Molecular epidemiology and evolution of Haemophilus influenzae

Journal Pre-proof Molecular influenzae

epidemiology

and

evolution

of

Haemophilus

Shuxian Wen, Donghua Feng, Dingqiang Chen, Ling Yang, Zhenbo Xu PII:

S1567-1348(20)30037-X

DOI:

https://doi.org/10.1016/j.meegid.2020.104205

Reference:

MEEGID 104205

To appear in:

Infection, Genetics and Evolution

Received date:

23 September 2019

Revised date:

20 January 2020

Accepted date:

21 January 2020

Please cite this article as: S. Wen, D. Feng, D. Chen, et al., Molecular epidemiology and evolution of Haemophilus influenzae, Infection, Genetics and Evolution(2019), https://doi.org/10.1016/j.meegid.2020.104205

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© 2019 Published by Elsevier.

Journal Pre-proof

Molecular epidemiology and evolution of Haemophilus influenzae Shuxian Wen1, Donghua Feng1, Dingqiang Chen2, Ling Yang1*, Zhenbo Xu3,4,5* 1

Department of Laboratory Medicine, The First Affiliated Hospital of Guangzhou Medical

University, Guangzhou, Guangdong, China 2

Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University,

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Guangzhou, Guangdong, China School of Food Science and Engineering, Guangdong Province Key Laboratory for Green

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Processing of Natural Products and Product Safety, South China University of Technology,

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Guangzhou 510640, China

Department of Microbial Pathogenesis, University of Maryland, Baltimore 21201, USA

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Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human

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Health (111 Center), Guangzhou, P.R. China

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*Corresponding author: Ling Yang, M. D.

Address: Department of Laboratory Medicine, First Affiliated Hospital of Guangzhou Medical

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University, Guangzhou 510120, China. Tel: +86-20-83062158

Fax: +86-20-83062158

E-mail address: [email protected]

* Corresponding author: Zhenbo Xu, Ph.D. Mailing address: School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, P.R. China. Tel: +86-20-87113252 Fax: +86-20-87113252 E-mail address: [email protected]

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Journal Pre-proof Abstract Haemophilus influenzae remains a common cause of illness in children worldwide. H. influenzae type b is the leading cause of bacterial meningitis in children before introduction of vaccination and is a common cause of pneumonia, epiglottis and septic arthritis. Since the implementation of the Hib conjugate vaccine, the non-typeable H. influenzae has rapidly decreased in respiratory and invasive infections in children and adults. However, the rate of antibiotic resistance of H. influenzae varies with region and period and is usually on the rise. In

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this review, typing of H. influenzae, virulence factors and resistance will be dissertated.

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Key words: Haemophilus influenzae; typing; virulence; resistance

Introduction

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Haemophilus influenzae is a pleomorphic Gram-negative coccobacillus which is responsible

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for a wide variety of airway mucosal infections and invasive diseases such as bacterial meningitis. The nasopharyngeal carriage rate of H. influenzae was high in young children and the notification

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rate was highest for patients <1 month of age (Wang et al., 2017; Whittaker et al., 2017). H. influenzae can be differentiated into encapsulated (typable) and nonencapsulated (nontypeable)

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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, nontypeable H. influenzae (NTHi) has detected more frequently in respiratory tract infection and invasive infection in children and adults for the rapid reduction of Hib infection. However, interest in H. influenzae 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.

Typing of H. influenzae Serologic capsule typing and molecular capsule typing Serologic capsule typing which is also called standard slide agglutination serotyping (SAST) 2

Journal Pre-proof is the traditional method to classify H. influenzae 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 nonencapsulated. 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

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for molecular capsule typing by PCR (Kroll et al., 1989; Satola et al., 2007; Davis et al., 2011).

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However, the method is not that suitable for researches with a large number of isolates and may

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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

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present in all capsular types and bexB in region I encodes protein in capsule exportation is more

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reliable for the capsule typing than bexA because no bexB partial deletions was reported yet (Davis et al., 2011). In addition, high throughput hybridization-based methods is an attractive and

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efficient alternative for screening large numbers of H. influenzae strains. Unfortunately, none of the available molecular typing methods can detect complete capsule deletions.

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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 invasive disease (Kilian et al., 1976).

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 advantages of MLST comparing with other typing methods. Isolates characterized in different laboratories are comparable by MLST, and the allelic profiles of strains and 3

Journal Pre-proof epidemiological information can be stored in the database which is available on the Internet (Meats et al., 2013; Chen et al., 2018). Table 1. Comparison of different typing methods Typing methods

advantage

shortcoming

serologic capsule typing

traditional and classic

poor accuracy

molecular capsule typing

sensitive and specific than SAST

complicated operation and can not detect complete capsule deletions

biotyping

associated with specific diseases or serotype

multilocus sequence typing

precise, results are comparable and storable

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（SAST）

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Virulence factors of H. influenzae

complicated operation and high cost

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low commonality

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There are such a lot of virulence factors involve in the pathological process of H. influenzae infection. For instance, the capsule polysaccharide protects H. influenzae from being

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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. Capsule polysaccharide The capsule polysaccharide is a major virulence factor in the pathogenesis 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 4

Journal Pre-proof 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 proteins 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). Hia adhesin 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.

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Variants with a low level of Hia may facilitate their escape from killing by anti-Hia antisera. In

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addition, HMW adhesins are also phase-variably expressed (Atack et al., 2015).

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Biofilms

Biofilms are communities of microorganisms attached to a surface. The formation of biofilms

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is influenced by bacteria pathogens self-factors such as pili, protein, DNA, lipooligosaccharide,

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quorum sensing system, and two-component signaling system (Murphy et al., 2002; Webster et al., 2006; Unal et al., 2012; Vogel et al., 2012). Biofilms protect bacteria from environmental, host and

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chemical stressors, and may enhance tolerance to antimicrobial. There are several mechanisms explaining antimicrobial susceptibility affected by biofilms. Firstly, the glycocalyx excludes and/or

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influences the access of antimicrobial 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 nutrients 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 antimicrobial susceptibility (Brown and Gilber, 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). Nontypeable 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 Gilber, 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 5

Journal Pre-proof control of NTHi dissemination, for it plays an increasingly important role in H. influenzae infections. 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 destroy 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

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biotype of H. influenzae. Serotypes a, b, d, and f secrete primarily type 1 protease, while serotypes

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c and e secrete only type 2 protease. NTHi yields one of the three protease types (Eton et al., 2017). IgA proteases may contribute to bacterial infection through mechanisms like stimulating

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production of proinflammatory cytokines, altering tumor necrosis factor α signaling, and

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mediating intracellular persistence (Lorenzen et al., 1999; Eton et al., 2017). In addition, IgA

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protease cleavage fragments in sputum samples from adults with COPD was identified, providing direct evidence for existence of IgA proteases in the human respiratory tract, which suggested that

influenzae (Murphy. 1999).

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IgA proteases may be important virulence factors of mucosal bacterial pathogens such as H.

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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 infection sites of H. influenzae (Roop et al., 2016).

Epidemic characteristics of H. influenzae H. influenzae is associated with acute otitis media, sinusitis, pneumonia and invasive disease 6

Journal Pre-proof such as meningitis and septicemia. Children and elder people are more commonly affected by H. influenzae infection, 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 preschool-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 conjugate

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vaccine was introduced to US since 1988 and was introduced to Germany in 1990, followed by

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England in 1992 (Zielen et al., 1992; Adams et al., 1993; Marshall et al., 2014). In 1993, the

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incidence of Hib invasive infections among children aged less than 5 years has declined by 97% from 1987 in the United States (Adams et al., 1993). In Germany, the morbidity rate of meningitis

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due to Hib among children has a substantial decline within two years (23 per 100000 to 6 per

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100000) 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.

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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

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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 government (Sakata et al., 2017). While in China and many other developing 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 infection 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 7

Journal Pre-proof the developing countries, where incidence of invasive infection was much higher than that of the developed countries (Wang et al., 2017; Shrestha et al. 2015). H. influenzae type b was responsible for more than 80% of invasive H. influenzae infections before Hib vaccination was implemented, which is the most virulent serotype (Peltola 2000). Along with the widely introduction of Hib vaccination, the incidence of Hib infection has led to great reduction with relative increase in occurrence of the other serotypes and NTHi strains (Whittaker et al., 2017). Moreover, the invasive infections are more often attributed to unencapsulated strains and a constant increase has seen in invasive NTHi worldwide (Schotte et

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al., 2019; Van et al., 2014). In North America, the current epidemiological data suggest that

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invasive Hia disease predominantly affects Indigenous communities. In addition, ST23 is

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responsible for most invasive 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

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cases caused by H. influenzae, approximately 54.6% isolates were NTHi during 2007 to 2014, far

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exceed the serotype a (23.1%) and b (8.3%) (Tsang et al., 2017). Epidemiological investigations of invasive diseases in the post-conjugate vaccine era from Europe and United States revealed that

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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

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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 another 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.

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 including 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 less than 5% in several countries to 67.9% in 8

Journal Pre-proof 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 et al., 2016).

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TEM-1 positive H. influenzae was disseminated worldwide while ROB-1 positive H. influenzae

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was mainly found in Canada, the USA and Mexico during1999 to 2003. Prevalences of TEM-1

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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

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difference between the two beta-lactamases. The ROB-1 subpopulation showed increased cefaclor

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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

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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

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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 responsible (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 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 9

Journal Pre-proof 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 respiratory 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,

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and wide variations exist in different countries (Ubukata et al., 2001; Qin et al., 2012; Schotte et

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al., 2019). For instance, only 6.8% of the H. influenzae isolates were BLNAR during 2013 to 2016

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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

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influenzae seemed change after then.

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have declined by the availability of Hib conjugate vaccine, the epidemiology and resistance of H.

Fluoroquinolones-resistant H. influenzae

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Fluoroquinolones (FQs), was frequently used as antimicrobial therapy in respiratory tract infections in adults. FQs-resistant H. influenzae was first reported in 1993 and the first report of

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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. influenzae 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 pneumonia has been reported, which should rise the public concern in preventing the dissemination of FQs-resistant H. influenzae (Vila et al., 1999). 10

Journal Pre-proof 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

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substitutions in gyrA may remain in the susceptible range according to the current breakpoints in

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CLSI (Ho et al., 2004). Nalidixic acid can be used for the detection of decreased susceptibility to

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quinolones 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

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clinics yet.

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Other antibiotic resistance in H. influenzae

Macrolides is widely used to treat with NTHi-related respiratory infections, although

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low-level intrinsic resistance to macrolide has been reported. There are some principle resistance mechanisms of macrolides in H. influenzae which including the acquisition of the drug efflux

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pump encoded by resistance gene mefA, the overexpression of chromosomal multidrug efflux pumps, the acquisition of 23S rRNA methylase 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 tetracycline resistance in H. influenzae via

plasmid-mediated chloramphenicol acetyltransferase production was most frequently reported and the acquired resistance of the H. influenzae transcipients was retransferable (Van et al., 1977). Besides, a relative permeability barrier due to the loss of an outer membrane protein was proposed in chloramphenicol resistance (Burns et al., 1985).

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 11

Journal Pre-proof 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 molecular 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 respond to new epidemiology trend

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of NTHi strains and other serotypes of H. influenzae worldwide.

Acknowledgements

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This work is supported by the research grants from the Natural Science Foundation of China

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(No. 81974318), Guangdong Province Science and Technology Innovation Strategy Special Fund (No. 2019B020209001), Natural Science Foundation of Guangdong Province (No s.

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Medicine (No. 20191206).

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2018A030310170 and 2018A030313279) and the Guangdong Bureau of Traditional Chinese

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Conflict of Interest form

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The authors report no conflicts of interest

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Highlights

1. Several common typing methods for Haemophilus influenza were introduced. 2. The virulence factors and epidemiological characteristics of Haemophilus influenza were discussed.

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3. The resistance of Haemophilus influenza to three antibiotics were introduced.

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