The Association between Transformation Ability and Antimicrobial Resistant Potential in Haemophilus influenzae

Abstract

The prevalence of quinolone low-susceptible Haemophilus influenzae has increased in Japan. Low quinolone susceptibility is caused by point mutations in target genes; however, it can also be caused by horizontal gene transfer via natural transformation. In this study, we examined whether this horizontal gene transfer could be associated with resistance to not only quinolones but also other antimicrobial agents. Horizontal transfer ability was quantified using the experimental transfer assay method for low quinolone susceptibility. Further, the association between horizontal transfer ability and resistance to β-lactams, the first-choice drugs for H. influenzae infection, was investigated. The transformation efficiency of 50 clinical isolates varied widely, ranging from 10² to 10⁶ colony forming unit (CFU) of the colonies obtained by horizontal transfer assay. Efficiency was associated with β-lactam resistance caused by ftsI mutations, indicating that strains with high horizontal transfer ability acquired quinolone low-susceptibility as well as β-lactam resistance more easily. Strains with high transformation efficiency increased the transcript level of comA, suggesting that enhanced com operon was associated with a high DNA uptake ability. Overall, this study revealed that the transformation ability of H. influenzae was associated with multiple antimicrobial resistance. Increase in the number of strains with high horizontal transformation ability has raised concerns regarding the rapid spread of antimicrobial-resistant H. influenzae.

INTRODUCTION

Treatment for infections caused by Haemophilus influenzae, a major causative bacterium of otitis media and sinusitis in paediatrics, has been mainly used β-lactams as the first-choice drug.¹⁾ Quinolones have also been used as alternative agents in cases of β-lactam resistant strains and/or recurrent infections after treatment with β-lactams.^1,2) However, the use of quinolones has increased owing to the increasing prevalence of resistant strains.^2,3) Notably, strains with low susceptibility or resistance to quinolones have emerged recently and are increasing.^4–8) The mechanism of low quinolone susceptibility in H. influenzae is known to be developed by point mutations in DNA gyrase, and then in topoisomerase IV, similar to those in other Gram-negative bacteria.^9–12) Further, the horizontal transfer of these target genes for quinolone low susceptibility by transformation was recently reported as an alternative mechanism for the development and spread of quinolone resistance.^13,14)

Transformation ability facilitates sharing of antimicrobial resistance and pathogenicity among H. influenzae, by the uptake of surrounding extracellular DNA fragments originating from close species.^15–17) Transformation is initiated by the recognition of a nine-base uptake signal sequence (USS, 5′-AAGTGCGGT-3′) on DNA; the DNA fragments are then taken up into bacterial cells and integrated into the chromosome by recombination.^18,19) Horizontal transfer of quinolone low susceptibility also occurs via this mechanism.¹⁴⁾ According to previous reports, the horizontal transfer efficiency of quinolones with low susceptibility differed depending on the strain,^13,14) suggesting that this horizontal transfer ability might be related to the ease of developing quinolone resistance. In addition, as β-lactam resistance was also developed by point mutations in targeting genes,²⁰⁾ development of resistance to β-lactams might also be related to horizontal transfer ability. In this study, therefore, we examined the association between horizontal transformation ability and antimicrobial resistance in H. influenzae.

MATERIALS AND METHODS

Bacterial Strains

Fifty quinolone-susceptible H. influenzae isolates obtained from a Japanese hospital in 2018 were used for this study.²¹⁾ For the horizontal gene transfer assay, H. influenzae Rd and 2017-22B, which was suggested to be highly transferable strains in a previous study,^13,14) were used as control strains. The bacterial strains were cultured overnight on chocolate agar at 37 °C.

In Vitro Horizontal Gene Transfer Assay

An in vitro horizontal gene transfer assay was performed as previously reported.^14,22) Briefly, H. influenzae was cultured in brain heart infusion broth supplemented with 15 mg/L of hemin and nicotinamide adenine dinucleotide (NAD) (sBHI broth) until it reached the logarithmic growth phase (OD₆₀₀ = approx. 0.25). Subsequently, the culture was spotted onto sBHI agar. After drying, PCR products (30 ng) were overlaid onto the bacteria. The plates were incubated overnight at 37 °C. The grown bacteria were scraped into sBHI broth and spread onto a chocolate agar plate supplemented with 10 µg/mL nalidixic acid (NA, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to select the quinolone low-susceptible strains.

Classification of Amino Acid Substitution in Penicillin-Binding Protein 3 (PBP3)

Amino acid substitutions in penicillin-binding protein 3 (PBP3) were classified by sequencing ftsI as described previously.²⁰⁾ In cases where the strain harboured R517H or N526K and M377I, S385T, or L389F in FtsI, the strain was classified as mutation positive.

Quantification of Transcript Level

The tested strains were cultured in sBHI broth until they reached the logarithmic growth phase, and then used for RNA extraction using a Monarch Total RNA Miniprep Kit (New England BioLabs, Ipswich, MA, U.S.A.). The extracted RNA was quantified using a Quantus Fluorometer and QuantiFluor RNA System (Promega, Madison, WI, U.S.A.). RNA was converted to cDNA using High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.). DNA amplification and fluorescence detection were performed using SYBR Green Master Mix (Thermo Fisher Scientific) and a Light Cycler (Roche, Switzerland). Amplification was initiated by incubation at 95 °C for 10 min, followed by 40 PCR cycles at 95 °C for 10 s, 52 °C for 10 s, and 72 °C for 10 s.

Statistical Analysis

Statistical significance was evaluated using EZR²³⁾ and GraphPad prism ver.10 (Graph Pad, La Jolla, CA, U.S.A.). p < 0.05 was considered statistically significant.

RESULTS

Quantification of Horizontal Transformation Ability in Clinical Isolates

A previous report on quinolone resistance (low quinolone susceptibility) suggested that horizontal transfer efficiency might vary among clinical isolates,^13,14) indicating that quantifying horizontal transfer ability was possible by utilising an experimental transfer assay for quinolone resistance. Therefore, 50 quinolone-susceptible clinical isolates, which were all isolated as quinolone low-susceptible isolates from a hospital in a year,²¹⁾ were tested using a horizontal transfer assay with a mutated gyrA fragment (30 ng). As shown in Supplementary Fig. 1, nalidixic acid (NA)-resistant colonies were obtained from 36 strains (72.0%). The number of colonies ranged from 10² to 10⁶ colony forming unit (CFU). The number of NA-resistant colonies of seven strains was higher than that of the laboratory strain Rd, suggesting that these had a high horizontal transfer ability.

β-Lactam-resistant H. influenzae is mainly categorised into either β-lactamase-producing ampicillin-resistant H. influenzae (BLPAR) or β-lactamase-non-producing ampicillin-resistant H. influenzae (BLNAR). BLNAR are prevalent and account for 20–50% of H. influenzae cases in Japan.^2,24) The mechanism of BLNAR involves mutations in ftsI, which encodes penicillin-binding protein 3, a target site for cephalosporins and penicillins. This means that β-lactam resistance occurs through mutation in drug targeting site like quinolones. According to previous studies in Japan, most quinolone low-susceptible clinical isolates were also BLNAR.^7,25) Therefore, we hypothesised that strains showing high-level transformation ability had already acquired mutated ftsI through horizontal transfer or were likely to acquire mutated ftsI in future. To verify this hypothesis, the association between ftsI mutations resulting in amino acid substitutions and transformation ability was analysed. Clinical isolates were classified into two groups according to amino acid substitutions involved in β-lactam resistance (Supplementary Table 2). Forty and ten strains were classified into the amino acid substitution-positive and -negative group, respectively (Figs. 1A, B). The number of NA-resistant colonies obtained in the positive group was significantly higher than that in the negative group (Fig. 1C). These results indicate that strains with high horizontal transfer ability could easily develop resistance to β-lactams as well.

Fig. 1. Association between Transformation Ability and FtsI Amino Acid Substitutions

(A, B) Bar graphs indicate the CFU/mL of nalidixic acid-resistant bacteria obtained using the horizontal gene transfer assay. Values represent the mean + standard error (S.E.) obtained from at least three independent experiments. (A) and (B) indicate amino acid substitution-negative and -positive groups for FtsI, respectively. (C) Scatter plot showing nalidixic acid-resistant bacteria in A and B. The p-value was calculated using a Mann–Whitney U test.

Contribution of the com Operon to Horizontal Transfer

The horizontal transfer of H. influenzae is driven by the com operon.²⁶⁾ To investigate the differences in horizontal transfer ability among strains, the transcript levels of comA, the first gene of the com operon, in randomly selected strains were compared. The comA transcript levels of highly transferable strains, 2018-Y41 and 2017-22B, were significantly higher than those of H. influenzae Rd (Fig. 2). In contrast, the low-transferable strain, 2018-Y24, showed significantly lower comA levels (Fig. 2). The transcript levels of comA in 2018-Y30 (low-transferable strain) and Y39 (high-transferable strain) tended to be low and high, respectively, but no significant differences were observed. These results indicated that strains with high-level transformation ability could arise by enhanced transcript levels of com genes. Other mechanisms may also affect transfer ability.

Fig. 2. Quantification of comA Transcript Level

Transcript levels were evaluated using real-time PCR. The bar graph indicates the transcript levels of comA. The value was relative to the transcript level of Rd. 2018-Y24 and 2018-Y30 were low transferable strains whereas 2018-Y39, 2018-Y41, and 2017-22B were high transferable strains. Values represent the mean + S.E. obtained from at least three independent experiments. p-Value was calculated using a Kruskal–Wallis test with Bonferroni' correction.

DISCUSSION

H. influenzae has been developing antimicrobial resistance to various agents.¹²⁾ In particular, resistance to β-lactams is highly prevalent, with a resistance rate of over 50% in Japan.^7,24) The mechanisms underlying the antimicrobial resistance in H. influenzae are diverse and complex.¹²⁾ H. influenzae has an inherently flexible gene transfer ability associated with resistance to quinolones.^14,16) In this study, the transformation efficiency evaluated by the transfer assay of quinolone resistance was associated with the presence of ftsI mutations involved in β-lactam resistance, indicating that strains with high horizontal transformation ability could easily acquire not only low susceptibility to quinolone as well as β-lactam resistance.

There are two β-lactam resistance mechanisms in H. influenzae: production of β-lactamase²⁷⁾ and acquisition of mutations in ftsI.^20,27,28) Since the 2000s, the number of resistant strains harbouring mutations in ftsI, also known as BLNAR, has increased in Japan.^2,24) The development of antimicrobial resistance is related to the use of antimicrobial agents.^29,30) The prevalence of BLNAR in Japan is presumed to be related to the high use of 3rd generation cephalosporins.³¹⁾ Because for H. influenzae, horizontal transfer is an important inherent system for adapting to and evolving in the environment, acquiring exogenous mutated ftsI horizontally is one way to adapt to the environment. Accordingly, a highly transferable strain may be selected based on the selective pressure of antimicrobial agents. Under such circumstances, it is easier for the selected strains to acquire resistance via horizontal transfer and spread it rapidly. Further, since macrolide resistance is known be caused by mutations,³²⁾ horizontal transfer ability could also be associated with macrolide resistance acquisition.

Horizontal transfer of H. influenzae is driven by the com operon.²⁶⁾ The transcript level of comA was found to be enhanced in strains with high-level horizontal transfer ability, suggesting that the enhanced com operon was associated with high DNA uptake ability. The detection of this enhancement could be a feasible method for detecting strains with potential ability for antimicrobial resistance.

This study has two limitations. First, we investigated the relationship between horizontal transfer ability and antimicrobial resistance by focusing on β-lactam resistance. Because we used strains isolated in Japan, where BLNAR is highly prevalent, isolates from other countries may show a different relationship. Further, the association between macrolides and transfer ability could not be evaluated owing to the small number of resistant strains. However, as macrolide-resistant strains are also β-lactam-resistant according to a previous report, transfer ability might be involved in resistance acquisition.²⁴⁾ Second, the transcript level of the com operon alone was insufficient to explain the high horizontal transfer ability observed. Because horizontal transformation of H. influenzae involves steps such as DNA recognition, DNA uptake, and recombination of DNA with the genome, horizontal transfer efficiency may be affected. Therefore, the difference in the horizontal transfer ability was considered to be affected by other factors.

Overall, this study revealed that the transformation ability of H. influenzae was associated with multiple antimicrobial resistance. Increase in the number of strains with high horizontal transformation ability has raised concerns regarding the rapid spread of antimicrobial-resistant H. influenzae. Although quinolone resistance is currently not a significant clinical concern, high-level quinolone-resistant strains have already emerged, in addition to low-susceptible strains. In Japan, where highly transferable strains have been selected by the usage of broad-spectrum antimicrobial agents, there is a major concern that quinolone-resistant H. influenzae may spread more rapidly.

Acknowledgments

We appreciate Prof. Hidemasa Nakaminami and Dr. Naoki Hara for providing clinical isolates and helpful discussions, and Mr. Tomokazu Ando for providing technical assistance.

Funding

This work was supported by JSPS KAKENHI Grant Number 23KJ1955 (to E.T.) and the Research Institute of Meijo University (to T.W.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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