Longitudinal Trend in Antimicrobial Susceptibility among Haemophilus influenzae: A Single-Centre Study in Japan

Abstract

Haemophilus influenzae presents significant concerns regarding antimicrobial resistance. The circumstances surrounding H. influenzae have been changing owing to changes in antimicrobial usage. In the present study, to determine the current situation of H. influenzae, the antimicrobial susceptibility trends were investigated. In total, 21 clinical isolates were analyzed. Antimicrobial susceptibility measured using the broth dilution method was compared with that reported in previous studies at the same hospital. Quinolone low-susceptible isolates were further characterized by multi-locus sequence typing. Upon comparing the susceptibility data in 2022 with those in the past 15 years, the number of β-lactamase nonproducing ampicillin-resistant isolates was decreased. Regarding recent changes, β-lactam-susceptible isolates were found to have gradually increased every year. However, β-lactamase-producing isolates did not decrease. In particular, the ratio of β-lactamase-producing amoxicillin and clavulanic acid-resistant isolates in 2022 was the highest among all the years studied. Moreover, quinolone low-susceptible isolates were still present, suggesting that these isolates could have been indigenized in the community. Furthermore, a β-lactamase-producing amoxicillin and clavulanic acid-resistant isolate was found to have emerged among the quinolone low-susceptibility isolates. At first glance, these findings indicate that antimicrobial resistance has decreased among H. influenzae in clinical settings. However, β-lactamase-producing isolates and quinolone low-susceptible isolates have remained at a constant rate in the community.

INTRODUCTION

Haemophilus influenzae is the major cause of acute otitis media, sinusitis, and respiratory infections in children. These infections are treated with antimicrobial agents.¹⁾ However, antimicrobial-resistant strains are prevalent and have become a serious concern. In particular, β-lactam resistance among H. influenzae is serious.^2–5) WHO has listed ampicillin-resistant H. influenzae as a medium risk in the prioritization of pathogens to guide the research and development of new antibiotics.⁶⁾ Furthermore, β-lactam-resistant H. influenzae tends to acquire resistance to other antimicrobial agents, macrolides, and quinolones, which are important alternative therapeutic agents.^7,8) In H. influenzae, β-lactam resistance is caused by either the production of β-lactamase or amino acid substitutions in penicillin-binding protein 3 (PBP3).^9–11) β-Lactam-resistant H. influenzae clinical isolates have either one or both of these mechanisms,²⁾ and are accordingly called β-lactamase-producing ampicillin-resistant (BLPAR), β-lactamase nonproducing ampicillin-resistant (BLNAR), and β-lactamase-producing amoxicillin and clavulanic acid-resistant (BLPACR) strains. By contrast, the β-lactam-susceptible strains are referred to as β-lactamase nonproducing ampicillin-susceptible (BLNAS). Previous epidemiological reports have indicated that BLNAR is prevalent in Japan, whereas BLPAR is prevalent in the United States.^2,3,7,8,12) BLNAR prevalence was linked to the usage of third-generation cephalosporins.^13,14) Since the 2015 World Health Assembly, many countries have struggled with antimicrobial resistance (AMR). Among these, one of the aims is the reduction of wide-spectrum agents such as third-generation cephalosporins, macrolides, and quinolones.¹⁵⁾ Accordingly, the usage of these antimicrobial agents is decreasing.^14,16) These factors can affect AMR. Continuous monitoring is required to evaluate its effects on antimicrobial susceptibility. Therefore, we have been investigating the antimicrobial susceptibility of H. influenzae as a representative of community pathogens since 2007.^7,17) In the present study, we investigated the 15-year short-term changes in antimicrobial susceptibility.

MATERIALS AND METHODS

Bacterial Isolate

In total, 21 H. influenzae isolated in 2022 at the Yokohama Rosai Hospital were used. This study was approved by the Ethics Committee of Yokohama Rosai Hospital (Case No. 2018-66-2). These isolates were collected based on the same criteria used in previous studies.^7,18,19) These isolates were identified as H. influenzae in routine tests using Vitek 2 (BioMérieux SA, L’Etoile, France) and were sent to the laboratory at Meijo University without any patient identification data. In all, 17 of these 21 isolates (80.9%) were obtained from pediatric patients. Four isolates died during transportation or storage, but their DNA was recovered. All isolates were isolated from noninvasive infections such as respiratory diseases and otitis media. Furthermore, the strains used in previous studies conducted at the same hospital were also used for comparison.^7,17) Haemophilus influenzae ATCC 49247 and Rd were used as quality controls for antimicrobial susceptibility testing.

Antimicrobial Susceptibility

Antimicrobial susceptibility was measured using the broth dilution method according to the Clinical and Laboratory Standard Institute (CLSI) guidelines.²⁰⁾ Antimicrobial agents were obtained from Tokyo Chemical Industry, Tokyo, Japan (amoxicillin, ceftriaxone, cefotaxime, clarithromycin, azithromycin, levofloxacin, and meropenem), FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan (ampicillin and tosufloxacin), and Combi-Blocks, San Diego, U.S.A. (clavulanic acid and sulbactam). Susceptibility and resistance were determined as per the breakpoint specified by the CLSI guidelines.²⁰⁾ Quinolone low-susceptible isolates were defined at a levofloxacin minimum inhibitory concentration (MIC) of 0.063–2 µg/mL according to a previous study.²¹⁾

Multi-Locus Sequence Typing

Multi-locus sequence typing (MLST) was performed as previously described.²²⁾ Amplicons of each gene were sequenced by Eurofins Genomics (Tokyo, Japan).

Amino Acid Substitutions of Quinolone-Resistant-Determining Regions in GyrA and ParC

Amino acid substitutions in quinolone-resistant-determining regions (QRDRs) were estimated using nucleotide sequences of gyrA and parC according to the methods of previous reports.^7,23)

Statistical Analysis

The differences between each year were analyzed using Fisher’s exact test and Bonferroni correction with EZR ver. 1.68 software.²⁴⁾ p-Values were considered statistically significant at p < 0.05.

RESULTS AND DISCUSSION

Antimicrobial Susceptibility Trends over 15 Years

To clarify the long-term trends of antimicrobial susceptibility, the MIC of clinical isolates in 2022 was measured and compared every 5 years based on previous studies at the same hospital^7,18,19) (Table 1). The proportion of β-lactam-resistant and clarithromycin-non-susceptible isolates was decreased in 2022. In particular, the number of ampicillin-resistant isolates decreased significantly (p <0.01 vs. 2017), suggesting that H. influenzae clinical isolates tended to become susceptible to therapeutic agents. Focusing on β-lactam resistance, the phenotype was determined based on CLSI criteria (Fig. 1A). In 2007, 2012, and 2017, BLNAR isolates accounted for a large proportion (more than 50%), whereas they were significantly decreased in 2022 (p <0.01 vs. 2017), contributing to a decrease in the β-lactam resistance ratio.

Table 1. Antimicrobial Susceptibility of Haemophilus influenzae Clinical Isolates in 15 Years

Agent	2007 (n = 16)					2012 (n = 24)
	MIC50	MIC90	MIC range	S%	R%	MIC50	MIC90	MIC range	S%	R%
Amoxicillin	—	—	—	—	—	4	32	0.25– ≥ 64	—	—
Amoxicillin–clavulanic acid	8	16	0.5–16	37.5	62.5	4	8	0.25–16	58.3	41.7
Ampicillin	4	8	0.25–8	25.0	68.8	4	16	0.25– ≥ 64	25.0	66.7
Ampicillin–sulbactam	—	—	—	—	—	—	—	—	—	—
Ceftriaxone	0.25	0.25	≤0.063–0.25	100	—	0.25	0.25	≤0.063–0.5	100	—
Cefotaxime	1	2	≤0.063–2	100	—	0.5	1	≤0.063–2	100	—
Meropenem	0.125	0.5	≤0.063–1	93.8	—	0.125	0.25	≤0.063–0.5	100	—
Clarithromycin	8	8	4–8	100	0	8	16	2–16	79.2	0
Azithromycin	1	2	0.5–2	100	—	1	4	0.5–4	100	—
Levofloxacin	≤0.063	≤0.063	≤0.063	100	—	≤0.063	≤0.063	≤0.063	100	—
Tosufloxacin	—	—	—	—	—	≤0.063	≤0.063	≤0.063	—	—

Agent	2017 (n = 57)					2022 (n = 17)
	MIC50	MIC90	MIC range	S%	R%	MIC50	MIC90	MIC range	S%	R%
Amoxicillin	8	≥64	0.5– ≥ 64	—	—	1	4	0.25– > 64	—	—
Amoxicillin–clavulanic acid	4	16	0.25–16	54.4	45.6	1	4	0.25–8	94.1	5.9
Ampicillin	16	≥ 64	0.25– ≥ 64	8.8	89.5	1	4	0.125– > 64	52.9	23.5
Ampicillin–sulbactam	8	16	0.25–32	21.1	78.9	1	4	0.125–8	76.7	23.5
Ceftriaxone	0.125	0.25	≤0.063–0.5	100	—	0.125	0.25	≤0.063–0.25	100	—
Cefotaxime	0.5	2	≤0.063–4	94.7	—	1	1	≤0.063–1	100	—
Meropenem	0.25	0.5	≤0.063–1	96.5	—	0.25	0.25	≤0.063–0.25	100	—
Clarithromycin	8	32	2– ≥ 64	57.9	10.5	4	4	2–4	100	0
Azithromycin	1	2	0.5–16	96.5	—	0.5	0.5	0.25–1	100	—
Levofloxacin	0.016	0.032	0.004–1	100	—	0.008	0.5	0.008–0.5	100	—
Tosufloxacin	0.008	0.016	0.004–2	—	—	0.008	2	0.008–2	—	—

MIC, minimum inhibitory concentration; S%, percent of susceptible isolates; R%, percent of resistant isolates; —, breakpoint was not defined or not tested. The data from 2007, 2012, and 2017 were referred from previous studies.^7,17)

Fig. 1. β-Lactam-Resistant Phenotype of Haemophilus influenzae Clinical Isolates

Changes over 15 years (A) and in the recent 4 years excluding 2020 and 2021 (B).

Antimicrobial Susceptibility Trend between 2017 and 2022

To clarify recent trends, susceptibility was compared between 2017 and 2022 (Table 2). Unfortunately, clinical isolates from 2020 and 2021 could not be obtained because of the coronavirus disease 2019 (COVID-19) pandemic during this period. As well as long-term trends, susceptibility to several agents tended to improve. Regarding β-lactam-resistant phenotypes, recent changes indicated that BLNAS was gradually increasing (Fig. 1B).

Table 2. Antimicrobial Susceptibility of Haemophilus influenzae Clinical Isolates in Recent Years

Agent	2017 (n = 57)					2018 (n = 62)
	MIC50	MIC90	MIC range	S%	R%	MIC50	MIC90	MIC range	S%	R%
Amoxicillin	8	≥ 64	0.5– ≥ 64	—	—	16	64	0.5– > 64	—	—
Amoxicillin–clavulanic acid	4	16	0.25–16	54.4	45.6	8	32	0.5–32	33.9	66.1
Ampicillin	16	≥ 64	0.25– ≥ 64	8.8	89.5	8	64	0.5– > 64	11.3	82.3
Ampicillin–sulbactam	8	16	0.25–32	21.1	78.9	8	16	0.25–32	22.6	77.4
Ceftriaxone	0.125	0.25	≤0.063–0.5	100	—	0.25	0.5	≤0.063–1	100	—
Cefotaxime	0.5	2	≤0.063–4	94.7	—	1	4	≤0.063–8	79.0	—
Meropenem	0.25	0.5	≤0.063–1	96.5	—	0.25	0.5	≤0.063–2	90.3	—
Clarithromycin	8	32	2– ≥ 64	57.9	10.5	8	16	2–32	61.3	8.1
Azithromycin	1	2	0.5–16	96.5	—	2	4	0.125–8	98.4	—
Levofloxacin	0.016	0.032	0.004–1	100	—	0.016	0.5	0.008–1	100	—
Tosufloxacin	0.008	0.016	0.004–2	—	—	0.016	2	0.008–2	—	—

Agent	2019 (n = 39)					2022 (n = 17)
	MIC50	MIC90	MIC range	S%	R%	MIC50	MIC90	MIC range	S%	R%
Amoxicillin	2	>64	0.25– > 64	—	—	1	4	0.25– > 64	—	—
Amoxicillin–clavulanic acid	2	4	0.25–8	92.3	7.7	1	4	0.25–8	94.1	5.9
Ampicillin	2	>64	0.125– > 64	35.9	35.9	1	4	0.125– > 64	52.9	23.5
Ampicillin–sulbactam	2	4	0.125–4	82.1	17.9	1	4	0.125–8	76.7	23.5
Ceftriaxone	0.125	0.25	≤0.063–0.25	100	—	0.125	0.25	≤0.063–0.25	100	—
Cefotaxime	0.5	1	≤0.063–0.5	100	—	1	1	≤0.063–1	100	—
Meropenem	≤0.063	0.125	≤0.063–0.25	100	—	0.25	0.25	≤0.063–0.25	100	—
Clarithromycin	4	8	0.25–8	100	0	4	4	2–4	100	0
Azithromycin	0.5	1	≤0.063–1	100	—	0.5	0.5	0.25–1	100	—
Levofloxacin	0.016	0.032	0.004–0.5	100	—	0.008	0.5	0.008–0.5	100	—
Tosufloxacin	0.008	0.032	0.008–2	—	—	0.008	2	0.008–2	—	—

MIC, minimum inhibitory concentration; S%, percent of susceptible isolates; R%, percent of resistant isolates; —, breakpoint was not defined or not tested. The data from 2017 were referred from a previous study.⁷⁾

Recent reports have described that the use of wide-spectrum third-generation cephalosporins was reduced because of the promotion of antimicrobial stewardship,^14–16) whereas that of penicillins tended to increase,^25,26) indicating that this circumstance could be more advantageous for β-lactamase-producing isolates. These findings suggest that β-lactamase-producing isolates should be monitored closely.

Features of Quinolone Low-Susceptible Isolates

The emergence of quinolone low-susceptible H. influenzae has been reported in pediatric patients in Japan.^7,21,27) Recently, similar isolates have also been identified in European countries.^28,29) Notably, these isolates can be detected using the EUCAST criteria but may be overlooked in routine clinical testing in Japan, where the CLSI criteria are predominantly used.²⁰⁾ To address this issue, a broad range of antimicrobial susceptibility testing was conducted. In 2022, 4 quinolone low-susceptible H. influenzae isolates were identified. These isolates were classified as ST422 (n = 3) and ST 1851 (n = 1) by MLST. The ST422 clone was previously reported as an outbreak clone in pediatric patients in 2018¹⁹⁾ (Supplementary Table 1). These findings indicated that the ST422 clone was still present after the COVID-19 pandemic and could be attaching and spreading in this community. These isolates have amino acid substitutions of Ser84Leu in GyrA and Ser84Ile in ParC, which were identical to those of the previous strains.^7,19) When comparing the antimicrobial susceptibility of these ST422 isolates, all of them showed features of BLNAR, similar to the isolates from previous studies (Table 3). In particular, one (2022-Y5) of these isolates was BLPACR. The β-lactamase gene in 2022-Y5 was located on an integrative and conjugative element, which facilitates gene transfer via conjugation (data not shown). A previous study demonstrated that ST422 isolates could acquire the β-lactamase gene through conjugation in vitro.³⁰⁾ Therefore, these results suggest that acquisition of the β-lactamase gene by the ST422 isolate can also occur in a clinical setting, potentially serving as a source for further dissemination of β-lactamase genes among ST422 strains. In pediatric patients, quinolone treatments are ineffective against quinolone low-susceptible H. influenzae.^7,31,32) Therefore, BLPACR and quinolone low-susceptible isolate should be regarded as a multidrug-resistant isolate, particularly in pediatric settings where therapeutic options are limited.

Table 3. Strain Information and Antimicrobial Susceptibility of Haemophilus influenzae ST422 Isolated in 2022

Strain	Department	Isolated site	Amino acid substitution		β-Lactamase gene	MIC (µg/mL)
			GyrA	ParC		AMX	AMC	AMP	SAM	CRO	CTX	MEM	CLR	AZM	LVX	TFX
2022-Y5	Pediatric	Sputum	Ser84Leu	Ser84Ile	blaTEM-1	>64	8	>64	8	0.125	0.5	0.125	4	0.5	0.5	1
2022-Y20	Pediatric	Pharynx	Ser84Leu	Ser84Ile	—	2	2	4	4	0.125	1	0.25	4	0.5	0.5	2
2022-Y21	Pediatric	Pharynx	Ser84Leu	Ser84Ile	—	2	2	4	4	0.125	1	0.25	4	0.5	0.5	2

MIC, minimum inhibitory concentration; AMX, amoxicillin; AMC, amoxicillin–clavulanic acid; AMP, ampicillin; SAM, ampicillin–sulbactam; CRO, ceftriaxone; CTX, cefotaxime; MEM, meropenem; CLR, clarithromycin; LXV, levofloxacin; TFX, tosufloxacin.

Limitations

This study has some limitations. First, as H. influenzae isolated from 1 hospital were investigated and the number of isolates was comparatively low because of the influence of COVID-19, the results and trends from this study may not correspond to all clinical settings. However, previous studies have shown that the trend of antimicrobial susceptibility was similar to that in other hospitals; furthermore, the isolation of quinolone-low susceptible ST422 was also reported in geographically separated hospitals in not only Japan but also other countries,^{19,27,29,30,33)} suggesting that the same or similar tendencies could be also observed in other settings. Second, isolates between 2020 and 2021 were not able to be collected and investigated. Several studies have reported that the COVID-19 pandemic affected community pathogens, including Streptococcus pneumoniae and H. influenzae.^34,35) However, the information on these community pathogens was limited during the pandemic of COVID-19. Therefore, the results of this study could be important data to understand the current situation even if the number of isolates was comparatively low.

CONCLUSION

Overall, this study indicates that, among H. influenzae in clinical settings, AMR to therapeutic agents decreased at first glance. However, β-lactamase-producing H. influenzae still existed at a constant rate. Furthermore, these results suggest that quinolone low-susceptible isolates were attaching and spreading in the community. Moreover, the emergence of a β-lactamase-producing isolate among the indigenized quinolone low-susceptible clones raises concerns regarding the emergence of multidrug-resistant H. influenzae. As long-term and continuous monitoring indicates a more accurate trend, continued epidemiological studies are needed to monitor the situation.

Acknowledgments

The authors thank Ms. Ruri Ota, Ms. Yurina Takabayashi, and Mr. Tomokazu Ando for providing technical assistance.

Funding

This study was supported by the Research Institute of Meijo University (T. W.) and the Takeda Science Foundation (T. W.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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