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ORIGINAL RESEARCH

High Prevalence of Group III-Like Mutations Among BLPACR and First Report of Haemophilus influenzae ST95 Isolated from Blood in China

Jiansheng Lin1 School of Public Health of Fujian Medical University, Fuzhou, People’s Republic of China;2 Microbiology Laboratory, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of ChinaORCID Iconhttps://orcid.org/0000-0002-1370-9217View further author information
ORCID Icon,
Yinna Wang2 Microbiology Laboratory, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of ChinaView further author information
,
Chunli Lin2 Microbiology Laboratory, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of ChinaView further author information
,
Ran Li3 Department of Anesthesiology, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, People’s Republic of ChinaCorrespondence[email protected]
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&
Gaoxiong Wang1 School of Public Health of Fujian Medical University, Fuzhou, People’s Republic of China;4 Research Administration Office, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of ChinaCorrespondence[email protected]
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Pages 999-1008 | Received 04 Dec 2022, Accepted 14 Feb 2023, Published online: 17 Feb 2023

Abstract

Purpose

We aimed to evaluate antibiotic resistance and molecular epidemiological characteristics of non-invasive Haemophilus influenzae (H. influenzae) from pneumonia patients and analyze the whole genome of one invasive H. influenzae isolated from blood in pediatric patients.

Methods

Antibiotic susceptibility was tested using the turbidimetric method. β-lactamase-producing and serotyping genes were evaluated via multiplex polymerase chain reaction (PCR), and ftsI was amplified using high-fidelity PCR. Lastly, whole genome sequencing (WGS) was conducted using Illumina HiSeq and PacBio sequencing technology.

Results

We observed that the ampicillin (AMP) and amoxicillin/clavulanate (AMC) resistance rates of non-invasive H. influenzae were as high as 99.06% (after adjustment) and 49.53%, respectively. The β-lactamase gene of 106 AMP-resistant strains was blaTEM-1. Group III-like mutation accounted for 71.15% of β-lactamase-positive, AMC-resistant (BLPACR) strain mutants. The novel Asn-526→His mutation was present in one β-lactamase-negative AMP-susceptible (BLNAS) strain. Non-invasive H. influenzae strains all belonged to non-typeable H. influenzae (NTHi). In contrast, the invasive H. influenzae 108 isolated from blood in China belonged to H. influenzae type b (Hib). It belonged to sequence typing ST95 and exhibited sensitivity to all 11 antibiotics. Three prophages were identified, and the capb loci of the H. influenzae strain 108 revealed regions I–III exist in duplicate; however, complete deletion of IS1016 was only present in one of the copies.

Conclusion

Non-invasive H. influenzae NTHi with β-lactamase-positive was highly prevalent. Notably, group III-like mutations had increased prevalence among BLPACR strains. H. influenzae belonging to Hib and ST95 was first reported to cause sepsis in China.

Introduction

H. influenzae, a gram-negative bacillus that commonly colonizes the nasopharynx of humans, is an opportunistic pathogen that can cause upper respiratory tract infection, pneumonia, asthma, other respiratory diseases, and severe systemic infections such as purulent meningitis and sepsis.Citation1,Citation2 Invasive H. influenzae disease is defined as a H. influenzae infection isolated from sterile sites such as blood, pleural fluid, peritoneal fluid, cerebrospinal fluid etc. Children under five years of age, especially those under one year of age, are more likely to develop invasive H. influenzae disease. It can also occur in individuals without any underlying diseases. It is more often severe, with a high mortality rate or various sequelae left in survivors.Citation2,Citation3 Non-invasive H. influenzae disease has a better prognosis; however, the disease burden remains high owing to its high occurrence in children.

H. influenzae is divided into two types: encapsulated type and non-encapsulated type. There are six serotypes: a, b, c, d, e, and f, often abbreviated as Hia, Hib, Hic, Hid, Hie, and Hif, respectively. NTHi is non-encapsulated and does not agglutinate with either type of serum. In a few cases, the encapsulated strains ceased producing capsules to adapt to the environment. Therefore, simple serotyping will be misjudged as NTHi, and capsular gene testing is required. In recent years, Hib infections have become rare with the widespread introduction of the Hib vaccine. Previous population-based studies in Hefei and Nanning, China, found that the incidence of Hib meningitis was 10.66 and 0.98 cases/100,000 children <5 years of age in the pre- and post-vaccine eras, respectively.Citation4,Citation5 However, the proportion of Hia and Hif in the infected population in some regions has increased,Citation3,Citation6 and NTHi has become the most common pathogen of H. influenzae disease globally.Citation7 The gene sequence IS1016 has been associated with an increased capacity to cause severe infections. It is usually present in encapsulated strains but is sometimes harbored by NTHi.Citation8 In nasopharyngeal colonization, NTHi can easily exchange large DNA fragments between strains. This exchange is done to obtain new virulence factors to enhance its ability to resist human neutrophils and complement-mediated bactericidal effects.Citation9,Citation10 Consequently, NTHi is already the most common cause of invasive H. influenzae disease in children under one year of age.Citation11

The preferred drugs for children infected with H. influenzae include oral ampicillin (AMP), amoxicillin/clavulanate (AMC), and an intravenous infusion of cefotaxime (CTX) and ceftriaxone. With the widespread use of antibiotics, the increasing trend of the H. influenzae resistance rate to various antibacterial drugs has received extensive clinical attention. There are two main mechanisms of β-lactam antibiotic resistance in H. influenzae. One mechanism is the inactivation of AMP by hydrolysis of β-lactamase (TEM-1 or ROB-1 type encoded by blaTEM-1 or blaROB-1 gene, respectively). H. influenzae with β-lactamase-positive AMP resistance is called β-lactamase-positive, AMP-resistant (BLPAR). The other mechanism is that an amino acid site mutation of the penicillin-binding protein (PBP3) encoded by ftsI causes changes in the spatial conformation, which reduces its affinity for AMP. H. influenzae with ftsI mutation for AMP resistance without producing β-lactamase is called β-lactamase negative, AMP resistant (BLNAR).Citation12 AMC-resistant strain with β-lactamase-positive is called β-lactamase-producing AMC-resistant (BLPACR), often occurring in ftsI mutations.Citation13

A recent multicenter study in China showed that the resistance rates of H. influenzae to AMP was increasing year by year, and there were statistically significant differences in the resistance rates of all antibiotics in different regions except chloramphenicol.Citation14 Since the introduction of the Hib vaccine in Fujian, China, in 2003, there has been no research on antibiotic resistance and molecular epidemiological characteristics of H. influenzae. Therefore, in this study, we investigated the antibiotic resistance and molecular characterization of H. influenzae isolated from pediatric children in Quanzhou, China, to provide a basis for the prevention and treatment of H. influenzae disease.

Materials and Methods

Strains

A total of 108 H. influenzae cases were collected from hospitalized children in Quanzhou Women’s and Children’s Hospital from July 2021 to August 2022. The mean age of the children was 1.56±0.16 years. There were 77 boys and 31 girls, and the sex ratio was 2.48. The non-invasive H. influenzae inclusion criteria were <10 squamous epithelium and >25 white blood cells (WBC) in each low-power field of view to avoid the collection of colonized H. influenzae. Gram-negative bacilli were seen in the WBC. In addition, there were evident clinical features of H. influenzae community-acquired pneumonia. Invasive H. influenzae inclusion criteria was that H. influenzae was cultured from double blood culture bottles. Repeated isolates from the same infection sites of the same patients were excluded. Among the 108 H. influenzae strains, 106 (98.15%) non-invasive strains were isolated from sputum, one (0.92%) non-invasive strain from bronchoalveolar lavage fluid, and one (0.92%) named invasive H. influenzae 108 from the blood of an unvaccinated pediatric patient. This pediatric patient was clinically characterized by fever, headache, and vomiting. During the first three days of hospitalization, ceftazidime (CAZ) was used for anti-infection. According to 1.151×109/L WBC in cerebrospinal fluid, positive culture for H. influenzae and the 135.12 mg/L value of C-reactive protein, vancomycin + meropenem were given for the next 3 days. After 6 days of treatment, the symptoms improved significantly and he was discharged from the hospital. H. influenzae strains were stored at −80℃ in BHI-sheep blood-20% glycerol storage solution. This study complied with the Declaration of Helsinki, and ethical permission for this study was approved by the Quanzhou Women’s and Children’s Hospital ethics committee (2021 Ethical Review No. 16). Guardians provided written informed consent on behalf of the minors. H. influenzae strains were identified by VITEK 2 bioMérieux bacterial identification instrument. H. influenzae ATCC 49247 was the quality control strain.

Antibiotic Susceptibility Test

Antibiotic susceptibility testing was performed using the turbidimetric method (similar to microdilution) of the ATB HAEMO CLSI (12) strip from bioMérieux. Antibiotics that were evaluated include AMP, AMC, Cefoxitin (CEP), Cefaclor (CEC), CAZ, Cefotaxime (CTX), Ofloxacin (OFX), Rifampicin (Rif), Chloramphenicol (CHL), Tetracycline (TCY), and Cotrimoxazole (SXT). First, H. influenzae colonies were cultured for 24 hours, and the bacterial suspension was adjusted to 0.5 McF concentration (0.85% NaCl). Subsequently, 50 µL of bacterial suspension was transferred to ATB S medium. After mixing, 135 µL of suspension was added to each well of the ATB HAEMO CLSI (12) reagent strip. The strip was then placed in a CO2 incubator for 20 h. Finally, the results were read automatically using an ATB instrument. The results were interpreted based on the breakpoints recommended by the CLSI M100-S23 2013 committee for H. influenzae. The quality control strain used was H. influenzae ATCC 49247.

Detection of β-Lactamase-Producing Genes and Serotype Capsular Genes

β-lactamase-producing genes and serotype capsular genes were assessed by the Multiplex PCR method. Bacterial DNA was extracted according to the instructions of the TIANamp Bacteria DNA Kit. The PCR amplification primer sequences and product lengths of β-lactamase-producing genes (blaTEM-1 and blaROB-1) and serotype capsular genes (a–f) are listed in Table S1. Multiplex PCR reagents were purchased from Vazyme. The total reaction volume of 50 µL consisted of 7 µL RNase-free ddH2O, 25 µL 2× Multiplex buffer, 5 µL 10× primer mix (Premix all amplification primers so that the concentration of each primer is 1 µM), 2 µL template DNA, 1 µL Multiplex DNA polymerase, and 10 µL 5× Multiplex GC Enhancer. The thermal cycle was programmed for 5 min at 95℃ for pre-denaturation, followed by 35 cycles of 30s at 95℃ for denaturation, 90s at 60℃ for annealing, and 90s at 72℃ for extension; a final extension was conducted for 10 min at 72℃. The PCR products were observed using agarose gel electrophoresis.

Amplification and Sequencing of fstI Gene

The ftsI gene was amplified by high-fidelity PCR. The PCR amplification primer sequences and product lengths of ftsI are listed in Table S1. High-fidelity PCR was purchased from Vazyme. The total reaction volume of 50 µL consisted of 20 µL ddH2O, 25 µL 2× Phanta Max Master Mix, 2 µL primer F, 2 µL primer R, and 1 µL template DNA. The thermal cycle was programmed for 3 min at 95℃ for pre-denaturation, followed by 32 cycles of 15s at 95℃ for denaturation, 15s at 58℃ for annealing, 60s at 72℃ for extension; a final extension was conducted for 5 min at 72℃. The PCR products were observed using agarose gel electrophoresis.

Subsequently, the PCR products were sent to Fuzhou Shangya Biological Co., Ltd. for Sanger sequencing. Amino acids 330–530 of PBP3, including the highly conserved motifs Ser-Thr-Val-Lys (STVK), Lys-Thr-Gly (KTG), and Ser-Ser-Asn (SSN), were compared with the corresponding sequence of H. influenzae Rd KW20. The PBP3 mutation patterns were determined according to the rules reported in the literature:Citation15 Group I was defined as Arg-517→His-517. Group II as Asn-526→Lys-526). Group II was further divided into four subgroups. IIa: only Asn-526→Lys-526; IIb: Asn-526→Lys-526 and Ala-502→Val-502; group IIc: Asn-526→Lys-526 and Ala-502→Thr-502; group IId: Asn-526→Lys-526 and Ile-449→Val. Group III was defined as Met-377→Ile, Ser-385→Thr, Leu-389→Phe, and Asn-526→Lys). Group III-like was defined as Met-377→Ile, Ser-385→Thr, and/or Leu-389→Phe in the SSN motif, Arg-517→His and Thr-532→Ser in the KTG motif, Asp-350→Asn in the STVK motif; and finally, Ser-357→Asn.

Whole Genome Sequencing (WGS)

The H. influenzae 108 isolated from blood was sent to Majorbio Bio-Pharm Technology Co., Ltd. for WGS. In this study, whole genome de novo sequencing was performed using the second-generation Illumina HiSeq and third-generation PacBio sequencing methods. No less than 100× Illumina sequencing data of the genome and 100× PacBio sequencing data were provided to ensure a complete and accurate assembly while avoiding the loss of small plasmid (<15 kb) information. Further, this information ensured that the complete genome contained the plasmid. The genome coverage of H. influenzae 108 strain was 100% for this sequencing. The Hicap tool (https://github.com/scwatts/hicap), online bioinformatic tools (http://www.genomicepidemiology.org/), the comprehensive antibiotic resistance database (https://card.mcmaster.ca/), and Abricate v.0.8 (https://github.com/tseemann/abricate) with the VFDB database (http://www.mgc.ac.cn/Vfs/) were used to analyze the serotypes, sequence types, resistomes, and virulomes. A >80% for gene identity was considered as the threshold for predicting virulence genes. In addition, PHASTER (https://phaster.ca/) was used to predict the prophage regions. A CLC sequence viewer was used to investigate single nucleotide polymorphisms (SNPs) of the cap loci, the complete deletion of the putative virulence-enhancing IS1016, and the partial deletion of bexA. 16S rRNA was utilized for performing phylogenetic analyses using megax software. The evolutionary relationship was inferred using maximum likelihood method.

Nucleotide Sequence

The complete sequences of H. influenzae 108 was deposited in GenBank under the accession number: PRJNA900629.

Results

Analysis of Drug Resistance in H. influenzae

We observed that the AMP resistance rate of non-invasive H. influenzae was as high as 91.59%, followed by the SXT resistance rate of 71.96%, CEC of 60.74%, CAZ of 56.07%, AMC of 49.53%, and CEP of 46.73%. CTX, OFX, Rif, and CHL were not resistant, as presented in . Only 2 non-invasive H. influenzae strains (1.87%) showed multidrug-resistance (MDR). Their MDR pattern was resistance to β-lactam antibiotics (AMP, AMC, CEP, CEC, CAZ), TCY and SXT. One BLNAS strain and the invasive H. influenzae 108 was sensitive to all 11 antibiotics.

Table 1 Antibiotic Resistance of 107 Non-Invasive H. influenzae Strains

Analysis of β-Lactamase-Producing Genes in 107 Non-Invasive H. influenzae Strains

The β-lactamase gene of 98 AMP-resistant strains were all blaTEM-1 genes but not blaROB-1. BLNAR strains were not detected. The instructions of the H. influenzae drug susceptibility test kit recommend verifying the AMP-susceptible strains to confirm the absence of β-lactamase. Therefore, the β-lactamase-producing gene of AMP-sensitive strains was detected. Notably, the blaTEM-1 gene was detected in eight of the nine isolates. Only one strain was confirmed as β-lactamase-producing gene negative. Finally, the AMP resistance rate was corrected to 99.06%.

Mutation Analysis of ftsI in 53 BLPACR Strains and One BLNAS Strain, and Serotype Analysis of 107 Non-Invasive H. influenzae Strains

We identified eight mutational patterns from 52 BLPACR strains; however, one BLPACR strain expressed no mutations (). Thirty-seven strains were group III-like, accounting for 71.15% of BLPACR strain mutants. Eleven strains belonged to group III, of which four were combined with group IIb or group IIc. Two strains belonged to group IIc. The untyped patterns of the two BLPACR strains were Asp-350→Asn, Ser-357→Asn, Met-377→Ile, and Ser-385→Thr. In one BLNAS strain, the mutation pattern was Asp-350→Asn, Ser-385→Thr, Leu-389→Phe, and Asn-526→His.

Table 2 Different Amino Acid Substitution Patterns Identified in Part of PBP3 from 53 BLPACR Strains and One BLNAS Strain

All 107 non-invasive H. influenzae strains belonged to NTHi because the six serotype genes were negative.

Whole Genome Analysis of Invasive H. influenzae 108

WGS of invasive H. influenzae 108 revealed the genome size to be 1,903,516 bp, comprising 1798 protein-coding sequences, 20 rRNAs, and 57 tRNAs, with a GC content of 38.1%. No plasmids were present in the strains. Three prophages were identified: one intact, two questionable (). The prophages 1 (questionable), located at 478,683–513,213, is 34.5 Kb in length and contains 54 proteins. The prophages 2 (intact), located at 1,089,401–1,145,733, is 56.3 Kb in full length and contains 61 proteins. The prophages 3 (questionable), located at 1,303,040–1,319,103, is 16 Kb in full length and contains 22 proteins. No exogenous antimicrobial resistance or putative virulence genes were present in the prophage regions.

Figure 1 The locations of the prophages on the chromosome of the strain Regions 1 to 3 represent prophages 1 to 3; Blue, red and green represent questionable, incomplete, and intact, respectively.

Figure 1 The locations of the prophages on the chromosome of the strain Regions 1 to 3 represent prophages 1 to 3; Blue, red and green represent questionable, incomplete, and intact, respectively.

The invasive H. influenzae 108 were discovered to belong to Hib, and its sequence type belonged to ST95. β-lactamase-producing genes and ftsI mutations were not detected. However, the LpsA gene for intrinsic peptide antibiotic resistance and the EF-Tu R234F amino acid mutation, which mediates felfamycin resistance, were present. According to the analysis of 16S rRNA phylogenetic tree, H. influenzae 108 was the closest to H. influenzae NCTC11394 and H. influenzae M3918 ().

Figure 2 Phylogenetic analysis between the H. infuenzae strains The 17 H. infuenzae genomes available on the NCBI database and H. influenzae strain 108 isolated in this study.

Figure 2 Phylogenetic analysis between the H. infuenzae strains The 17 H. infuenzae genomes available on the NCBI database and H. influenzae strain 108 isolated in this study.

Comparative analysis of the capb loci of the H. influenzae 108 and the H. influenzae 146 references (GenBank accession number: PQJI00000000.1) revealed regions I, II, and III exist in duplicates. However, IS1016 complete deletion rather than partial deletion was identified upstream of the region I in only one of the duplicate copies (). Compared with the multiple mutations in the SNPs analysis of the capb locus from reference H. influenzae 146, no mutations were identified in the H. influenzae 108.

Figure 3 Cap locus structure identified in H. influenzae strain 108 Arrows indicate the direction of transcription and translation, and squares indicate the deletion of a part of the gene.

Figure 3 Cap locus structure identified in H. influenzae strain 108 Arrows indicate the direction of transcription and translation, and squares indicate the deletion of a part of the gene.

There are 79 predicted related virulence genes, as displayed in Table S2. These virulence genes can be divided into four categories: capsule gene (20 genes), lipopolysaccharide (LPS) biosynthesis-related genes (45 genes), nonspecific virulence factors (6 genes), defensive virulence factors (3 genes), and offensive virulence factors (5 genes).

Discussion

H. influenzae, a common pathogen of community-acquired pneumonia, was majority isolated from the sputum of the lower respiratory tract. In this study, we observed a 99.06% AMP resistance rate, which was higher than the 61.4% reported by a Chinese multicenter study in 2016,Citation16 and the 13% in ParaguayCitation17 but similar to the 91.1% reported by another Chinese study in 2022.Citation18 This increasing trend may be attributed to the 99.06% detection rate of the β-lactamase gene in this study. Except for AMC, the resistance rates of CEC, CTX, OFX, Rif, TCY, and SXT were consistent with those reported in another Chinese study in 2022.Citation18 Compared with other reports of CHL low resistance,Citation18 no CHL resistance was observed in this study. A multi-center study in China showed that the major multidrug resistance pattern was resistant to β-lactams, macrolides, and sulfonamides,Citation14 which was inconsistent with the MDR pattern in this study. The MDR detection rate in this study was much lower than 12.8% reported in China.Citation19 MDR may exhibit a unique resistance pattern in a low prevalence state at Quanzhou.

Given that CHL, OFX, and TCY are contraindicated in clinical use for pediatric patients, CTX and Rif, with a 100% sensitivity rate, can be the first choice for empirical medication. The high AMC resistance rate of 49.53% observed in this study was associated with the reduced drug sensitivity caused by the high PBP3 mutation rate. Therefore, it is not recommended that AMC be the drug of choice for treatment.

Chromosome-mediated overproduction of dihydrofolate reductase is the primary resistance mechanism of SXT. SXT resistance rate is also high, close to 72%; therefore, it is not recommended for empirical treatment.

The β-lactamase genes identified in this study were all blaTEM genes, consistent with a previous Chinese study.Citation20 Furthermore, eight strains carrying β-lactamase-producing blaTEM-1 genes were detected from nine AMP-sensitive strains to clarify the detection efficiency of the ATB HAEMO CLSI (12) strip for AMP sensitivity. The missed detection rate was 8/107 (7.48%), which exhibits effective false negative prevention.

The primary resistance-conferring mutations are in the transpeptidase domain of PBP3. These mutations decrease the binding of β-lactam antibiotics to PBP3 under the selective pressures of different β-lactam antibiotics.Citation21,Citation22 BLNAR strains had reduced sensitivity to AMC and its combinations with beta-lactamase inhibitors and second-generation cephalosporins.Citation23 However, no BLNAR strains were identified in this study. The detection rate of BLPACR strains was 49.53%, which was higher than the 15.4% reported in another Chinese study,Citation24 23% in a British Columbian study,Citation25 and 32.1% in a Korean study.Citation26 The MICs of CTX for group III-like strains were at least 10 times higher than fully susceptible isolates.Citation22 Moreover, group III and III-like H. influenzae strains were more resistant to β-lactam antibiotics than group I and II strains.Citation26,Citation27 BLPACR strains were the main group III and III-like strains in this study, which is consistent with previous literature.Citation26,Citation28 However, group III-like BLPACR strains exhibited a high prevalence. One BLPACR strain was observed to have no variation in the amino acid sequence 350–532 of PBP3. We speculated that this strain might carry resistance mutations in other regions of PBP3 or other β-lactam resistance genes. Asn-526→His in the KTG motif, with Asp-350→Asn, Ser-385→Thr, and Leu-389→Phe were first reported in one BLNAS strain, which may slightly reduce or not affect the MIC values of β-lactam antibiotics.

With the introduction of the H. influenzae vaccine, the serotypes of strains causing respiratory diseases are mainly NTHi.Citation28 In this study, we observed that all non-invasive strains were NTHi, consistent with the strain isolated from the lower respiratory tract previously reported in China.Citation29 However, BLPACR NTHi strains are highly prevalent in Korea.Citation26 We also observed that BLPACR NTHi strains were popular in China, thereby drawing attention to the increasing β-lactam resistance.

Compared with the reference genome of H. influenzae 146 belonging to Hib, this H. influenzae 108 strain’s genome is relatively large but does not carry a plasmid containing the blaTEM-1B gene and has no mutations in ftsI, which may explain the lack of β-lactam antibiotic resistance. Diversification due to genome rearrangement has been reported to contribute to the global spread of H. influenzae.Citation30 Compared with the strains reported to carry four complete prophages and one incomplete prophage,Citation31 the strains in this study carried fewer prophages, resulting in less phenotype diversity. In the pre-vaccination era, H. influenzae ST95, which belonged to Hib and was transferred by healthy Japanese children, exhibited resistance to AMP and TCY.Citation32 In the post-Hib vaccine era, some invasive H. influenzae Hib and ST-95 isolated from Canada exhibited no resistance, whereas some demonstrated resistance to AMP owing to β-lactamase production.Citation33 H. influenzae Hib and ST-95 isolated from children with bacterial meningitis are typical for strains circulating in Russia.Citation34 H. influenzae Hib and ST-95 have been isolated from the respiratory tract in China.Citation35 In this study, we identified H. influenzae Hib and ST-95 isolated from Chinese children’s blood for the first time, which demonstrated no resistance to eleven antibiotics. H. influenzae NCTC11394 and H. influenzae M3918 isolated from USA also belonged to Hib, which is the same as H. influenzae 108 serotype. Although H. influenzae NCTC11394 belonged to ST6 through MLST analysis by us, the scores of 6 of the 7 housekeeping genes of its ST were consistent with those of H. influenzae 108 and H. influenzae 108 belonged to ST-6 complex. This further indicates that the two strains are related in evolution.

The capsular polysaccharide of H. influenzae Hib is a major virulence factor and a crucial antigen for Hib vaccination.Citation36 The capb locus, which consists of three functionally distinct regions (I to III), is responsible for generating the type b capsule.Citation37 Multiple copies of the capb locus exhibit strong virulence and can result in increased polysaccharide production. Another large category of virus-related genes are those involved in LPS synthesis.Citation38 The LPS of H. influenzae may play an important role in the pathogenesis of meningitis by destroying the blood-brain barrierCitation39 and septicemia.Citation40 The strains in this study also comprised invasiveness LPS biosynthesis genes, lic1 and lic2, consistent with Cardosoa et al, suggesting that these genes exist in invasive strains.Citation37 We also observed that H. influenzae Hib and ST-95 caused severe septicemia in unvaccinated children, highlighting the indispensable and crucial role of vaccines.

Conclusions

In this study, AMP and AMC were not suitable for empiric medication due to high drug resistance rate. MDR was in a very low prevalence state at this region. BLPACR NTHi strains dominated by group III-like showed high prevalence. H. influenzae Hib and ST-95 was isolated from blood for the first time in China. In addition, genomic surveillance study conducted to monitor changes in the molecular epidemiology of invasive H. influenzae infection was important.

Disclosure

The authors declare that they have no competing interests.

Acknowledgments

We expressed our appreciation to the patient and her parents in this study.

Additional information

Funding

This research was funded by Quanzhou Municipal Science and Technology Plan Project (Grant No: 2021N089S).

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