitol phosphate (PRP). To increase immunogenicity and induce
immune memory, several conjugate vaccines were developed through covalent
linkage of PRP to a carrier protein. Four conjugated vaccines were found safe and
were introduced into routine immunization programs worldwide.
While the introduction of conjugate vaccine against Hib has had a substantial
impact on Hib infection, over the past 20 years, sparse reports of cases of
invasive disease after Hib vaccination have been published. Booy et al. identified
two kinds of vaccine failures: apparent (early) and true (late). True failures were
defined as Hib invasive disease occurring either >1 week after a child up to the
age of 12 months received at least two doses of the vaccine, or >2 weeks after a
single dose was received by a child >12 months of age. Hib invasive infections
that occurred within one week after the administration of one or two doses of
vaccine were considered apparent vaccine failures. Thus, in the present report,
both cases represent apparent (early) vaccine failures.

 The apparent vaccine failure" was a known phenomenon of the early

polysaccharide vaccine, but relatively rare when attributed to conjugate
vaccine.
Already in 1901, Wright coined the term negative phase" to describe the
decrease in bactericidal activity; he observed 1 to 21 days after
administration of typhoid vaccine. This phenomenon of postimmunization
provocative disease was also confirmed in early studies of conjugated
and unconjugated Hib vaccines which reported that subjects with
preexisting anticapsular antibodies showed a decrease in antibody
concentrations after immunization. The nadir in antibody decline was
reached 2-3 days after immunization, and concentrations normalized by
day 7. The magnitude of the decline was negatively correlated with the
preimmunization concentration. This decrease is presumed to occur with
all 4 available Hib conjugate vaccines. Some authors attributed these
findings to the formation of a complex between the vaccine antigens and
the preexisting serum antibodies, which induces a transient decline in
antibody concentration. This could pose a risk of invasive disease if it
occurs during a period of asymptomatic colonization with Hib.

 In order to understand whether the individual having received the

Hib vaccine is adequately protected against the organism, the level
of anti-PRP antibodies should be assessed. The exact mechanism
underlying the invasive infection in our patients could not be
determined because the concentration of Hib antibodies was not
measured in either case before or after immunization. However,
these cases are reported to serve as a reminder to clinicians of the
risk, albeit rare, of invasive Hib disease in the short period after
successful immunization. Clinicians should bear this possibility in
mind when starting empiric antibiotic treatment in children who
present with signs of infection within a week of receiving the
vaccine. Large-scale studies that focus on this time frame are still
needed.

 References
J. Eskola, Foresight in medicine: current challenges withHaemophilus influenzaetype b conjugate vaccines,Journal of Internal Medicine, vol. 267, no.
3, pp. 241250, 2010.View at PublisherView at Google ScholarView at Scopus
R. Dagan, D. Fraser, M. Roitman et al., Effectiveness of a nationwide infant immunization program againstHaemophilus influenzaeb,Vaccine, vol. 17,
no. 2, pp. 134141, 1999.View at PublisherView at Google ScholarView at Scopus
R. Booy, P. T. Heath, P. E. M. Slack, N. Begg, and E. Richard Moxon, Vaccine failures after primary immunisation withHaemophilus influenzaetype-b
conjugate vaccine without booster,The Lancet, vol. 349, no. 9060, pp. 11971202, 1997.View at Google ScholarView at Scopus
R. Singleton, L. Hammitt, T. Hennessy et al., The AlaskaHaemophilus influenzaetype b experience: lessons in controlling a vaccine-preventable
disease,Pediatrics, vol. 118, no. 2, pp. e421e429, 2006.View at PublisherView at Google ScholarView at Scopus
K. D. Cowgill, M. Ndiritu, J. Nyiro et al., Effectiveness ofHaemophilus influenzaetype b conjugate vaccine introduction into routine childhood
immunization in Kenya,The Journal of the American Medical Association, vol. 296, no. 6, pp. 671678, 2006.View at PublisherView at Google Scholar
View at Scopus
M. T. Osterholm, J. H. Rambeck, K. E. White et al., Lack of efficacy of Haemophilus b polysaccharide vaccine in Minnesota,The Journal of the American
Medical Association, vol. 260, no. 10, pp. 14231428, 1988.View at Google ScholarView at Scopus
A. E. Wright, On the changes effected by anti-typhoid inoculation in the bactericidial power of the blood; with remarks on the probable significance of
these changes,The Lancet, vol. 158, no. 4072, pp. 715723, 1901.View at Google ScholarView at Scopus
C. D. Marchant, E. Band, J. E. Froeschle, and P. H. McVerry, Depression of anticapsular antibody after immunization withHaemophilus influenzaetype b
polysaccharide-diphtheria conjugate vaccine,Pediatric Infectious Disease Journal, vol. 8, no. 8, pp. 508511, 1989.View at Google Scholar
View at Scopus
R. S. Daum, G. R. Siber, G. A. Ballanco, and S. K. Sood, Serum anticapsular antibody response in the first week after immunization of adults and infants
with theHaemophilus influenzaetype b-Neisseria meningitidisouter membrane protein complex conjugate vaccine,Journal of Infectious Diseases, vol.
164, no. 6, pp. 11541159, 1991.View at Google ScholarView at Scopus
S. K. Sood and R. S. Daum, Disease caused byHaemophilus influenzaetype b in the immediate period after homologous immunization: immunologic
investigation,Pediatrics, vol. 85, no. 4, part 2, pp. 698704, 1990.View at Google ScholarView at Scopus

Research Note
Vaccine effectiveness of the pneumococcalHaemophilus influenzaeprotein D conjugate vaccine
(PHiD-CV10) against clinically suspected invasive pneumococcal disease:
a cluster-randomised trial

DrA A Palmu, MDPress enter key for correspondence information

,J Jokinen, PhD
,H Nieminen, MD
,R Syrjnen, MD
,E Ruokokoski, MSc
,T Puumalainen, MD
,M Moreira, MD
,L Schuerman, MD
,D Borys, MD
,ProfT M Kilpi, MD
Published Online:07 August 2014

 Summary
Background
Vaccine effectiveness of pneumococcal conjugate vaccines
against culture-confirmed invasive pneumococcal disease has
been well documented. In the Finnish Invasive Pneumococcal
disease (FinIP) trial, we reported vaccine effectiveness and
absolute rate reduction against laboratory-confirmed invasive
pneumococcal disease (confirmation by culture or antigen or
DNA detection irrespective of serotype). Here, we assessed
vaccine effectiveness of PHiD-CV10 against clinically suspected
invasive pneumococcal disease in children by use of diagnoses
coded in hospital discharge registers.

 Methods
For this phase 3/4 cluster-randomised, double-blind trial, undertaken
between Feb 18, 2009, and Dec 31, 2011, in municipal health-care
centres and the Tampere University Vaccine Research Centre (Finland),
we randomly assigned (2:2:1:1) 78 clusters into PHiD-CV10 three plus
one, PHiD-CV10 two plus one, control three plus one, control two plus
one groups (26:26:13:13 clusters) to give PHiD-CV10 in either three plus
one or two plus one schedule (if enrolled before 7 months of age; infant
schedules), two plus one (if enrolled between 7 and 11 months; catch-up
schedules), and two doses at least 6 months apart (if enrolled between
12 and 18 months; catch-up schedules). Children were eligible if they
had not received and were not anticipated to receive any of the study
vaccines and had no general contraindications to vaccinations. We
collected all inpatient and outpatient discharge notifications from the
national hospital discharge register with International Classification of
Diseases (ICD) 10 diagnoses compatible with invasive pneumococcal
disease or unspecified sepsis, and verified data with patient files. We
excluded invasive pneumococcal disease cases confirmed by positive
culture or DNA/RNA detection from normally sterile body fluid. The
primary objective was to estimate vaccine effectiveness against all

 Findings
We enrolled 47366 children. On the basis of ICD-10 diagnoses, we
recorded 264 episodes of register-based non-laboratory-confirmed
invasive pneumococcal disease or unspecified sepsis, of which 102 were
patient-file verified non-laboratory-confirmed invasive pneumococcal
disease. The vaccine effectiveness was 50% (95% CI 3263) in the 30
527 infants with three plus one and two plus one schedules combined
and the absolute incidence rate reduction was 207 episodes per 100000
person-years (95% CI 127286). The vaccine effectiveness against the
patient-file verified non-laboratory-confirmed invasive pneumococcal
disease was 71% (95% CI 5283) in infant three plus one and two plus
one schedules combined. The absolute rate reduction was 142 episodes
per 100000 person-years (95% CI 91191) in infant cohorts.

 Interpretation
This vaccine-probe analysis is the first report showing
the effect of pneumococcal conjugate vaccines on
clinically suspected invasive pneumococcal disease.
The absolute rate reduction was markedly higher
compared
with
laboratory-confirmed
invasive
pneumococcal disease, which implies low sensitivity of
the
laboratory-based
case
definitions
and
subsequently
higher
public
health
effect
of
pneumococcal conjugate vaccines against invasive
pneumococcal disease than previously estimated.
Funding
GlaxoSmithKline Biologicals SA and National Institute
for Health and Welfare (THL), Finland.

Haemophilus ducreyi

Haemophilus ducreyi
Causative agent of chancroid or soft chancre
(STD), highly contagious
Specimens should be collected from base of
lesion, inoculated directly to enriched media
and held for 5 days
Gram stain appears as groups of coccbacilli that
resemble a school of fish or railroad tracks
Requires only X factor to grow

Haemophilus ducreyi
A fastidious gram-negative bacillus is a wellknown cause of Chancroid
A sexually transmitted pathogen, associated
with the sexual transmission of human
immunodeficiency virus (HIV).
More recently it has been identified as the
etiologic agent in chronic skin ulcers in the
South Pacific region.

Chancroid
Once commonly seen in sexually transmitted
diseases (STD) clinics across Africa, Asia, and
Latin America.
Since 2000 with the widespread use of
syndromic approaches to the management of
bacterial STDs, chancroid has been rapidly
declining as a significant cause of genital ulcers
and may have been eliminated in some parts of
eastern and southern Africa.
Prevalence of chancroid among patients with
genital ulcers, in sub-Saharan Africa declined

 (2005)H. ducreyi- newly identified as a cause of

chronic skin ulcers in children living in yaws-endemic
areas of the South Pacific region.
First described in 1989, cause of a lower extremity
chronic ulcer in an adult male.
The cases reported from surveys of chronic skin ulcers
in children conducted in 2013 in Papua New Guinea
and the Solomon Islands describe a non-sexually
transmitted lower extremity chronic ulcer that is
similar in clinical appearance to genital chancroid
without marked regional lymphadenopathy or bubo
formation.It appears to respond to standard

Diagnosis
H. ducreyiinfection is difficult to isolate in
culture can be confused with:
syphilis
genital herpes viral infection
yaws in children.

Specific selective enriched culture media which

are available only in specialized reference
laboratories have sensitivities ranging from
less than 50% to over 80%.
Non-culture diagnostic antigen detection tests

Treatment
ANTIMICROBIAL THERAPY
STD
treatment
azithromycin,ceftriaxone,ciprofloxacin,erythromycin
Treatment regimens for chronic ulcers in children caused byH.
ducreyihave
not
been
studied,
hence
recommended
antimicrobial treatment options at this time cannot be
recommended.Nevertheless, it appears that single-oral-dose
azithromycin 30 mg/kg may be effective in children
MICROLIDES
QUINOLONES
ENDPOINTS OF MONITORING THERAPY
VACCINES
Currently, there are noH. ducreyivaccines available

Prevention or Infection Control

Prompt identification and treatment of cases with simple
syndromic management algorithms, accompanied by treatment
of all recent sexual contacts within the previous 14 days
Sensitive and appropriate counseling and prevention education
messages.
Eradication of infection in persons who are sources of multiple
infections, such as sex trade workers, can be effective in
controlling chancroid outbreaks.
Properly used condoms can also reduce the transmission of
chancroid.

Case Study
Chancroid in Sheffield
A report of 22 cases diagnosed by isolating Haemophilus ducreyi in a
modified medium
S HAFIZ,* G R KINGHORN,t AND M G McENTEGART* From the *Department
of Medical Microbiology, University of Sheffield Medical School, and the
Department of Genitourinary Medicine, Special Clinic, Royal Infirmary,
Sheffield
SUMMARY
Haemophilus ducreyi, is generally considered to be very fastidious and its
isolation, maintenance, and detailed study very demanding. In this study
a modified medium was developed, which allowed the organism to be
isolated morefrequently than previously would have been expected.
Twenty-two cases of chancroid wereconfirmed by the isolation of H ducreyi
in 160 patients with genital ulceration examined over aone-year period.
Cases were apparently unrelated, and in only five was there a history of
recentsexual contact abroad. Concurrent infection with other sexually
transmitted diseases was presentin 18 (81 - 8%) patients, and in 14 (63'6G) both H ducreyi and herpes simplex virus were isolated from the same

References
I. Ducrey A. Recherches experimentales sur la nature intime du
principe contagieux du chancre mou. Annales de Dermatologie
et de Syphiligraphie, 1890; 1:56.
2. Bazancon F, Griffon V, Le Sourd L. Recherche sur la culture
du bacille de Ducrey. Press Med 1900; 2:385-90.
3. Himmel J. Des animaux vis-a-vis du bacille du chancre mou.
Annales de l'!nstitut Pasteur 1901; 15:928-40.
4. Stein R. Die Plattencultur der streptobacillen des ulcus molle.
Zentralbl Bakteriol (Orig B) 1908;46:664-70.
5. Teague 0, Deibert 0. The value of cultural method in the
diagnosis of chancroid. J Urol 1920;4:543-50.
6. Hewlett RT. Chancroid and Bacillus ducreyii. In: MRCSystem
of Bacteriology, Vol 2. London: HMSO 1929; 394-419.
7. Greenwald E. Chancroidal infection; treatment and diagnosis.
JAMA 1943;121:9-1 1.
8. Beeson P. B. Studies on chancroid. IV The Ducrey bacillus
growth requirements and inhibition by antibiotic agents. Proc
Soc Exp Biol Med 1946;61:81-5.
9. Deacon WE, Albritton DC, Olansky S, Kiplan W. A simplified
procedure for the isolation and identification of Haemophilus
ducreyi. J Invest Dermatol 1956; 26:399-406.
10. Heyman A, Beeson PB, Sheldson WH. Diagnosis of
chancroid. JAMA 1945; 129:935-8.
11. Kilian M. A taxonomic study of the genus Haemophilus with
the proposal of a new species. J Gen Microbiol 1976;93:9-62.
12. Tan T, Rajan VS, Koe SL, Tan NJ, Tan BH, Goh AJ. Chancroid:
A study of 500 cases. Asian J Infect Dis 1977; 1:27-8.
13. Khoo R, Sng EH, Goh AJ. A study of sexually transmitted
diseases in 200 prostitutes in Singapore. Asian J Infect Dis
1977; 1:77-9.
386
14. Hammond GW, Lian CJ, Wilt TC, Albritton WL, Ronald AR.
Determination of the hemin requirement of Haemophilus
ducreyi: evaluation of the prophyrin test and media used in
satellite growth. J Clin Microbiol 1978;7:243-8.
15. Sottnek FO, Biddle JW, Kraus SJ, Weaver RE, Stewart JA.
Isolation and identification of Haemophilus ducreyi in a
clinical study. J Clin Microbiol 1980; 12:170-4.
16. Kellogg DS, Peacock WL, Deacon WE, Brown L, Pirkle CI. N
gonorrhoeae. I Virulence genetically linked to colonial
variation. J Bacteriol 1963; 85:1273-9.
17. Morton RS. In: Gonorrhoea. London, Philadelphia, and
Toronto: WB Saunders and Co Ltd, 1977; 71, 100, and 247.
18. Alergant CD. Chancroid. Practitioner 1972;209:624-7.
19. Gaison A, Heaton CL. Chancroid: alias the soft chancre. Int J
Dermatol 1975; 14:188-97.
20. Lykke-Olesen L, Pedersen TG, Larsen L, Gaarslev K.
Epidemic of chancroid in Greenland 1977-1978. Lancet 1979;
i: 654-5.
21. Kerber RE, Rowe CE, Gilbert KR. Treatment of chancroid.
Arch Dermatol 1969; 100:604-7.
22. Nayyar KC, Stolz E, Michel MF. Rising incidence of chancroid
in Rotterdam. Br J Vener Dis 1979; 55:439-41.
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treatment of chancroid. J Trop Med Hyg 1974; 77:55-60.
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25. Anomymous, Sexually

Research Notes
ABSTRACT
Haemophilus ducreyicauses chancroid and has
recently been shown to be a significant cause of
cutaneous lesions in tropical or subtropical regions
where yaws is endemic. Here, we report the draft
genome assemblies for 11 cutaneous strains
ofHaemophilus ducreyi, isolated from children in
Vanuatu and Ghana.

 GENOME ANNOUNCEMENT
Haemophilus ducreyiis a fastidious Gram-negative
bacterium that causes chancroid, a sexually transmitted
disease characterized by painful genital ulcers. The
global prevalence of chancroid has declined significantly
in the past decade due to syndromic management of
genital ulcer disease. There have been sporadic reports
of cutaneous lesions due to nonsexual transmission
ofH.ducreyi, but recent surveys, as part of the WHO
yaws eradication program, have shown a high
prevalence in the South Pacific islands and Ghana.

Very little is known aboutH.ducreyistrains responsible for

cutaneous lesions in children. To better understand the
genetic differences between genital and cutaneous strains
ofH.ducreyifrom different geographic locations, we
performed whole-genome sequencing on cutaneous strains
isolated in 2014 and 2015 from children in Vanuatu and
Ghana during yaws surveys.
Lesion swabs were streaked onto Columbia agar plates
containing 1% hemoglobin (BBL, Franklin Lakes, NJ, USA),
0.2% activated charcoal (Sigma-Aldrich, St. Louis, MO, USA),
5% fetal bovine serum (Atlanta Biologicals, Atlanta, GA, USA),
and 1% IsoVitaleX (BBL), and incubated in a sealed paint can
(candle jar) under CO2conditions. Plates were transferred to
the laboratory and incubated for 48h at 33C under

In Vanuatu, all bacterial colonies were scraped off

primary plates, transferred to a transport medium, and
transported on ice packs to the WHO Collaborating
Centre for STD, Sydney.H.ducreyiwas isolated on
Columbia agar plates and identified by 16S rRNA
sequencing. In Ghana, bacteria from primary plates or
suspectedH.ducreyicolonies were frozen in storage
medium containing 1% proteose peptone no. 3 (BD,
Franklin Lakes, NJ, USA) and 0.8% glycerol and shipped
to the CDC for identification using biochemical tests and
PCR.

DNA was extracted using the ArchivePure DNA cell/tissue kit (5

PRIME, Inc., Gaithersburg, MD, USA) following the manufacturers
guidelines. Whole-genome sequencing was conducted using the
PacBio RSII platform (Pacific Biosciences, Menlo Park, CA, USA)
with P6-C4 and P6 v2-C4 chemistry. A single-molecule real-time
(SMRT) cell was used to sequence each genome, andde
novoassembly of the genomes was conducted using the
hierarchical genome assembly process (HGAP3, SMRTAnalysis
version 2.3.0) workflow, which included consensus-polishing
using Quiver (9). Sequences were annotated using the NCBI
Prokaryotic Genome Annotation Pipeline.

Colored Plate

Microscopic image ofHaemophilus ducreyi,

the bacteria species responsible for chancroid infections

Haemophilus and Other Fastidious Gramnegative Rods

The fastidious group of gram-negative bacilli
include:
Haemophilus
HACEK( Haemophilus, Actinobacillus, Cardiobacteria, Eikenella
& Kingella)

Legionella
Bordetella
Pasteurella
Brucella
Francisella

Haemophilus influenzae
Misnamed originally thought to cause the
flu
Now know that flu is caused by viruses
In some cases of flu, H. influenzae is
secondary infection

Haemophilus influenzae: VIRULENCE FACTORS

Capsule
Antiphagocytic

IgA Protease
Cleaves IgA on mucosal surfaces

Lipid A
Effects ciliated respiratory epithelium

Pili
Attachment

Haemophilus influenzae:
CLINICAL INFECTIONS: TYPABLE STRAINS
Acute epiglottitis or laryngotracheal infection in small
children
Can cause airway obstruction neeading immediate tracheostomy

Cellulitis/arthritis
cheek and upper extremities

Meningitis
Children under 6 years

Contagious, vaccine has decreased incidence

Pneumonia/septicemia
In children

Conjunctivitis pink eye

very contagious

Haemophilus Influenzae:
Clinical Infections: nontypable strains
Otitis media
Children 6 months- 2 years
Sinusitis
Pneumonia, bronchitis
In adults
These sites are all in proximity to respiratory tract

Haemophilus influenzae
Haemophilus species require growth factors:
X-factor ( hemin)
Heat-stable substance
Present in RBC and released with degradation of
hemoglobin

V-factor (NAD: nicotinamide adenine

dinucleotide)
Heat- labile
Found in blood or secreted by certain organisms

Haemophilus influenzae
H. influenzae satellitism
around and between the
large, white, hemolytic
staphylococci.
This occurs when
another organism
produces V factor as a biproduct.

Haemophilus influenzae
Gram Stain Morphology
Usually very small pleomorphic gram negative cb or rod
May be able to observe a halo around the organism
Gram stain can be enhanced by extending time for
safranin to 2 minutes OR substitute carbolfuschin for
safranin

Haemophilus influenzae

Direct smear of H. influenzae in CSF in a case of meningitis. Note the TINY intracellular
and extracellular pleomorphic gram-negative bacilli.
Remember to look for capsules surrounding the rod.

Haemophilus influenzae
Colony Morphology
No growth on BAP or
MAC
On CA:
semi-opaque, graywhite
convex, mucoid.

Haemophilus influenzae: IDENTIFICATION

Gram stain
Gram negative cocco-baccillus
Catalase +
Oxidase +
X and V factor strips or disks
Quad plates
Rapid ID Panels
NHI cards- automated

Haemophilus influenzae:
Identification

This organism would be identified as H. influenzae because it is using

both X and V factors.

Haemophilus Species: Identification

This organism would be identified as H. parainfluenzae because

it is using V factor only.

Treatment
Antibiotic therapy
Historically ampicillin was the drug of choice.
However, resistance has developed due to
production of beta-lactamase or altered
penicillin binding proteins and cell wall
permeability
Susceptibility testing can be performed by
disk diffusion, broth dilution or E-test
Primary antibiotics include cefotaxime or
ceftriaxone

Haemophilus aegyptius

Haemophilus aegyptius
Koch-weeks bacilli
Disease:
Acute contagious conjunctivitis
Brazilian purpuric fever

Normal flora of human upper respiratory tract.

Appearance: resembles H. influenzae except
colonies are smaller at 48 hrs.

General Characteristics
Virulence Factors

Gram (-)
pleomorphic
coccobacilli/rods,
Nonmotile
facultatively
anaerobic

Capsule
adherence by fimbriae
and
other structures
outer membrance
protein
LPS

Clinical Infections

Associated with an acute,

contagious conjunctivitis ("pink
eye")
Primarily in small children, and
Brazilian Purpuric Fever

Lab Diagnosis:
Specimen
Processing and Isolation

Common specimens:
conjunctivae swabs
Special media:
CHOC agar w/ 1% IsoVitaleX
or Vitox (required due to
fastidious nature).

Lab Diagnosis:
Microscopic
Morphology
Varies from small gram (-)
coccobacilli to long filaments,
and often stains faint pink
An acridine orange or
methylene blue stain may help
in detection of Haemophilus
spp.

Lab Diagnosis:
Colony
Morphology
SBA, CHOC, and MAC are often
used (no growth on SBA or MAC
with pure Haemophilus culture)
Aegyptius resembles H.
influenzae on CHOC with a
small, translucent, tannish,
moist, smooth, and convex
appearance, accompanied by a
"mousy" or bleach-like odor

Lab Diagnosis:

Lab Diagnosis:
Lab ID

X and V Requirements:
requires both X (hemin)
and
V
(NAD) factors.

Lab ID

Porphyrin Test:
like H. influenzae, H.
aegyptius is NEGATIVE
under UV-light in the
Porphyrin Test

Lab Diagnosis:
Lab ID
Biochemical Tests:
(Same as H.
influenzae)
Positive - Oxidase, Catalase,
Glucose, Xylose, and Nitrate
Negative - Hemolysis, CO2
growth enhancement, Indole,
ONPG, Sucrose, Mannose,
Mannitol, Maltose, Lactose, and
Esculin

Mode of Transmission
Person to person spread by
contaminated resporatory
droplets.
Certain infections maybe
caused by persons endogenous
strains.

Acute Contagious Conjunctivitis

A contagious inflammation of the conjunctiva caused
by
Haemophilus aegypticus
Secretions must be handled with extreme care to
prevent its spread
Popularly known as pinkeye

Signs and Symptoms

Itchy eyes
Tearing
Redness
Light sensitivity

Treatment

Idoxuridine solution

and ointment
Vidarabine ointment
Trifluridine solution

Brazilian Purpuric Fever

Severe systemic disease
caused by H. influenzae
biogroup aegyptius
Characterized by recurrent
or concurrent conjunctivitis,
high
fever,
vomiting,
petechial/purpural
rash,
septicemia,
shock
and
vascular collapse mortality
rate can be up to 70%
within 48 hours of onset

Case Study
Abstract: The Brazilian Purpuric Fever (BPF) is a systemic disease with
many clinical features of meningococcal sepsis and is usually preceded by
purulent conjunctivitis.
The illness is caused by Haemophilus influenza biogroup aegyptius, which
was associated exclusively with conjunctivitis. In this work construction of
the las gene, hypothetically responsible for this virulence, were fusioned
with ermAM cassette in Neisseria meningitidis virulent strains and had its
DNA transfer to non BPF H. influenzae strains. The effect of the las transfer
was capable to increase the cytokines TNF and IL10 expression in Hec-1B
cells line infected with these transformed mutants (in eight log scale of
folding change RNA expression). This is the first molecular study involving
the las transfer to search an elucidation of the pathogenic factors by
horizontal intergeneric transfer from meningococci to H. influenzae.

 Introduction: The Brazilian Purpuric Fever (BPF) is a fulminant

pedriac disease caused by Haemophilus influenzae biogroup aegyptius
(Hae). BPF was first described just over a decade ago when an
outbreak emerged in several locations in the Sao Paulo State, Brazil.
The illness has many clinical features of meningococcal sepsis as high
fever, vomiting, abdominal pain, rapid progression of petechiae and
vascular collapse. These symptons generally manifest between 7-10
days after an episode of purulent conjunctivitis (1985; 1986; 1987a;
1987b). The disease may be more common than expected. Since the
clinical picture is very similar to the meningoccocal septicemia,
possible cases of BPF could be misreported. Currently, BPF is a disease
requiring mandatory reporting in Brazil, because BPF agents may
potentially lead to new outbreaks.

 Before the emerging of BPF, Hae was a bacterium

only associated with conjunctivitis, pro,ducing
seasonal and epidemic infection in hot climates
(Pittman and Davis 1950). Little is known of the
determinants of Hae virulence or the pathogenesis
of infection in BPF. Potential virulence factors such
as pilus proteins and lipopolysaccharide have been
investigated in the infant rat model (Rubin and St
Geme 1993) and in vitro with endothelial cells
(Quinn and others 1995; Weyant and others 1994),

 The horizontal transfer of virulence genes has a

major role in the evolution of bacterial
pathogens and since the natural genetic
exchange between Haemophilus influenzae and
Neisseria meningitidis was already described
(Kroll and others 1998), the highly virulent
``meningococcal phenotype in Hae may be
result of the genetic transfer from the
meningococcus to Hae-BPF.
One possible explanation for the emergence of
the invasive Hae strains is an occurrence of a

 Constructions of the las gene from Hae were

transfer to non BPF H. influenzae strains and
the inflammatory effects were analyzed and
measured in an endothelial cellular system in
vitro. This work is the first molecular study
involving the las transfer to search an
elucidation of the pathogenic factors by
horizontal intergeneric transfer between
meningococci and H. influenzae.
Material and Methods Bacterial
conditions: The Neisseria meningitidis and

 Genetic transfer assays of las gene from N. meningitidis to H. influezae

The fusioned mutant LG2 was used as DNA donor to performed in vitro
transformation of H. influenzae RdKw20 and H. influenzae lac strains.
The transformation process was performed according specifications
already described (Taha and others 1998) adapted for H. influenzae
using the BHI liquid medium (supplemented with haemin and NAD at
10 g/mL). The H. influenzae strains were growth in chocolate agar and
suspensions of DO 600 in supplemented BHI were made. The 5 g of
DNA from LG2 strain was added in the bacterial suspensions and then
incubated at 37 C in atmosphere of 5% of CO2 by 5 hours.
Hec1b cell culture and adhesion assay Hec1B cells line were grown in
plastic flasks (25 cm2 ) with RPMI 1640 medium (Cultilab, Campinas,
SP Brazil), supplemented with 2% L-glutamine, 120 g/mL of garamycin
and 10% inactivated fetal bovine serum (comH plete medium). The
tests for adhesion in cells followed specifications already described
(Pereira RFC 2011).

 Statistical analysis: The data from each assay

were statistically analyzed using Turkeys test
compared with a control sample and p < 0.05 was
considered significant. All experiments were
performed in triplicate and the data shown in the
graphs and in the table represent the means
standard errors. Results For the production of the
las::ermAM gene aiming the detection of the las
transfer was used the template strain Hae11116
(GenBank GI: 14994100). The construction of

 Discussion: The use of the increase of cytokines in septic shock

characterization is normally done in N. meningitidis pathologies. The
implication of TNF and interleukines, such as IL10, in meningococci sepsis
is well described by many authors (Bjerre and others 2004; Brandtzaeg and
others 2001; Jacobs and Tabor 1990). In this work the use the Hec-1-B cells
line, which had already used as model of TNF verification in meningococcal
infection (Pereira RFC 2011; Taha 2000), for the immunological analysis in
the transfer process of the las gene from meningoccci strains to H.
influenzae not associated with the purpura fulminans process.
Nevertheless, the study of the immune response of H. influenzae biotype
aegyptius causing the Brazilian purpuric fever had never been described
before in vitro. The strains involved in the first BPF outbreak Hae254,
Hae258 and Hae284 were able to activate the expression of IL10 and TNF
in Hec-1B cells.

 It is very important to reinforce that the gene

acquisition was mediated by an environmental
action. The BPF outbreak occurred in the
Southest Brazilian region where the primitive
agriculture was performed with a great
emission of carbon dust from sugar cane burn.
The atmosphere is similar to the one described
to N. meningitidis using nanostructures
mimicking the action of dust and particles
formed by silica and carbon in atmosphere
(Hollanda and others; Mattos and others).

 Acknowledgments: All the authors declare have no conflicts of interest in this

work. This work was supported by FAPESP (number 2008/56777-5) and CNPq
(number 575313/2008-0). Thanks for the English revision for Julia N. Varela.
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children-Brazil. MMWR Morb Mortal Wkly Rep 34:217-219. (1986) Brazilian
purpuric fever: Haemophilus aegyptius bacteremia complicating purulent
conjunctivitis. MMWR Morb Mortal Wkly Rep 35:553-554. (1987a) Brazilian
purpuric fever: epidemic purpura fulminans associated with antecedent
purulent conjunctivitis. Brazilian Purpuric Fever Study Group. Lancet 2:757-761.
(1987b) Haemophilus aegyptius bacteraemia in Brazilian purpuric fever.
Brazilian Purpuric Fever Study Group. Lancet 2:761-763. Bjerre A, Brusletto B,
Hoiby EA, Kierulf P, Brandtzaeg P (2004) Plasma interferon-gamma and
interleukin-10 concentrations in systemic meningococcal disease compared
with severe systemic Gram-positive septic shock. Crit Care Med 32:433-438.

 Brandtzaeg P, Bjerre A, Ovstebo R, Brusletto B, Joo GB, Kierulf P (2001)

Neisseria meningitidis lipopolysaccharides in human pathology. J Endotoxin Res
7:401-420. Brenner DJ, Mayer LW, Carlone GM, Harrison LH, Bibb WF,
Brandileone MC, Sottnek FO, Irino K, Reeves MW, Swenson JM and others
(1988) Biochemical, genetic, and epidemiologic characterization of
Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) strains
associated with Brazilian purpuric fever. J Clin Microbiol 26:1524-1534. Davis J,
Smith AL, Hughes WR, Golomb M (2001) Evolution of an autotransporter:
domain shuffling and lateral transfer from pathogenic Haemophilus to
Neisseria. J Bacteriol 183:4626-4635. Hollanda LM, Cury GC, Pereira RF,
Ferreira GA, Sousa A, Sousa EM, Lancellotti M (2011) Effect of mesoporous
silica under Neisseria meningitidis transformation process: environmental
effects under meningococci transformation. J Nanobiotechnology 9:28.

 Jacobs RF, Tabor DR (1990) The immunology of sepsis and meningitis-cytokine

biology. Scand J Infect Dis Suppl 73:7-15. Kroll JS, Wilks KE, Farrant JL, Langford
PR (1998) Natural genetic exchange between Haemophilus and Neisseria:
intergeneric transfer of chromosomal genes between major human pathogens.
Proc Natl Acad SciUSA 95:12381- 2385. Lancellotti M, Pace F, Stehling EG,
Villares MC, Brocchi M, Silveira WD (2008) Ribotyping, biotyping and capsular
typing of Haemophilus influenzae strains isolated from patients in Campinas,
southeast Brazil. Braz J Infect Dis 12:430-437. Li MS, Farrant JL, Langford PR,
Kroll JS (2003) Identification and characterization of genomic loci unique to the
Brazilian purpuric fever clonal group of H. influenzae biogroup aegyptius:
functionality explored using meningococcal homology. Mol Microbiol 47:11011111. Mattos IB, Alves DA, Hollanda LM, Ceragiogli HJ, Baranauskas V,
Lancellotti M Effects of multi-walled carbon nanotubes (MWCNT) under
Neisseria meningitidis transformation process. J Nanobiotechnology 9:53.

 McIntyre P, Wheaton G, Erlich J, Hansman D (1987)

Brasilian purpuric fever in central Australia. Lancet
2:112. Overbergh L, Kyama CM, Valckx D, Debrock
S, Mwenda JM, Mathieu C, DHooghe T (2005)
Validation of real-time RT-PCR assays for mRNA
quantification in baboons. Cytokine 31:454-458.
Pereira RFC AD, Jacinto RK, Hollanda LM, Verinaud
LMC, Machado CML and Lancellotti M (2011)
Effects of Neisseria meningitidis Infection in Tumor
Glioblastoma Cell Line NG97: Respiratory Pathogen

 Rubin LG, St Geme JW, 3rd (1993) Role of lipooligosaccharide in virulence of the
Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius for
infant rats. Infect Immun 61:650-655. Santana-Porto EA, Oliveira AA, da-Costa MR,
Pinheiro A, Oliveira C, Lopes ML, Pereira LE, Sacchi C, Araujo WN, Sobel J (2009)
Suspected Brazilian purpuric fever, Brazilian Amazon region. Emerg Infect Dis
15:675-676. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the
comparative C(T) method. Nat Protoc 3:1101-1108. Strouts FR, Power P, Croucher NJ,
Corton N, van Tonder A, Quail MA, Langford PR, Hudson MJ, Parkhill J, Kroll JS and
others Lineage-specific virulence determinants of Haemophilus influenzae biogroup
aegyptius. Emerg Infect Dis 18:449-457. Taha MK (2000) Neisseria meningitidis
induces the expression of the TNF-alpha gene in endothelial cells. Cytokine 12:21-25.
Taha MK, Morand PC, Pereira Y, Eugene E, Giorgini D, Larribe M, Nassif X (1998) Pilusmediated adhesion of Neisseria meningitidis: the essential role of cell contactdependent transcriptional upregulation of the PilC1 protein. Mol Microbiol 28:11531163.

 Virata M, Rosenstein NE, Hadler JL, Barrett NL,

Tondella ML, Mayer LW, Weyant RS, Hill B, Perkins
BA (1998) Suspected Brazilian purpuric fever in a
toddler with overwhelming Epstein-Barr virus
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Quinn FD, Utt EA, Worley M, George VG, Candal FJ,
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Research Note
Haemophilus influenzae: using comparative genomics to accurately identify
a highly recombinogenic human pathogen
Abstract
Background: Haemophilus influenzaeis an opportunistic bacterial
pathogen that exclusively colonises humans and is associated with both
acute and chronic disease. Despite its clinical significance, accurate
identification ofH. influenzaeis a non-trivial endeavour.H.
haemolyticuscan be misidentified asH. influenzaefrom clinical specimens
using selective culturing methods, reflecting both the shared environmental
niche and phenotypic similarities of these species. On the molecular level,
frequent genetic exchange amongstHaemophilusspp. has confounded
accurate identification ofH. influenzae, leading to both false-positive and
false-negative results with existing speciation assays.

 Results: Whole-genome single-nucleotide

polymorphism data from 246 closely related
globalHaemophilusisolates, including 107
Australian isolate genomes generated in this
study, were used to construct a whole-genome
phylogeny. Based on this phylogeny,H.
influenzaecould be differentiated from closely
related species. Next, aH. influenzae-specific
locus,fucP, was identified, and a novel TaqMan
real-time PCR assay targetingfucPwas designed.

 Conclusions: This study is the first of its kind to

use large-scale comparative genomic analysis
ofHaemophilusspp. to accurately delineateH.
influenzaeand to identify a species-specific
molecular signature for this species.
ThefucPassay outperforms existingH.
influenzaetargets, most of which were identified
prior to the next-generation genomics era and
thus lack validation across a large number
ofHaemophilusspp. We recommend use of

 Background: The Gram-negativeHaemophilusspp. bacteria comprise a diverse

group containing at least 12 currently recognised species, all of which are
commensal or pathogenic to humans or animals.Haemophilus influenzaeis the
best-known member of this genus, particularly serotybe b (Hib), the leading cause
of invasive bacterial disease in children prior to the introduction of the first
licensed Hib conjugate vaccine in 1987. In regions where Hib vaccination has been
implemented, the spectrum of severe Hib disease is now close to eradication.
OtherH. influenzaeserotypes (a; c-f), and nonencapsulated, nontypeableH.
influenzae(NTHi), which are not targeted by the Hib vaccine, are now recognised
as important causes of primarily mucosal acute and chronic infections. NTHi is a
common coloniser of the upper respiratory tract in healthy individuals but can
cause otitis media, conjunctivitis, sinusitis, and lower respiratory infections in
children, exacerbations of chronic obstructive pulmonary disease (COPD) and
cystic fibrosis (CF) in adults, and sepsis in neonates and immunocompromised
adults. Although far less common thanH. influenzae, otherHaemophilusspecies
also have the potential to cause human disease includingH. haemolyticus,H.
parainfluenzae,H. aegyptius(a biogroup ofH. influenzae),H. pittmaniae,H.
parahaemolyticusandH. paraphrohaemolyticus

 Misidentification of near-neighbourHaemophilusspecies asH.

influenzaehas broad-ranging implications for clinical diagnosis,
reported carriage rates and assessment of disease outcomes from
antibiotic or vaccine clinical trials. Microbiological differentiation
ofH. influenzaefrom other species has conventionally relied upon
colonial morphology, haemin and NAD (X and V factor)
dependence, and for capsular strains, serotyping using various
methods. For NTHi, identification relies on the absence of capsule
and is thus more challenging than capsulatedH. influenzae. In
2007, Murphy and colleagues were the first to report the
misidentification of non-haemolytic strains ofH. haemolyticusas
NTHi. These strains are phenotypically indistinguishable from NTHi
and represent the only otherHaemophilusspp. for which X and V
factor dependence is a diagnostic criterion.

 Numerous genetic methods for discriminatingH. influenzaefrom other species have

been
described.
However,
accurate
delineation
ofH.
influenzaefrom
otherHaemophilusspecies using genetic methods has proven challenging.
Recombination betweenH. influenzaeand otherHaemophilusspp., particularlyH.
haemolyticus, has confounded molecular speciation attempts, especially in the
absence of genomic data. Binks and colleagues recently assessed the ability of a
number of published and novel PCR-based methods to discriminate NTHi from closelyrelatedHaemophilusspecies compared withrecAand 16S rRNA gene sequencing, and
reported that an assay targeting sequence diversity within thehpdgene, which
encodes for Protein D, was superior to other molecular signatures. Additionally,
thehpd#3assay was specific forH. influenzaewhen compared against a panel of
common respiratory bacteria and has been used to quantifyH. influenzaedirectly
from clinical specimens. However, we recently reported the absence ofhpdin a
proportion ofH. influenzaeisolates, which was only identified following wholegenome sequencing analysis of 20 NTHi isolates. This finding highlights both the
limitation ofhpdforH. influenzaedetection and the requirement for genomic data
spanning a comprehensiveHaemophilusdataset to identify a gold standard
molecular signature.

 Here, we describe a large-scale comparative

genomics approach comprising 246 closely
related,
globalHaemophilusspp.
isolates
to
identify loci unique toH. influenzae. One of these
loci,fucP, was used to develop a real-time PCR
assay targetingH. influenzae.PCR screening of
thefucPassay across 59 genome-sequenced
AustralianHaemophilusspp. isolates and 35
clinically relevant species demonstrated 100%
specificity towardsH. influenzae.

 Methods
Ethics statement: Whole-genome analysis of the isolates in this study
was covered by the Human Research Ethics Committee of the Northern
Territory Department of Health and Menzies School of Health Research,
approval numbers 07/63 and 07/85, and the Princess Margaret Hospital
for Children Ethics Committee, approval number 1295/EP.
Bacterial isolates: A total of 511 isolates were examined in this study,
the
majority
of
which
wereHaemophilusspp.
(Table1
).Haemophilusisolates originated from a wide range of clinical sites,
clinical conditions and geographic regions. Samples were obtained from
nasopharyngeal swabs, sputum, bronchoalveolar lavage, throat, or
blood specimens, and include isolates sourced from either healthy
carriers or cases of otitis media, bronchiectasis, protracted bacterial
bronchitis, chronic obstructive pulmonary disease and bacteraemia.

 Phenotypic selection of the AustralianHaemophilusspp. isolates was

undertaken prior to molecular speciation. Only clinically-derived isolates that
were both X and V factor-dependent and that failed to react with capsular
antisera using the Phadebact Haemophilus coagglutination test (MKL
Diagnostics, Sweden) were selected for further analysis. Of the 107
AustralianHaemophilusisolates that underwent genome sequencing, 18 were
not speciated by molecular methods, 15 were speciated using 16S rDNA
sequencing, and the remainder underwent molecular speciation usinghpdbased methods.
Isolates were subcultured for purity through a minimum of three passages
prior to DNA extraction. The Qiagen DNeasy kit (Qiagen, Doncaster, VIC,
Australia) was used for DNA extraction according to the manufacturers
instructions, with enzymatic pre-treatment as described previously. DNA was
quality-checked for purity and extraction efficiency using a NanoDrop 2000
spectrophotometer (Thermo Fisher Scientific, Scoresby, VIC, Australia). All
DNA samples were diluted 1:100 in molecular-grade H 2O prior to PCR.

 H. influenzaewhole-genome sequencing
Paired-end genomic data for the Australian isolates were generated using the HiSeq or
MiSeq platforms (Illumina, Inc., San Diego, CA, USA), and were sequenced by Macrogen
Inc. (Geumcheon-gu, Seoul, Republic of Korea). The comparative genomics pipeline
SPANDx v2.6 was used to analyse theHaemophilusgenome. Input data for SPANDx were
already in paired-end Illumina format, except for publicly available reference genomes,
which were first converted to synthetic paired-end Illumina reads with ART (version
VanillaIceCream) using quality shifts of 10. The closed NTHi 86-028NP genome was used
as the reference for short-read alignment mapping. Synthetic reads for 86-028NP were
included as a control.
Phylogenetic analysis
Species
boundaries
forH.
influenzaeandH.
haemolyticuswere
establisheda
posteriorifollowing phylogenetic reconstruction of 63,447 high-confidence orthologous,
core genome, bi-allelic single-nucleotide polymorphisms (SNPs) identified across 246
closely relatedHaemophilusgenomes. Genomes from more distantly related species (H.
parainfluenzae,H.
haemoglobinophilus,H.
parahaemolyticusandH.
paraphrohaemolyticus) were excluded from this analysis to maximise the core genome
size. No additional SNP filtering (e.g. to exclude recombined regions) was performed.
Maximum parsimony trees were generated using PAUP* 4.0b10]; bootstrapping was
based on 200 replicates. Trees were visualised using FigTree v1.4.0

 Identification ofH. influenzae-specific signatures

We deliberately chose not to pursue SNPs forH. influenzaespeciation due to the high risk
of eventually encountering a homoplastic event. This risk is greatly increased in highly
recombinogenic species likeH. influenzae.Instead, we aimed to identify discrete,H.
influenzae-specific genetic loci for assay design. To identify such loci, the coverageBed
module of BEDTools v2.18.2, which is part of the SPANDx pipeline, was applied across the
246 closely relatedHaemophilusgenomes as described previously, using default settings.
This tool performs presence/absence analysis of Illumina reads for all genomes against
the reference genome. MS Excel 2013 was used to visualise presence/absence outputs.
CandidateH. influenzae-specific loci were identified by filtering for regions with 100%
read coverage in the target species but with <50% coverage in outgroup strains. Using
this approach, three candidate loci 4kb were identified. One locus,fucP (NTHI0865in
86-028NP), which encodes L-fucose permease, was chosen for real-time PCR assay design
as this region was highly conserved amongst the 201H. influenzaegenomes. The
coverageBED output was also used to assess presence/absence of the following
previously reportedH. influenzae-specific targets:fucK(NTHI0870in 86-028NP,
hap(NTHI0354)hpd(NTHI0811)iga(NTHI1164),lgtC(NTHI0365),ompP2(NTHI0225)
andompP6(NTHI0501).

 H. influenzaereal-time PCR assay

ThefucPassay was used to complement ourhpdresults due to the recent observation
that some AustralianH. influenzaestrains lack full-lengthhpd, and can therefore be
erroneously genotyped with this assay.
Unlabelled primers fucP-F (5GCCGCTTCTGAGGCTGG) and fucP-R (5-AACGACATTACCAATCCGATGG) (Sigma-Aldrich,
Castle Hill, NSW, Australia) were designed to generate a 68-bp fragment. A TaqMan
probe (fucP-Probe: 5-6FAM TCCATTACTGTTTGAAATAC-MGBNFQ; Life Technologies,
Grand Island, NY, USA) was included to increase specificity and to provide a gold
standard PCR methodology for clinical specimens. Microbial discontiguous
MegaBLAST analysis of theH. influenzae fucPamplicon across 3,546 complete
microbial genomes, 10,247 Proteobacterial draft genomes and 1,734 complete
plasmid genomes (total: 15,527 genomes) confirmed locus specificity, with a 100%
match in allH. influenzae(including biogroupaegyptius) at both primer- and probebinding sites, and several primer and probe mismatches in the next closest species
match (the avian pathogenAvibacterium paragallinarum; two nucleotide mismatches
in the forward primer, four mismatches in the reverse primer and one mismatch in the
TaqMan probe). No otherHaemophilusspp. yielded a detectable BLAST result for
thefucPamplicon, indicating the absence of this locus in other species.

 Real-time PCRs were performed using the RotorGene 6000 (Qiagen,

Chadstone, VIC, Australia) and ABI PRISM 7900HT (Life Technologies,
Mulgrave, VIC, Australia) platforms. Each reaction contained 0.25M of
each primer, 0.1M of probe and 1L genomic DNA. For the RotorGene
6000 instrument, 1X Platinum PCR SuperMix (Life Technologies) was
used to a total reaction volume of 10L. For the 7900HT instrument, 1X
TaqMan Universal Master Mix (Life Technologies) and 384-well plates
were used, enabling 5L reaction volumes. The 317 isolates tested by
PCR in this study (Table1; includes 46H. influenzaeand 13H.
haemolyticusthat were also subjected to whole-genome sequencing)
were assessed in duplicate, and all runs contained appropriate positive
control and no-template control reactions. For both instruments,
thermocycling was carried out as follows: 50C for 2min, 95C for
10min, followed by 45cycles of 95C for 5sec (15sec for the ABI
PRISM) and 60C for 5sec (1min for the ABI PRISM). The green/FAM
channels were used for fluorescence detection.

 Results and discussion: This study is the first to use extensive wholegenome sequence data from globalHaemophilusisolates to identify and
design a highly accurate molecular assay targetingH. influenzae.Based
on microbiological characteristics alone,H. influenzae, and particularly
NTHi, cannot always be differentiated from non-haemolyticH.
haemolyticus or closely related fuzzyHaemophilusspecies. Molecular
methods are therefore essential for accurate identification ofH.
influenzae. However, assay design has conventionally been thwarted by
high
levels
of
recombination
betweenH.
influenzaeand
otherHaemophilusspecies,
and
has
even
been
documented
betweenHaemophilusandNeisseria meningitidis. Compounding this
issue is the lack of rigorous, comparativein silicoanalysis of putative
molecular signatures using large-scale whole-genome sequence data.
These inherent obstacles with accurateH. influenzaespeciation have
likely led to underreporting of false-positive and false-negative results
for this clinically important bacterium.

 To address this issue, we combined whole-genome data generated for 107

Australian strains by our laboratory with all closely relatedHaemophilusspp.
genome data available in the public domain, including 87 unique NTHi
genomes from De Chiaraet al., to identify a highly specific signature forH.
influenzae.Species boundaries were first establisheda posterioriusing
phylogenetic analysis of 246Haemophilusgenomes. Based on this analysis,
201 of these strains were identified asH. influenzae, 32 asH.
haemolyticusand 13 as possible novel fuzzyHaemophilusspecies. This
phylogeny was highly similar to a recent whole-genome phylogeny
constructed using 97 predominantly NTHi strains. Amongst the 107
AustralianHaemophilusspp.
isolates
subjected
to
whole-genome
sequencing, 89 had prior speciation data by PCR-based methods.
Interestingly, the genome phylogeny reassigned two NTHi isolates that had
previously been identified by 16S rDNA PCR (n=1) orhpd#3PCR (n=
1)asH. haemolyticus, and reassigned 10 NTHi, oneH. haemolyticus, and
two equivocal isolates as a potentially novel fuzzyHaemophilusspecies.

 Following species delineation on a whole-genome SNP level, loci specific

toH. influenzaewere located. Core genome analysis of the 201H.
influenzaegenomes found that 936kb was conserved amongst allH.
influenzaestrains, represented by 100% read coverage across allH.
influenzaegenomes; however, across the larger 246Haemophilusgenome
dataset, only a minute fraction (12kb) was unique toH. influenzae. This
very low prevalence ofH. influenzae-specific loci exemplifies the inherent
difficulties in molecular speciation of this bacterium, particularly in lieu of
whole-genome data. Four loci, ranging from 1 to 6kb in size, were identified
asH. influenzae-specific. A 4kb locus, part of a fucose transport and
degradation operon, was selected for assay design due to high sequence
conservation across allH. influenzaeisolates. Within this locus we targeted
the L-fucose permease-encoding gene,fucP. L-fucose permease is a pHdependent major facilitator superfamily transporter that uptakes L-fucose, a
substrate that can act as a sole carbon source for bacteria. In the human
host, the fucose operon may impart a competitive advantage and virulence
potential toH. influenzae, as has been documented inCampylobacter jejuni.

 Following its design andin silicovalidation (as detailed in Methods),

thefucPTaqMan real-time PCR assay was screened for specificity against 35
bacterial species (comprising 40 isolates) of clinical relevance. As expected,
onlyH. influenzaeamplified using thefucPassay. A further selection of 212
nasopharyngeal, bronchoalveolar lavage and throat isolates, previously
designatedH. influenzaeorH. haemolyticususing thehpdPCR high-resolution
melt (HRM) assay, were screened using thefucPassay; 124/137 (91%)hpddefinedH. influenzaeisolates and 0/75hpd-definedH. haemolyticusisolates
amplified with thefucPassay. Subsequent genomic analysis of 10 of the 14
presumptive NTHi isolates that failedfucPPCR confirmed they are neitherH.
influenzaenorH. haemolyticus, but rather represent a possible novel
fuzzyHaemophilusspecies. An additional threeHaemophilusisolates (H18,
H40 and H180) not screened with thehpdHRM assay also grouped with this
fuzzyHaemophilusspecies based on whole-genome phylogenetic analysis.
The remaining four isolates were not whole-genome sequenced in this study,
but we suspect that they will also group with the fuzzy,fucPnegative,hpdHRMH. influenzae-positive isolates. We plan to genome-sequence
these strains in the future to confirm their phylogenetic placement.

 Conclusions: We have used a large-scale

genomic approach to characterise the highly
recombinogenicHaemophilusspp., including 107
new isolates from Australia. This approach
enabled accurate delineation ofH.
influenzaefrom morphologically identical nearneighbour species. Using extensive genomic data,
we next designed and validated a real-time PCR
assay targeting thefucPlocus inH. influenzae,
which provided 100% specificity for this
bacterium bothin silicoand across a diverse
bacterial DNA panel.ThefucPTaqMan assay

 Declarations
Acknowledgements: This project was funded by the Channel 7 Childrens
Research Foundation (13699) and the Australian National Health and Medical
Research Council (grant no. 1023781). We are grateful to the Rebecca L. Cooper
Medical Research Foundation for provision of the NanoDrop 2000
spectrophotometer. HSV and L-AK are supported by NHMRC Career Development
Fellowships 1024175 and 1061428, and RLM is supported by NHMRC Frank Fenner
Early Career Fellowship 1034703.
We would like to thank the families who participated in these studies and for their
continued support of our research. We wish to acknowledge Daniel J. Morton,
University of Oklahoma Health Sciences Center, for the provision of a bloodderivedH. haemolyticusisolate. We would also like to thank the Menzies
Respiratory, Ear Health Research and Child Health Laboratory Teams and
particularly Professor Amanda Leach for clinical swabs, clinical data, and
laboratory support.

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