Infection, Genetics and Evolution 80 (2020) 104205

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Infection, Genetics and Evolution

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Review article

Molecular epidemiology and evolution of Haemophilus influenzae T

a a b a,⁎ c,d,e,⁎,1
Shuxian Wen , Donghua Feng , Dingqiang Chen , Ling Yang , Zhenbo Xu
a
Department of Laboratory Medicine, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China
b
Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
c
School of Food Science and Engineering, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of
Technology, Guangzhou 510640, China
d
Department of Microbial Pathogenesis, University of Maryland, Baltimore 21201, USA
e
Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), Guangzhou, China

A R T I C LE I N FO A B S T R A C T

Keywords: Haemophilus influenzae remains a common cause of illness in children worldwide. H. influenzae type b is the
Haemophilus influenzae leading cause of bacterial meningitis in children before introduction of vaccination and is a common cause of
Typing pneumonia, epiglottis and septic arthritis. Since the implementation of the Hib conjugate vaccine, the non-
Virulence typeable H. influenzae has rapidly decreased in respiratory and invasive infections in children and adults.
Resistance
However, the rate of antibiotic resistance of H. influenzae varies with region and period and is usually on the rise.
In this review, typing of H. influenzae, virulence factors and resistance will be dissertated.

1. Introduction 2. Typing of H. influenzae

Haemophilus influenzae is a pleomorphic Gram-negative coccoba-
cillus which is responsible for a wide variety of airway mucosal infec-
tions and invasive diseases such as bacterial meningitis. The naso-
pharyngeal carriage rate of H. influenzae was high in young children
and the notification rate was highest for patients < 1 month of age
(Wang et al., 2008; Whittaker et al., 2017). H. influenzae can be dif-
ferentiated into encapsulated (typable) and nonencapsulated (non-
typeable) based upon the presence or absence of capsule. Encapsulated
H. influenzae (serotype a to f), especially the H. influenzae type b (Hib),
were used to be one of the most common causes of lower respiratory
infection. Since the Hib conjugate vaccine was implemented, non-
typeable H. influenzae (NTHi) has detected more frequently in re-
spiratory tract infection and invasive infection in children and adults
for the rapid reduction of Hib infection. However, interest in H. influ-
enzae was also declined by the reduction of Hib infection, which has led
to the underestimate of NTHi and other serotypes of H. influenzae.
Antimicrobial resistance rate of H. influenzae is varied from areas and
periods, with an overall increased trend. In this review, molecular
epidemiology and evolution of H. influenzae will be classified.
2.1. Serologic capsule typing and molecular capsule typing
Serologic capsule typing which is also called standard slide agglu-
tination serotyping (SAST) is the traditional method to classify H. in-
fluenzae into encapsulated and nonencapsulated. Strains may fail to
react with typing sera for inaccuracies in performing and interpreting
slide agglutination tests, capsule-deficient variants caused by partially
deletion of bexA and deletion of the entire capsule locus in a previously
serotypeable strain, which will misclassify encapsulated and none-
ncapsulated. To improve the accuracy of typing, molecular capsule
typing by PCR according to the three functionally defined regions of the
capsule encoding gene cap locus is recommended. The cap locus is
composed of three distinct regions, from I to III. Capsule common gene
bexA in region I and capsule specific (types a to f) genes in region II of
the cap locus are used for molecular capsule typing by PCR (Kroll et al.,
1989; Satola et al., 2007; Davis et al., 2011). However, the method is
not that suitable for researches with a large number of isolates and may
misclassify encapsulated and nonencapsulated for the deletions of bexA.
Thus, a bexB-based method to differentiate true NTHi strains from
typeable strains was developed. Region I genes are present in all cap-
sular types and bexB in region I encodes protein in capsule exportation
is more reliable for the capsule typing than bexA because no bexB
partial deletions was reported yet (Davis et al., 2011). In addition, high

⁎
Corresponding authors at: Department of Laboratory Medicine, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China.
E-mail addresses: jykresearch@126.com (L. Yang), zhenbo.xu@hotmail.com (Z. Xu).
1
Mailing address: School of Food Science and Engineering, South China University of Technology, Guangzhou 510,640, P.R. China.

https://doi.org/10.1016/j.meegid.2020.104205
Received 23 September 2019; Received in revised form 20 January 2020; Accepted 21 January 2020
Available online 22 January 2020
1567-1348/ © 2020 Elsevier B.V. All rights reserved.
S. Wen, et al.
throughput hybridization-based methods is an attractive and efficient
alternative for screening large numbers of H. influenzae strains. Un-
fortunately, none of the available molecular typing methods can detect
complete capsule deletions.
2.2. Biotyping
H. influenzae can be defined into eight different biotypes (I-VIII)
according to the possible combinations of the presence or absence of
indole, urease and/or ornithine decarboxylase production (Schotte
et al., 2019). H. influenzae isolates of different biotypes occasionally
associated with specific diseases has been reported and some serotype
strains were seem to related with distinct biotypes. For instance, the
majority of serotype b isolates are biotype I, especially those from in-
vasive disease (Kilian, 1976).
2.3. Multilocus sequence typing (MLST)
Multilocus sequence typing offers an unambiguous and precise
method for characterizing strains of bacterial pathogens by sequencing
internal fragments of seven housekeeping genes. There are great ad-
vantages of MLST comparing with other typing methods. Isolates
characterized in different laboratories are comparable by MLST, and the
allelic profiles of strains and epidemiological information can be stored
in the database which is available on the Internet (Meats et al., 2003;
Chen et al., 2018) (Table 1).
2.4. Virulence factors of H. influenzae
There are such a lot of virulence factors involve in the pathological
process of H. influenzae infection. For instance, the capsule poly-
saccharide protects H. influenzae from being attacked by leukocyte. The
pili promotes adhesion and participate in biofilm formation which is of
great help for protection and antibiotic resistance. IgA proteases can
destroy the protection of mucosal barrier by IgA and cause mucosal
bacterial pathogens infection. Macrophage survival factor is more
common seen in disease-related H. influenzae strains and may protects
H. influenzae from being phagocytosed and killed by macrophages.
Now, a detailed introduction will be followed.
2.5. Capsule polysaccharide
The capsule polysaccharide is a major virulence factor in the pa-
thogenesis of diseases caused by encapsulated bacteria pathogens. It is
an important defense of outer environment for bacteria and may help
against leukocyte phagocytosis. In addition, capsule promotes bacterial
adherence and leads to infections. Six capsule types (a to f) have been
reported in H. influenzae, and the capsule synthesis is encoded by capB
genes in two copies in the chromosome. The loss of the capsule
synthesis associated copy may lead to a capsule-deficient mutants
(Meats et al., 2003; Kostyanev and Sechanova, 2012). In most of NTHi
strains, adhesion to the host epithelial cells is mediated by two main
groups of adhesion proteins, HMW1/HMW2. HMW1 and HMW2 pro-
teins are highly homologous glycoproteins presenting on the bacterial
surface and are encoded by loci hmw1 and hmw2 (Kostyanev and
Sechanova, 2012; Atack et al., 2015; Rempe et al., 2016). Hia adhesin
Table 1
Comparison of different typing methods.
Typing methods Advantage
Serologic capsule typing (SAST) Traditional and classic
Molecular capsule typing Sensitive and specific than SAST
Biotyping Associated with specific diseases or serotype
Multilocus sequence typing Precise, results are comparable and storable
2
Infection, Genetics and Evolution 80 (2020) 104205
can be found in approximately 25% of the NTHi strains, which is a key
factor in initial colonization of the nasopharynx. The analogue of Hia
presenting in encapsulated H. influenzae is called Hif. Recent study
claimed that, Hia expression may phase vary to escape the immune
response against NTHi. Variants with a low level of Hia may facilitate
their escape from killing by anti-Hia antisera. In addition, HMW ad-
hesins are also phase-variably expressed (Atack et al., 2015).
2.6. Biofilms
Biofilms are communities of microorganisms attached to a surface.
The formation of biofilms is influenced by bacteria pathogens self-fac-
tors such as pili, protein, DNA, lipooligosaccharide, quorum sensing
system, and two-component signaling system (Murphy and Kirkham,
2002; Webster et al., 2006; Unal et al., 2012; Vogel et al., 2012). Bio-
films protect bacteria from environmental, host and chemical stressors,
and may enhance tolerance to antimicrobial. There are several me-
chanisms explaining antimicrobial susceptibility affected by biofilms.
Firstly, the glycocalyx excludes and/or influences the access of anti-
microbial agents to the underlying organisms. Secondly, the surface
regions of the glycocalyx and outlying cells react with the chemically
reactive antimicrobial agents. Thirdly, limited availability of key nu-
trients within the biofilm forces a slowing of the specific growth rate
and lead to a dominance of relatively dormant cells at the base of the
biofilm. Finally, attachment to surfaces causes the cells to derepress/
induce genes associated with a sessile existence which may affect an-
timicrobial susceptibility (Brown and Gilbert, 1993; Reimche et al.,
2017). Biofilms formation bacteria such as H. influenzae are important
cause of otitis media in children and lower respiratory tract infection in
adults with chronic obstructive pulmonary disease (COPD). Nontype-
able H. influenzae, is a common biofilms formation bacteria while it
does not express a capsule, in addition, treatment failure caused by
biofilms formation NTHi has been reported (Brown and Gilbert, 1993;
Mohd-Zain et al., 2012). Along with the decline of Hib infections after
introduction and coverage of the Hib conjugate vaccine, we should
attach importance to the control of NTHi dissemination, for it plays an
increasingly important role in H. influenzae infections.
2.7. IgA proteases
IgA proteases are produced by bacterial pathogens that colonize and
infect at human mucosal surfaces, including H. influenzae. IgA proteases
can cleave the heavy chain of human IgA1 in the hinge region to de-
stroy the protection of mucosal barrier by IgA (Poulsen et al., 1992).
Three types of IgA protease were discovered in H. influenzae, cleaving
different site of the hinge region of human IgA1 and IgA proteases types
are associated with the capsular serotype but not with the biotype of H.
influenzae. Serotypes a, b, d, and f secrete primarily type 1 protease,
while serotypes c and e secrete only type 2 protease. NTHi yields one of
the three protease types (Eton et al., 2017). IgA proteases may con-
tribute to bacterial infection through mechanisms like stimulating
production of proinflammatory cytokines, altering tumor necrosis
factor α signaling, and mediating intracellular persistence (Lorenzen
et al., 1999; Eton et al., 2017). In addition, IgA protease cleavage
fragments in sputum samples from adults with COPD was identified,
providing direct evidence for existence of IgA proteases in the human
Shortcoming
Poor accuracy
Complicated operation and cannot detect complete capsule deletions
Low commonality
Complicated operation and high cost
S. Wen, et al.
respiratory tract, which suggested that IgA proteases may be important
virulence factors of mucosal bacterial pathogens such as H. influenzae
(Murphy et al., 2015; Murphy 1999).
2.8. Macrophage survival factor
There is a novel virulence factor called macrophage survival factor,
msf, reported in H. influenzae. It belongs to the Sel1-like repeats (SLRs)-
containing gene subfamilies, SlrVA, which has a significant association
with the disease isolates. SlrVA subfamily was encoded by slrV genes
located at a common chromosomal locus (SlrV locus 1). The msf was
significantly higher among disease isolates than carriage isolates and
was confirmed important in in vitro macrophage uptake and survival.
Strains lacking slrV gene can be quickly phagocytosed and killed by
macrophages. Moreover, the msf was proposed to survive within human
host cells and contributes to persistence and/or trafficking to new in-
fection sites of H. influenzae (Roop et al., 2016).
2.9. Epidemic characteristics of H. influenzae
H. influenzae is associated with acute otitis media, sinusitis, pneu-
monia and invasive disease such as meningitis and septicemia. Children
and elder people are more commonly affected by H. influenzae infec-
tion, especially by Hib. In addition, pregnancy was associated with a
greater risk of invasive H. influenzae infection (Collins et al., 2014). Hib
infections was the leading cause of bacterial meningitis among pre-
school-aged children before the introduction of conjugate vaccines in
the early 1900s (Adams et al., 1993).
In recent years, the incidences of invasive diseases caused by H.
influenzae have been decreasing rapidly with the availability of the Hib
vaccination worldwide and the epidemiology of invasive H. influenzae
has changed (Adams et al., 1993; Marshall et al., 2014). The Hib con-
jugate vaccine was introduced to US since 1988 and was introduced to
Germany in 1990, followed by England in 1992 (Zielen et al., 1992;
Adams et al., 1993; Marshall et al., 2014). In 1993, the incidence of Hib
invasive infections among children aged < 5 years has declined by 97%
from 1987 in the United States (Adams et al., 1993). In Germany, the
morbidity rate of meningitis due to Hib among children has a sub-
stantial decline within two years (23 per 100,000 to 6 per 100,000)
after the vaccine was introduced (Zielen et al., 1992). The morbidity of
H. influenzae infection was greatly affected by the introduction and
coverage of the Hib conjugate vaccine. However, the Hib conjugate
vaccine was introduced to Asia far behind introductions in Europe and
America, especially in the developing countries, which has led to a wide
variation in epidemiology of H. influenzae around the world. The Hib
vaccination was available in Japan since 2008, but the vaccination rate
was low until it became a routine vaccination supported by the gov-
ernment (Sakata et al., 2017). While in China and many other devel-
oping countries, the Hib vaccination is still voluntary, resulting in a low
vaccination rate. The pooled overall coverage of Hib conjugate vaccine
was 54.9% in China, and coverage was higher in the east than in the
central and west parts of the country (Yang et al., 2019). In recent
years, the nasopharyngeal carriage rate of H. influenzae is 26.3% in
children younger than 5 years with acute upper respiratory tract in-
fection of China (Wang et al., 2008). While H. influenzae carriage was
detected in 37% of the children and Hib carriage rate was 3% in
Vietnam (Yoshida et al., 2013). In Nepal, H. influenzae (38.9%) was
reported as the leading cause of meningitis in young children (Shrestha
et al., 2015). Similar conditions are to be found in other developing
countries (Zaidi et al., 2010; Shenoy et al., 2016). Thus H. influenzae
infection is still a public health concern over the world, especially for
the developing countries, where incidence of invasive infection was
much higher than that of the developed countries (Wang et al., 2008;
Shrestha et al., 2015).
H. influenzae type b was responsible for > 80% of invasive H. in-
fluenzae infections before Hib vaccination was implemented, which is
3
Infection, Genetics and Evolution 80 (2020) 104205
the most virulent serotype (Peltola 2000). Along with the widely in-
troduction of Hib vaccination, the incidence of Hib infection has led to
great reduction with relative increase in occurrence of the other ser-
otypes and NTHi strains (Whittaker et al., 2017). Moreover, the in-
vasive infections are more often attributed to unencapsulated strains
and a constant increase has seen in invasive NTHi worldwide (Schotte
et al., 2019; Van et al., 2014). In North America, the current epide-
miological data suggest that invasive Hia disease predominantly affects
Indigenous communities. In addition, ST23 is responsible for most in-
vasive disease in North America and is the predominant clone described
on the H. influenzae MLST website (Tsang and Ulanova, 2017). While in
Canada, for all invasive cases caused by H. influenzae, approximately
54.6% isolates were NTHi during 2007 to 2014, far exceed the serotype
a (23.1%) and b (8.3%) (Tsang et al., 2017). Epidemiological in-
vestigations of invasive diseases in the post-conjugate vaccine era from
Europe and United States revealed that invasive H. influenzae disease is
predominantly caused by NTHi and serotype f (MacNeil et al., 2011;
Ladhani et al., 2012). In Asia, patients with H. influenzae associated
community-acquired respiratory infections caused by NTHi were as
high as 81% in China, during 2008 to 2009 (Qin et al., 2012). Biotype II
which is more related to NTHi is seen more frequently than other
biotype now (Schotte et al., 2019). Obviously, NTHi has become an-
other new burden in causing lower respiratory infection and invasive
diseases, highlighting the importance and emergency of investigating
the mechanisms of invasiveness in the absence of capsule.
2.10. Resistance of beta-lactams in H. influenzae
Infections caused by H. influenzae are usually treated with beta-
lactam antimicrobial agents, Aminopenicillins and cephalosporins are
the first choice for treatment of H. influenzae infections (Schotte et al.,
2019). Resistance mechanisms to beta-lactams in H. influenzae are in-
cluding enzymatic and nonenzymatic mechanism. The most common
resistant mechanism to beta-lactams in H. influenzae is production of
beta-lactmases. The beta-lactamase producing H. influenzae was 15.0%
overall but varied greatly by country, from < 5% in several countries to
67.9% in Taiwan (Farrell et al., 2005). In addition, significant increase
in beta-lactamase-producing isolates was observed (Tsang et al., 2017).
Nonenzymatic mechanism to beta-lactams in H. influenzae can be
mediated by modifications in cell permeability, defects in the autolytic
system and in overall peptidoglycan synthesis and metabolism, or
amino acid substitutions in penicillin-binding protein 3 (PBP3) encoded
by the ftsI gene, and alterations of the target PBPs is the most common
nonenzymatic mechanism involved in beta-lactmase resistance
(Clairoux et al., 1992).
ROB-1 and TEM-1, are distinct beta-lactamases confer high-level
resistance to ampicillin and other aminopenicillins, which have been
reported in H. influenzae (Sondergaard and Norskov-Lauritsen, 2016).
TEM-1 positive H. influenzae was disseminated worldwide while ROB-1
positive H. influenzae was mainly found in Canada, the USA and Mexico
during1999 to 2003. Prevalences of TEM-1 and ROB-1 beta-lactamase
producing H. influenzae were 90–95% and 5–10%, respectively. Isolates
with both enzymes were also reported (Scriver et al., 1994). There are
susceptibility difference between the two beta-lactamases. The ROB-1
subpopulation showed increased cefaclor MIC and resistance compared
with TEM-1 isolates (Scriver et al., 1994; Farrell et al., 2005). The
variation in cefaclor MICs in ROB-1-producing isolates may associated
with PBP3 substitutions and altered PBP3 in combination with TEM-1 is
well recognized in H. influenzae before (Tristram et al., 2010). Some
beta-lactamase-positive H. influenzae strains were negative for both
ROB-1 and TEM-1 genes suggesting either a mutation has occurred in
either or both ROB-1 and TEM-1 gene(s) to prevent detection by the
current methodology, or a previously undescribed enzyme is re-
sponsible (Farrell et al., 2005). ROB-1 and TEM-1 are located on large
integrative conjugative elements (ICEs) or carried on small plasmids,
thus the resistance to beta-lactams is horizontal transferable among
S. Wen, et al.
different strains (Leaves et al., 2000).
H. influenzae isolates resistant to ampicillin without producing an
beta-lactmase is called beta-lactmase-negative ampicillin resistance
(BLNAR). The mechanism basis of BLNAR is alteration of PBP3 protein,
encoded by the ftsI gene. Moreover, increasing resistance of Cefuroxime
in nonenzymatic H. influenzae is also linked to mutations in ftsI (Straker
et al., 2003). Genotypic characterization of BLNAR strains occurs
through sequencing of the amino acid substitutions presence of the ftsI
gene, which classified BLNAR strains into three groups (group I to III)
(Ubukata et al., 2001). Base on the level of resistance, BLNAR can be
divided into two groups, the high-level resistant group and the low-
level resistant group. The high-level resistant group was belonging to
the genotypic group III, which was commonly found in Asia. The low-
level resistant group, belonging to groups I and II, are widespread
around the world (Skaare et al., 2014).
In the early 21st century, BLNAR is still rare in China. While in
2008, 29.8% of H. influenzae associated community-acquired re-
spiratory tract infections were caused by BLNAR in Shanghai, and the
proportion was 22.1% in Guangzhou during 2016 to 2017 (Qin et al.,
2012; Chen et al., 2018). In recent years, significant increase of BLNAR
were documented worldwide, and wide variations exist in different
countries (Ubukata et al., 2001; Qin et al., 2012; Schotte et al., 2019).
For instance, only 6.8% of the H. influenzae isolates were BLNAR during
2013 to 2016 in Belgium (Schotte et al., 2019). On the other hand, the
BLNAR has been documented as high as 28.8% in Japan in
1997(Ubukata et al., 2001). Although H. influenzae infections causing
by Hib have declined by the availability of Hib conjugate vaccine, the
epidemiology and resistance of H. influenzae seemed change after then.
2.11. Fluoroquinolones-resistant H. influenzae
Fluoroquinolones (FQs), was frequently used as antimicrobial
therapy in respiratory tract infections in adults. FQs-resistant H. influ-
enzae was first reported in 1993 and the first report of FQs-resistance H.
influenzae in children was from Hong Kong in 2004 (Ho et al., 2004). In
recent years, FQs-resistant H. influenzae increased quickly and spread
worldwide, with a variety in epidemiology (Ho et al., 2004; Li et al.,
2004; Chang et al., 2010; Puig et al., 2015). In Taiwan, prevalence of
levofloxacin-resistance H. influenzae (the proportion was as high as
41.7%) was reported and cloning spread has occurred in the nursing
home residents (Chang et al., 2010). While in mainland China, only
2.7% ciprofloxacin-resistant H. influenzae were detected (Chen et al.,
2018). Nevertheless, the proportion of ciprofloxacin-resistance H. in-
fluenzae in Spain during 2000 to 2013 was low and remained stable
(Puig et al., 2015). No levofloxacin-resistant H. influenzae isolates were
found in Pakistan between 2009 and 2010 (Furqan and Paracha, 2014).
Although the FQs-resistant H. influenzae was not so common as the
BLNAR, levofloxacin treatment failure in H. influenzae relative pneu-
monia has been reported, which should rise the public concern in
preventing the dissemination of FQs-resistant H. influenzae (Vila et al.,
1999). Resistance to FQs is related to chromosome-mediated mutations
in the quinolone resistance–determining regions (QRDRs) of the genes
encoding DNA gyrase and topoisomerase IV, including gyrA, gyrB, parC
and parE (Georgiou et al., 1996). Amino acid substitutions in gyrA (at
Ser84 and Asp88) and parC (at Gly82, Ser84 and Glu88) are more
common than in gyrB and parE (Shoji et al., 2014).
In general, resistance to FQs developed in a stepwise manner, and
DNA gyrase gyrA was considered as the first step of the mechanism.
Increasing numbers of mutations in QRDRs was believed to enhance the
resistance of FQ (Puig et al., 2015). H. influenzae isolates with amino
acid substitutions in gyrA may remain in the susceptible range ac-
cording to the current breakpoints in CLSI (Ho et al., 2004). Nalidixic
acid can be used for the detection of decreased susceptibility to qui-
nolones in H. influenzae (Ho et al., 2004; Perez-Vazquez et al., 2004).
However, NAL screening was not applied routinely in determination of
the susceptibilities of H. influenzae in clinics yet.
4
Infection, Genetics and Evolution 80 (2020) 104205
2.12. Other antibiotic resistance in H. influenzae
Macrolides is widely used to treat with NTHi-related respiratory
infections, although low-level intrinsic resistance to macrolide has been
reported. There are some principle resistance mechanisms of macro-
lides in H. influenzae which including the acquisition of the drug efflux
pump encoded by resistance gene mefA, the overexpression of chro-
mosomal multidrug efflux pumps, the acquisition of 23S rRNA methy-
lase encoded by resistance gene ermB and amino acid substitutions in
ribosomal proteins that lead to decreased affinity for macrolides
(Atkinson et al., 2017; Tsuji et al., 2018). Chloramphenicol and tetra-
cycline resistance in H. influenzae via plasmid-mediated chlor-
amphenicol acetyltransferase production was most frequently reported
and the acquired resistance of the H. influenzae transcipients was re-
transferable (Van et al., 1977). Besides, a relative permeability barrier
due to the loss of an outer membrane protein was proposed in chlor-
amphenicol resistance (Burns et al., 1985).
3. Future work
Along with the introduction of Hib conjugate vaccine, epidemiology
of H. influenzae has changed in recent years. NTHi and other serotypes
of H. influenzae has become more prevalence than Hib around the
world. As the overall coverage of Hib conjugate vaccine in developing
countries are not that common, H. influenzae is still a threaten pathogen
causing community acquired pneumonia and invasive diseases for
public health. Rapidly increased antibiotic resistance in H. influenzae
has led to treatment failure and which is causing serious burdens to the
publics. Continuous monitoring of the antibiotic resistance and mole-
cular evolution in H. influenzae is extremely necessary. To explore how
biofilm formation H. influenzae involves in the pathological process of
infection may help for clinical treatment. Moreover, constant vigilance
and precaution such as new vaccine development is necessary to re-
spond to new epidemiology trend of NTHi strains and other serotypes of
H. influenzae worldwide.
Declaration of Competing Interest
The authors report no conflicts of interest.
Acknowledgements
This work is supported by the research grants from the Natural
Science Foundation of China (No. 81974318), Guangdong Province
Science and Technology Innovation Strategy Special Fund (No.
2019B020209001), Natural Science Foundation of Guangdong Province
(Nos. 2018A030310170 and 2018A030313279) and the Guangdong
Bureau of Traditional Chinese Medicine (No. 20191206).
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