Unique and Ingenious Mechanisms Underlying Antimicrobial Resistance and Spread of Haemophilus influenzae

Abstract

Antimicrobial resistance (AMR) is a serious global concern. AMR pathogens are found in hospitals and communities. Haemophilus influenzae is a common pathogen associated with community-acquired infections. H. influenzae infections are usually treated with β-lactams, macrolides, and quinolones. However, the drug-resistant strains have emerged. The resistance mechanisms of H. influenzae are complex but are roughly characterized by the acquisition of a mutation in antimicrobial-targeting genes and exogenous resistant genes. Generally, the former cannot be transferred horizontally to a susceptible strain. However, several studies have demonstrated that, in the case of H. influenzae, both the former and the latter can be transferred horizontally. In this review, we provide an overview of the bacterial features and antimicrobial resistance of H. influenzae. We also summarize the unique and ingenious antimicrobial resistance mechanisms used by this pathogen based on the findings of recent studies. These are expected to facilitate the understanding of AMR pathogens in the community and develop strategies to combat infections.

1. INTRODUCTION

Antimicrobial resistance (AMR) is a major global concern. According to a report from the UK, deaths caused by AMR pathogens will be more than 10 million per year if actions are not taken.¹⁾ Multidisciplinary approaches are required to overcome AMR. The National Action Plan on AMR 2023–2027 of the government of Japan sets 6 goals, including public awareness and education, surveillance and monitoring, infection prevention and control, antimicrobial stewardship, research and development, and international cooperation.²⁾ Among these, the goals of research and development include developing novel methods for the treatment and elucidation of the mechanism of the emergence and transmission of AMR. However, the development of novel antimicrobial agents is declining.³⁾ The mechanisms underlying the emerging AMR are generally divided into 2 categories. One is caused by mutations in the antimicrobial-targeting genes. The other is the acquisition of exogenous resistance genes such as β-lactamase genes. However, the underlying mechanisms for each cause are complex and diverse.⁴⁾

AMR pathogens include both hospital- and community-associated pathogens. The AMR action plan also set targets for reducing the use of broad-spectrum agents such as quinolones, macrolides, and third-generation oral cephalosporins. These agents are often used for the treatment of community-associated infections, especially in clinics,⁵⁾ suggesting the importance of implementing antimicrobial stewardship in hospitals and clinics. In fact, the incidence of AMR pathogens among community-associated infections has been increasing.^6,7) For instance, in a report by the WHO, ampicillin-resistant Haemophilus influenzae and penicillin-non-susceptible Streptococcus pneumoniae are listed as medium priority.⁸⁾ These pathogens make infectious diseases difficult to treat. Among H. influenzae, the resistance to broad-spectrum antimicrobial agents is progressing rapidly.^9,10) Recent studies have revealed that H. influenzae has unique and ingenious mechanisms of AMR.^11,12) In this review, we focus on H. influenzae and summarize the mechanisms underlying its resistance and spread.

2. Haemophilus influenzae

H. influenzae is a major cause of acute otitis media, sinusitis, pneumonia, and bronchitis in the community, along with Streptococcus pneumoniae in both children and older adults.¹³⁾ Encapsulated H. influenzae, especially capsule type b, often caused infant invasive infections such as meningitis.^14,15) However, since the introduction of the H. influenzae type b (Hib) vaccine in the routine vaccine schedule, cases of meningitis have decreased drastically.^16–18) By contrast, almost all noninvasive infections are caused by non-typeable H. influenzae (NTHi), which cannot be inhibited by any vaccine at present. Furthermore, the ratio of H. influenzae in the causative pathogens of these infections has been relatively increasing because S. pneumoniae infections have decreased due to the introduction of the pneumococcal conjugate vaccine.¹⁹⁾ β-Lactams, macrolides, quinolones, and sulfamethoxazole/trimethoprim act on H. influenzae. In the clinical setting, the judgment for its use is usually in accordance with guidelines of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) but the criteria laid down by the 2 organizations are not always the same (Table 1).

Table 1. Criteria for Antimicrobial Susceptibility

	CLSI M100-33rd ed			EUCAST v13.1
	S	I	R	S ≤	R >
Ampicillin	≤1	2	≥4	1	1
Ampicillin/sulbactam	≤2/1	–	≥4/2	1	1
Amoxicillin/clavlanic acid	≤4/2	–	≥8/4	2	2
Ceftriaxone	≤2	–	–	0.125	0.125
Meropenem	≤0.5	–	–	2	2
Clarithromycin	≤8	16	≥32	–	≥32
Azithromycin	≤4	–	–	–	≥4
Levofloxacin	≤2	–	–	0.06	0.06

CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing; S, susceptible; I, intermediate; R, resistance.

Haemophilus spp. have natural transformation activity as well as Neisseria spp. and Streptococcus spp. etc.^20–22) These bacteria can take up extracellular dsDNA through type IV pilus family protein.^20,21) They can take up fragments of more than 50 kb.^20,23,24) Some DNA is replaced with genomic DNA strands by homologous recombination, whereas others are used for nucleic acid biosynthesis.²⁵⁾ This natural transformation system benefits bacteria from evolutionary aspects, allowing them to adapt to environmental changes faster and dietary aspects, allowing them to obtain essential materials.^26,27) However, this system can be deleterious in the case of DNA from dead cells and distant species or genera.²⁸⁾ Among the bacteria with natural transformation abilities, Haemophilus spp. and Neisseria spp. have unique transformation mechanisms. Most bacteria take up any available DNA from their surroundings but Haemophilus spp. and Neisseria spp. preferentially take up DNA from their species and close species by recognizing certain sequences.^29,30) In H. influenzae, the uptake signal sequence (USS) of the 9-mer AAGTGCGGT on the DNA fragment is recognized at the initial step of uptake.³¹⁾ The density of USS in the genome of H. influenzae is approximately 1/kb, whereas this in other bacterial genera is quite rare.³²⁾ This recognition contributes to the uptake specificity. Previous studies have shown that this natural transformation system is relevant to the emergence and spread of AMR.^12,33)

3. β-LACTAM RESISTANCE

β-Lactams are the first-choice antimicrobial agents for the treatment of H. influenzae infections.³⁴⁾ As β-lactams can be used safely in children, they are important therapeutic agents for the treatment of H. influenzae infections, most of which affect children. In H. influenzae, 2 mechanisms for β-lactam resistance are known. One is the production of β-lactamase, an enzyme that degrades β-lactams, including the so-called β-lactamase-producing ampicillin-resistant H. influenzae (BLPAR).^35–37) The other is the decreasing affinity for β-lactams due to amino acid substitutions in the target site penicillin-binding protein (PBP) 3, the so-called β-lactamase-non-producing ampicillin-resistant H. influenzae (BLNAR).^36,38,39) BLPAR shows high-level resistance to penicillins but is susceptible to other β-lactams, including cephalosporins because its β-lactamase is a TEM-type penicillinase which can degrade only penicillins (Table 2). In addition, β-lactamase activity can be inhibited by β-lactamase inhibitors, such as clavulanic acid and sulbactam. By contrast, BLNAR shows decreased susceptibility to both penicillins and cephalosporins^39,40) (Table 2). Isolates with both resistance determinants have already emerged, known as β-lactamase-producing amoxicillin-clavulanic acid-resistant (BLPACR) H. influenzae.

Table 2. Antimicrobial Susceptibility of β-Lactam-Resistant Haemophilus influenzae

Strain	ftsI mutation	Exogenous gene	MIC (μg/mL)
		blaTEM-1	AMX	AMC	CEF
Susceptible strain	–	–	0.25–0.5	0.25–0.5	0.03
BLNAR*	+	–	1–16	1–16	0.06–0.25
BLPAR	–	+	32– > 64	0.25–1	0.03–0.06
BLPACR	+	+	32– > 64	8–16	0.06–0.13

AMX, amoxicillin; AMC, amoxicillin-clavlanic acid; CEF, cefixime; BLNAR, β-lactamase-nonproducing ampicillin resistant; BLPAR, β-lactamase-producing ampicillin resistant; BLPACR, β-lactamase-producing amoxicillin-clavlanic acid resistant. *, including β-lactamase-nonproducing ampicillin intermediate resistant (BLNAI).

Generally, the emergence of resistant strains is strongly associated with the use of antimicrobial agents.^41,42) In North America, the isolation rate of BLPAR is higher than that in Japan.^6,43,44) In the United States, β-lactamase-producing strains account for more than 30% of clinical isolates since the 1990s.⁴⁵⁾ By contrast, the isolation rate of BLNAR is higher in Japan than in other countries. This proportion increased rapidly during the 2000s. In the 2010s, more than half of clinical isolates were of BLNAR.^{6,10,46–48)} Several studies have suggested that these differences could be caused by the increased usage of third-generation oral cephalosporins in Japan.^5,38) With the reconsideration of the use of antimicrobial agents, the usage of third-generation oral cephalosporins has decreased. Along with these trends, the BLNAR is decreasing as well.^6,49)

β-Lactamase of H. influenzae is located on either a small plasmid or an integrative conjugate element (ICE). In the case of the latter, β-lactamase gene can transfer horizontally through conjugative transfer.⁵⁰⁾ Although the transfer efficiency varies depending on the strain, it reaches a maximum of 10^–5 according to a previous report.⁵¹⁾ These results suggest that BLPAR is rapidly becoming prevalent. As the use of penicillins is increasing, as described above, attention should be paid to the trend of increasing BLPAR prevalence.

BLNAR harbors mutated fstI encoding PBP3 with amino acid substitutions. PBP3 is a peptidoglycan synthase associated with building a septum that consists of transglycosidase and transpeptidase regions.^52,53) Amino acid substitutions near the conserved sequences STVK, SSN, and KTG in the transpeptidase region (Fig. 1) contribute to β-lactam resistance in BLNAR.³⁹⁾ Furthermore, the accumulation of amino acid substitutions is linked to increased resistance levels.^39,54) Previously, it was thought that the resistance caused by mutations could not be transferred horizontally. However, Takahata et al. indicated that ftsI can be transferred horizontally by natural transformation in vitro.³³⁾ Furthermore, because genetical structures of ftsI in H. influenzae, H. haemolytius, and H. parainfluenzae are similar, the interspecies transfer of ftsI can occur.^55,56) Therefore, H. influenzae has diverse ftsI by transferring it to the same genus and undergoing repeated evolution.

Fig. 1. Antimicrobial Target and Resistance-Associated Amino Acid Substitutions

(A) Penicillin-binding protein 3 (FstI), a target of β-lactams; (B) DNA gyrase subunit A (GyrA) and DNA topoisomerase IV subunit A (ParC), targets of quinolones. USS, uptake signal sequence; USLS, uptake signal-like sequence; QRDR, quinolone resistance-determining region.

4. MACROLIDE RESISTANCE

In β-lactam-resistant strains, macrolides and fluoroquinolones are alternative options.³⁴⁾ In general, the antimicrobial activity of macrolides against Gram-negative bacteria is weaker than that against Gram-positive bacteria; however, azithromycin and clarithromycin show activity against H. influenzae.⁵⁷⁾ Owing to their low toxicity to children and broad-spectrum action, they are widely used. However, in Japan, an increase in macrolide-non-susceptible BLNAR isolates was reported after 2010.⁶⁾ These strains had nonsense or frameshift mutations in acrR, a negative regulator of the multidrug efflux pump AcrAB, which elevated AcrAB levels and conferred clarithromycin resistance⁵⁸⁾ (Table 3). In addition to the acrR mutation, a certain amino acid substitution in AcrB confers azithromycin non-susceptibility in a stepwise manner⁵⁹⁾ (Table 3). Thus, BLNAR easily becomes macrolide resistant through the acquisition of mutations.

Table 3. Antimicrobial Susceptibility of Macrolide-Resistant Haemophilus influenzae

Strain	Mutation		Exogenous gene	MIC (μg/mL)
	acrR*	acrB	mefA/E	CLR	AZM
Susceptible strain	–	–	–	4	0.5
Resistant strain 1	+	–	–	32	4
Resistant strain 2	+	+	–	32	8
Resistant strain 3	–	–	+	>64	>64

*, Nonsense or frameshift mutation; CLR, clarithromycin; AZM, azithromycin.

Among many bacteria, macrolide resistance is often mediated by exogenous resistance genes that are located on transposons.⁶⁰⁾ It had been controversial whether H. influenzae became resistant to exogenous resistance genes.^61,62) However, in Japan, the isolation of H. influenzae harboring mef A/E from pediatric patients was reported in 2 different hospitals.^63,64) These isolates showed higher resistance to macrolides than the acrR mutant (Table 3). Genetic analyses suggested that genetic elements, including resistance genes, could come from Streptococci which colonize the same niche as H. influenzae.⁶⁴⁾ Furthermore, similar isolates were reported in Norway and Belgium.^65,66) In particular, in Norway, an outbreak of mef A/E-positive H. influenzae occurred.⁶⁵⁾ mef A/E was located on ICE and could be transferred to susceptible strains through conjugation.⁶⁵⁾ To date, exogenous macrolide-resistant genes have not attracted significant attention. However, these reports suggest that macrolide-resistant strains are prevalent below the surface.

5. FLUOROQUINOLONE RESISTANCE

Quinolones inhibit DNA replication by acting on DNA gyrase and topoisomerase IV, which are essential enzymes for replicating DNA.^67,68) As described above, quinolones are important alternative agents for β-lactam-resistant H. influenzae as well as macrolides. Quinolones have greater bactericidal activity against Gram-negative bacteria than macrolides and are highly transferable to tissues. Therefore, they have often been used as empirical treatments for community-acquired respiratory infections in adults.^34,69) By contrast, quinolones, except for norfloxacin and tosufloxacin, are contraindicated in children because of their toxicity. Among these, tosufloxacin is the only respiratory quinolone that was introduced for the treatment of infections caused by AMR pathogens such as β-lactamase-producing H. influenzae and penicillin-resistant S. pneumoniae in 2010 in Japan.^34,70) The use of quinolones in pediatric settings has tended to increase, especially in community clinics.^5,71)

Quinolone resistance is caused by amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE, which encode the targets of quinolones.^72–75) In Gram-negative bacteria, including H. influenzae, a mutation occurs in gyrA and then in parC in a stepwise manner. Along with the acquisition of mutations, resistance levels gradually increase.⁷⁶⁾

H. influenzae originally showed high susceptibility to levofloxacin (MIC = 0.008–0.016 μg/mL). Upon amino acid substitution of the 84th serine in most cases in GyrA, the MIC increased to 0.125 μg/mL (Table 4). Furthermore, through an amino acid substitution, 84th serine in most cases, in ParC, MIC reached up to 0.5–1 μg/mL (Table 4). However, they are classified as “susceptible strain” according to CLSI guidelines (Table 1) despite the clearly deferent MIC with susceptible strains.⁷⁷⁾ Several studies have defined these strains as quinolone low-susceptible strains (MIC = 0.063–2 μg/mL).^48,78,79) However, the maximum blood concentration (C_max) of tosufloxacin was 0.96 ± 0.30 μg/mL for a common dosage for children according to the pharmacokinetics data collected from interview forms, suggesting that this dosage could be insufficient to kill quinolone low-susceptible strains harboring mutations in QRDRs. In fact, some studies have suggested that quinolone low-susceptible strains were resistant to tosufloxacin treatment both in vitro and in vivo.^48,80,81)

Table 4. Antimicrobial Susceptibility of Quinolone-low Susceptible Haemophilus influenzae

Strain	Mutation		MIC (μg/mL)
	gyrA	parC	LVX	TFX
Susceptible strain	–	–	0.008	0.004
Low-susceptible strain 1	+	–	0.125	0.125
Low-susceptible strain 2	+	+	0.5–1	2

LVX, levofloxacin; TFX, tosufloxacin.

In Japan, the number of quinolone low-susceptible strains isolated from children has been increasing gradually.^48,79,82) Recent studies have described the outbreaks and prevalence of quinolone low-susceptible ST422 clone in hospitals in Kanagawa, Tokyo, and Chiba.^79,82,83) High-level quinolone-resistant strains have also been isolated.^10,84–86) The mechanism of high-level resistance cannot be completely explained by mutations in target genes; other factors, such as drug efflux pumps, may also be related.⁸⁷⁾ Moreover, H. haemolyticus and H. parainfluenzae, which are closely related to H. influenzae, have also emerged as quinolone low-susceptible or highly resistant strains.^86,88,89)

Recent studies have demonstrated that quinolones with low susceptibility can be transferred horizontally through their inherent natural transformation system.¹²⁾ This transfer is relevant to the recognition of USS on the quinolone target genes¹²⁾ (Fig. 1). The efficacy of the horizontal transfer is higher than that of point mutations. Furthermore, horizontal transfer enables the simultaneous transfer of multiple genes¹¹⁾ and occurs across species such as H. haemolyticus to H. influenzae.⁹⁰⁾ Therefore, even if there are a few quinolone-resistant Haemophilus spp., there is a risk of the development and spread of quinolone-resistant H. influenzae in a short time. Considering the rapid increase in quinolone low-susceptible and -resistant strains in Japan, where quinolones are routinely used for all ages, similar situations may occur worldwide.

6. EFFICIENT SPREADING SYSTEM

Transformation is an efficient method of acquiring exogenous genes. As described above, H. influenzae has a unique natural transformation ability through the recognition of the USS, which decreases the risk of uptake of deleterious genes. Horizontal transfer by transformation contributes to the prevalence of BLNAR and quinolone resistance,^12,33,55) indicating that every means of resistance, including chromosomal mutations, can transfer horizontally in H. influenzae. According to recent studies, transformation efficiency varies among strains.¹²⁾ Moreover, a higher transformation ability is relevant to the acquisition of AMR. Thus, the transformation of H. influenzae is efficient for the spread of AMR (Fig. 2). In most cases, the emergence of AMR strains is initiated by the acquisition of mutations in target genes. Some strains have become more resistant to further mutations. Once resistant strains emerge, DNA fragments can also be released from these strains. These DNA fragments can confer resistance to susceptible strains when taken up by them. This pathway enables the rapid and efficient spread of resistant strains.

Fig. 2. Scheme of the Efficient Spreading Model of Haemophilus influenzae

7. CONCLUSION

Historically, H. influenzae acquired AMR in response to the introduction and increased use of antimicrobial agents. In Japan, it began with the prevalence of BLNAR, followed by the emergence and prevalence of macrolide and quinolone resistance. This rapid increase in prevalence may be related to transformational abilities. Resistance acquisition by transformation is a strategy for bacteria to adapt to their circumstances. Therefore, resistant bacteria may adapt easily, suggesting that the selection of resistant strains by antimicrobial use may lead to the simultaneous selection of highly adaptable strains. By repeating these selections, the bacterial community could form a knot of bacteria that are prone to acquiring resistance. The same phenomenon may occur in other bacteria with natural transformation abilities, such as Neisseria spp. Therefore, attention to the efficient spread of drug-resistant bacterial strains is necessitated.

Acknowledgments

The authors appreciated Prof. Yuji Morita and Prof. Hidemasa Nakaminami for giving us an opportunity to write this review.

Conflict of Interest

The authors declare no conflict of interest.

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