Haemophilus influenzae (Hi) is a gram-negative bacterium that has been established to contribute to a wide variety of airway mucosal infections and invasive diseases such as bacterial meningitis. Before the introduction of Hib conjugate vaccines, Hib was the commonest cause of bacterial meningitis in young children in European countries (1) and is also reportedly the second most common bacterial pathogen causing pneumonia in Chinese children (2). Nowadays, H. influenzae strains are classified as typeable or non-typeable Hi based upon the presence of the polysaccharide capsule antigen, which also helps characterize typeable Hi into 6 serotypes (a to f), and lack of a capsule are identified as NTHi (non-typeable H.influenzae). Since the introduction of a conjugate vaccine against H. influenzae type b (Hib), invasive diseases of H.influenzae are now usually attributed to NTHi (3). In addition, H.influenzae infection is influenced by many factors, including the environment, population density and climatic conditions (4, 5). Accordingly, continuous monitoring of local and worldwide epidemiology is critical to help develop future disease control strategies.

Antibiotic resistance surveillance provides valuable information to aid clinicians in selecting empirical treatment. β-lactam antibiotics are nowadays commonly used in the clinical treatment of H. influenzae. In many countries, these empirical drugs have been found to be resistant (6). Rates of ampicillin resistant H. influenzae from <5% in several countries to 67.9% have been reported such as Canada, Japan, Italy, Shanghai and Taiwan (7). The resistance of H. influenzae to ampicillin is mainly through two mechanisms. One of the reported main resistance mechanisms involves the production of β-lactamase in H.influenzae that hydrolyze β-lactam antibiotics. Thus, strains that acquire β-lactamase genes such as TEM-1 and ROB-1 are referred to as β-lactamase-positive ampicillin-resistant H. influenzae (BLPAR) strains. The other mechanism involves β-lactamase-negative ampicillin-resistance (BLNAR), which arises from alterations of penicillin-binding protein 3 (PBP3) resulting from ftsI gene mutations, leading to the decreased affinity of β-lactamase antibiotics for PBP3 (7, 8). BLNAR isolates may represent a significant risk due to their ability to develop third-generation cephalosporin resistance (9).

Therefore, the classification of drug resistance phenotypes is important for clinical treatment and understanding the transmission of drug resistance genes. In this study, we aimed to preliminarily investigated the serotype distribution, antimicrobial resistance, and molecular epidemiology of H. influenzae among hospitalized patients in Guiyang, Guizhou, China.

H. influenzae is a Gram-negative bacterium responsible for respiratory tract infections, especially in children (19). After the introduction of the Hib conjugate vaccine, the prevalence of invasive H. influenzae has declined, leading to a change in the epidemiology of H. influenzae. However, it should be borne in mind that Hib conjugate vaccines was promoted in the 1980's, Asia far behind its introduction in Europe and America, particularly in developing countries (20), which has led to large differences in the epidemiological patterns of H. influenzae around the world. All 196 Hi strains in this study were from respiratory specimens, sputum samples mainly from pediatric patients with pneumonia. Drug sensitivity test showed that resistance to ampicillin, cefaclor and azithromycin was 71.4, 36.7, and 34.2%, respectively. Our ampicillin and azithromycin resistance rates were relatively higher than those reported by the National Bacterial Resistance Monitoring Network 2014–2019 (21), while the resistance rates for the remaining antibiotics were relatively lower. Many factors, such as chronic disease, medications, regions or age, may possibly contribute to the diversity. The application of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines also led to many discrepancies. At present, it remains difficult to establish a “gold standard” method for detecting ampicillin resistance and a consensus definition of β-lactam resistance in H. influenzae (22). This increase in resistance emphasizes the need for careful selection of empirical medication.

The detection ampicillin resistance was 71.4%, resistance of the BLNAR strain to ampicillin-sulbactam, rifampin and cefaclor was higher than for the BLPAR strain. In comparison, the resistance of the BLPAR strain to azithromycin was higher than for the BLNAR strain (P < 0.05) (Table 1). After the addition of the enzyme inhibitor sulbactam, a decrease in drug resistance strains was observed, indicating that ampicillin-resistant strains in this region were mainly enzyme-producing bacteria, and their resistance genes were TEM-1 type, and no ROB-1 type, consistent with findings of a study by Qin et al. (23). Previously, BLNAR isolates were thought scarce in China (6). However, in our study, 18 isolates of BLNAR were identified from 196 test isolates (9.2%). A former report in Shanghai, China, and Beijing, China, the detection rate of BLNAR strains was 11.76 and 18.9% (24, 25). In other countries, especially, Japan, BLNAR strains prevalence was >40% (3). The mechanism of BLNAR resistance involves the transposition of amino acid sites of the ftsI gene on the cell wall of penicillin-binding protein 3, which changes the spatial conformation. Importantly, three important mutations have been documented near the conservative motif: STVK (Ser-Thr-Val-Lys), SSN (Ser-Ser-Asn) and motif (Lys-Thr-Gly) (26). The amino acid site replacement of ftsI gene in isolates was 57.7% (113/196), and BLNAR were mostly distributed in the class III-like group (Table 3), which contributed to a 10–60-fold increase in MIC values of cefixime and cefuroxime, causing higher resistance than groups I and groups II (27). It has been shown that isolates with significant PBP3 mutations are more resistant to β-lactam antibiotics, resulting in decreased affinity between bacteria and β-lactam antibiotics. This phenomenon explains the unaltered antimicrobial activity in response to β-lactamase inhibitors and first and second-generation cephalosporins (28). BLNAR strains, which can potentially develop resistance to ampicillin, first- and second-generation cephalosporins in this region, can lead to high resistance rates, raising awareness on the need to strengthen the epidemic surveillance of BLNAR strains.

It has been established that multiple drug resistance (MDR) is the resistance of organisms to three or more different antimicrobials (29). In the present study, one multi-resistant H. influenzae isolate exhibited resistance to β-lactamase antimicrobias (ampicillin and amoxilin-clavulanate), carbapenem (imipenem, meropenem and levofloxacin), tetracycline, chloramphenicol and ampicillin-sulbactam (Table 2), which a number of studies in Japan and Thailand have shown a gradual increase in MDR Hi (30, 31). The situation of H.influenzae mechanisms of multiple drug resistance is not clear, which may result from the interplay of multiple factors, including the production of β-lactamase or ftsI gene mutations, plasmid exchange, and efflux pumps.

All 196 H.influenzae isolated in this study were NTHi, and cannot be prevented by Hib vaccine. MLST is widely acknowledged to identify pathogen strains by sequencing seven housekeeping genes; it can compare isolates from different laboratories since the allele profiles, and epidemiological information of strains can be stored in databases on the Internet. This approach has become increasingly popular to study clonal pathogen expansion (18). An increasing body of evidence suggests a high level of genetic diversity among the isolates, related to the genetic heterogeneity of the NTHi strains (32, 33). Guizhou Province accounts for the largest proportion of CC107 and ST107 was the most common sequence type in this study which is different from other provinces of China (Shanghai, Guangzhou, Beijing) (24, 25, 34) and other countries (Italy, Malaysia, Canada, Japan) (35–38). However, CC107 appears in multiple countries suggesting no geographical connection (Figure 1). This finding demonstrates the geographic heterogeneity of H.influenzae strains that have evolved independently. Despite the genetic heterogeneity of the NTHi isolates, we observed an association between ampicillin resistance status and ST. In this regard, β-lactamase has been established to be mainly carried by ST107 and ST103, while some BLNAR isolates belong to ST12. ST103 and ST14 have been reported in Italy, Portugal, Ireland and are associated with invasive disease (39–41). ST14 has recently been documented in Sweden and is thought to be associated with increased virulence and persistence (42, 43) and which was included the most common CC3. The presence of ST103 and ST14 with loci mutations underscores the importance of surveillance studies, including molecular typing of these isolates.

Some limitations were noted in our study. First of all, the vaccination data of enrolled patients was not available. Moreover, PCR detection of pathogens was not carried out in patients with cold-like symptoms whose sputum culture was negative. Finally, our sampling was biased toward non-invasive bacterial isolates, as sputum samples were predominantly collected while other specimens such as blood were not. However, to the best of our knowledge, this is the first systematic investigation of H. influenzae colonization in the Guiyang area. Importantly, our study provides important information on potential high-risk genotypes to be tracked during epidemiological surveillance.

In conclusion, although data on the distribution of serotypes carrying H.influenzae isolates in our region prior to vaccination is unavailable, it is now clear that NTHi is overwhelmingly dominant and cannot be prevented by Hib vaccination. PBP3 mutations and β-lactamase production are prevalent in H. influenzae strains, leading to ampicillin resistance and reduced sensitivity to other β-lactamase antibiotics. Despite the high genetic diversity of NTHi, we found an association between ampicillin resistance status and specific clonal complexes; specific genotypes may have a higher potential for aggressive disease. To our knowledge, this is the first report of NTHi sequence typing in Guiyang. Indeed, monitoring NTHi colonization rates and prevailing genotypes will provide useful information for better understanding the evolution of H. influenzae disease and assist in vaccine development.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The study was approved by the Human Ethics Committee of the First People's Hospital of Guiyang (Approval No.No.G2020-S001) and complied with the Declaration of Helsinki. Before our team obtained the samples/isolates and conducted the study, any personal identifiers of the suspected H. influenzae-infected patients had been removed by the monitoring stations. So, the patient informed consent was waived by the Human Ethics Committee of the First People's Hospital of Guiyang.

YW, YZ, and JW conceived and designed the experiments and wrote the paper. YZ, XZ, and JC performed the experiments. YZ and YW analyzed the data. All authors contributed to the article and approved the submitted version.

This study was supported by [2019] Zhu wei jian ke ji he tong zi di 001, Zhu ke he tong [2020]-10-6 and Zhu ke he tong [2021]-43-25 from Science and Technology Department of Guiyang city of Guizhou Province, and was supported by Qian Ke He Zhi Cheng [2021] Yi Ban 440 from Science and Technology Department of Guizhou Province.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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