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Antimicrobial Chemotherapy
Research Article
15 February 2022

Quinolone Resistance Is Transferred Horizontally via Uptake Signal Sequence Recognition in Haemophilus influenzae

ABSTRACT

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA. Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC, respectively, contributed to the horizontal transfer of resistance in H. influenzae. Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

INTRODUCTION

Non-typeable Haemophilus influenzae (NTHi) is a common causative agent of community-acquired respiratory infections, sinusitis, and otitis media in the pediatric field. The β-lactams antibiotics are frequently selected as first-line agents for the treatment of H. influenzae infections, whereas respiratory quinolones can be used for empirical treatment without the identification of the causative pathogens (1). Moreover, the demand for quinolones has been growing due to an increase in the appearance of β-lactam-resistant strains among respiratory pathogens (24). In this sense, Japan approved the use of tosufloxacin for pediatric respiratory infections in 2010, further increasing the usage of quinolone (1). Accordingly, the amount of H. influenzae strains showing reduced susceptibility to quinolones with various genetic backgrounds has been increasing throughout Asia (47). In recent years, outbreaks caused by strains with low susceptibility to quinolones have been reported in Japan (7, 8). Furthermore, a high-level quinolone-resistant Haemophilus spp. strain has been recently isolated from pediatric patients (9), indicating that there is an urgent need to elucidate the mechanisms underlying the emergence of low susceptibility to quinolones in H. influenzae.
In general, quinolone resistance is developed by amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) (1012). For example, in Escherichia coli, the most well-characterized Gram-negative bacteria, amino acid substitutions in the QRDRs of GyrA result in strains that are less susceptible to quinolones (Ciprofloxacin [CIP] MIC = 0.5–2 μg/mL), and substitutions in the QRDRs of ParC confer resistance to quinolones (CIP MIC > 4 μg/mL) (13, 14). Based on Clinical and Laboratory Standard Institute (CLSI) guideline, the former strains were classified as intermediate or resistant, and the latter as resistant (14). The previous studies have suggested that quinolone resistance in H. influenzae has developed by a similar process (15); however, the mechanisms involved have not been fully explored yet.
In this study, we aimed to elucidate the acquisition mechanisms underlying H. influenzae low susceptibility to quinolones by performing phylogenetic analysis and in vitro resistance transfer assay in clinical H. influenzae isolates.

RESULTS

Phylogenetic analysis of H. influenzae strains based on gyrA sequence.

To clarify the genetic relationship among quinolone-low-susceptible H. influenzae strains, the clinical isolates harboring amino acid substitutions in the QRDRs of gyrA were selected and then the QRDRs of each isolate were compared (Fig. 1; Table S1 in the supplemental materials). The alignment results confirmed the presence of four amino acid substitutions (Ser 84 Phe or Leu, Asp88 Asn, Ala 117 Val, Glu 142 Lys) and multiple silent mutations (Fig. 1A). Furthermore, the phylogenetic analysis performed using the neighbor-joining method with the Tamura-Nei model indicated that the QRDRs were highly diverse across the isolates. Strains belonging to the same sequence type (ST) determined by multilocus sequence typing were classified into different clusters (Fig. 1B), suggesting the possibility of horizontal transfer of gyrA.
FIG 1
FIG 1 Phylogenetic analysis of H. influenzae strains harboring amino acid substitutions in QRDRs of gyrA. (A) Sequence alignment map. ST indicates the multilocus sequence typing. The matches and mismatches in the isolate sequences compared to H. influenzae Rd strain (Rd ST47) sequence are indicated by dots and nucleotide bases, respectively. The arrows represent the middle base of a codon that results in amino acid substitution. * indicates no change in amino acid. Color coding indicates strains of a same ST or clonal complex. (B) Dendrogram based on the nucleotide sequences shown in Fig. 1A, using Clustal Omega program and the neighbor-joining method. Color coding indicates strains of a same ST or clonal complex. * indicates putative outbreak strain.

Acquisition of resistance via horizontal recombination.

To verify our hypothesis that horizontal transfer of exogenous mutated gyrA is responsible for the low susceptibility of H. influenzae strains to quinolones, concentrated supernatants from quinolone-low-susceptible strains, 2018-Y40 (ST422) and 2018-Y38 (ST107), were mixed with H. influenzae Rd strain (ST47). The 2018-Y40 strain is an outbreak clone containing four prophages in its genome (8, 16), whereas ST107 is one of the major ST among the NTHi clinical isolates (4). Following culturing, many quinolone-low-susceptible (nalidixic acid-resistant) colonies were obtained (Fig. 2A). The sequencing of the resulting colonies confirmed their H. influenzae Rd origin and revealed that their QRDR of gyrA was replaced with that of the donor strain (Fig. 2B and C). The QRDRs of the quinolone-targeting genes gyrB, parC, and parE were not replaced. The quinolone susceptibility of the recombinant strains was 8-fold higher than that of Rd (Table S2).
FIG 2
FIG 2 Horizontal gene transfer and sequence analyses. (A) Representative result of supplemented brain heart infusion agar plates with 30 μg/mL of nalidixic acid following horizontal gene transfer assays. (B and C) Sequence maps showing partial QRDRs of gyrA from recipient, donor (2018-Y40 or 2018-Y38), and recombinant strains. (D) Bar graph indicating CFU/mL of resistant bacteria obtained following the horizontal gene transfer assays.
Phages, extracellular DNA, and outer membrane vesicles in the culture supernatant may act as putative determinants of recombination. To elucidate their involvement in the recombination process, a series of prophage knockout mutants were constructed, and horizontal gene transfer assays were performed in the presence of 1U DNase, 0.1% Triton X-100, or both. No resistant colonies were obtained when 1U DNase was added (Fig. 2D), suggesting that extracellular DNA plays an important role in horizontal gene transfer via recombination. Moreover, extracellular DNA was detected in the precipitated supernatant by nested PCR (data not shown); nevertheless, it could not be measured using the Qubit dsDNA BR assay kit, which has a quantification limit of 2 ng/μL (Thermo Fisher Scientific, Tokyo, Japan).

Involvement of the uptake signal sequence in horizontal gene transfer.

Haemophilus spp. are known to possess natural transformation ability and a short sequence (5′-AAGTGCGGT-3′) involved in gene uptake, also known as uptake signal sequences (17, 18). In particular, four (5′-GCGG-3′) are critical for gene transfer (19). Based on our analysis, the uptake signal sequence was found between 2,199 and 2,207 bp in gyrA sequence, which was located approximately 1,700 bp downstream the QRDR. To elucidate the role of the uptake signal sequence in recombination, various gyrA regions of the 2018-Y40 isolate, with or without the uptake signal, were amplified by PCR, and the amplicons were then used for the transfer assays, instead of culture supernatant. As shown in Fig. 3, only a few nalidixic-acid-resistant colonies were obtained by using the fragments without the uptake signal sequence, whereas a significant increase of resistant colonies was observed when fragments that included the uptake signal sequence were used, in particular those containing at least 50 bp of the upstream region of the uptake signal sequence (Fig. 3, Fig. S1). The gyrA sequence of eight resistant colonies, obtained using fragments c and d, showed that all the QRDRs were replaced with that of the fragments, indicating that horizontal gene transfer occurs by recognition of the gyrA uptake signal sequence (Fig. 3, Fig. S1).
FIG 3
FIG 3 Role of the uptake signal sequence in horizontal transfer of gyrA. Bar graph indicating CFU/mL of resistant bacteria obtained following the horizontal gene transfer assays. P value was calculated using Welch’s t test; P < 0.05 was considered statistically significant. The arrow indicates gyrA sequence. The red area in the sequence shows the QRDR and uptake signal sequence. Each fragment represents the PCR product of a different length that was used for horizontal gene transfer experiments. Fragment a does not contain an uptake signal sequence, whereas fragments b–d contain the uptake signal sequence.

Recombination of parC.

Previous reports have indicated that Gram-negative bacteria, including H. influenzae, become quinolone resistant owing to amino acid substitutions, first in the QRDRs of GyrA and then in that of ParC, in a stepwise manner (13, 15). To investigate the horizontal transfer of both gyrA and parC, in vitro horizontal gene transfer assays were performed using H. influenzae Rd and the culture supernatant of 2018-Y40 strain. For the selection of strains with both gyrA and parC substitutions, pipemidic acid was used, as described in our previous report (20). No pipemidic-acid-resistant colonies were obtained (Fig. S2). Therefore, we performed the assay using the Rd2018-Y40gyrA (Rd strain containing gyrA from 2018-Y40) strain, and obtained several pipemidic-acid-resistant colonies (Fig. 4, Fig. S3). These colonies showed a higher LVX MIC value than the Rd harboring mutated gyrA (Table S2). These findings indicated the occurrence of horizontal transfer of parC in a strain harboring the mutated gyrA, further reflecting the actual resistance mechanism in clinical settings.
FIG 4
FIG 4 Role of the uptake signal sequence in horizontal transfer of parC. Bar graph indicating CFU/mL of resistant bacteria obtained following the horizontal gene transfer assays. P value was calculated using Welch’s t test; P < 0.05 was considered statistically significant. The arrow indicates the parC sequence. The red area in the sequence shows the QRDR and uptake-signal-like sequence. Each fragment represents a PCR product with a difference in length of approximately 250 bp and was used for the horizontal gene transfer experiments. The fragments a–d do not contain the uptake signal-like sequence, whereas fragments e–g do contain it.
To explain the horizontal transfer mechanism of parC, we screened the parC sequence for the presence of the uptake signal sequence. However, no known uptake signal sequence was found in parC. Therefore, fragments of various length were amplified by PCR from the parC of 2018-Y40 strain and added to the Rd2018-Y40gyrA cultures. The resistant colonies were obtained using fragments of at least 1,250 bp (Fig. 4, Fig. S3). Moreover, resistant colonies obtained significantly increased when fragments of at least 1,500 bp were used, and further increase was detected with increasing fragment length (Fig. 4, P = 0.006 for fragment e, P = 0.001 for fragment f, P < 0.001 for fragment g). Four resistant colonies were picked up and their QRDRs of parC were sequenced. Three out of four colonies obtained with fragment d, and all of the colonies obtained with fragment e to g had a replaced parC, which meant that they were recombinant (Fig. S4). To confirm if the recombination of parC depends only on the fragment length, fragments of approximately 1,250 and 1,500 bp including the QRDR were constructed and examined; however, no recombinant colonies were obtained (Fig. S5). After detailed investigation of the parC sequence, a sequence similar to the uptake signal sequence (5′-AAGAGCGGT-3′), including four critical bases (5′-GCGG-3′), was found at ∼1,345 bp (Fig. 4, Fig. S3).The recombination efficiency of the fragment containing this region was significantly higher than that of other fragments (Fig. 4, Fig. S3), suggesting that the uptake signal-like sequence functions as an uptake signal sequence and plays a role in the recombination of parC.

Horizontal transfer in clinical isolates.

Haemophilus influenzae Rd is a laboratory strain known to have high competence ability (21). To confirm universality through H. influenzae, the clinical isolates that have various ST were tested. Although the transform ability was various, in all clinical isolates, nalidixic-acid-resistant colonies were obtained with fragment d, as used in Fig. 3 (Fig. 5A). In addition, pipemidic-acid-resistant colonies were obtained when fragment g, as used in Fig. 4, was added to each strain harboring a gyrA with amino acid substitutions (Fig. 5B). The LVX MIC value of these colonies increased to same level with a donor strain (Table S2). These findings suggest that horizontal transfer occurs not only in laboratory strain but also in clinical isolates.
FIG 5
FIG 5 Horizontal transfer in clinical isolates. (A) Bar graph indicating CFU/mL of nalidixic-acid-resistant bacteria obtained following the horizontal gene transfer assays using H. influenzae clinical strains and fragment d as used in Figure 3. (B) Bar graph indicating CFU/mL of pipemidic-acid-resistant bacteria obtained following the horizontal gene transfer assays using H. influenzae clinical strain harboring the mutated gyrA and fragment g as used in Figure 4. P value was calculated using Welch’s t test; P < 0.05 was considered statistically significant.

DISCUSSION

In this study, we analyzed the quinolone target genes and the horizontal transfer of quinolone resistance to understand H. influenzae quinolone-resistance mechanisms. Decreased susceptibility to quinolone is caused by amino acid substitutions in the QRDRs of GyrA and ParC, the quinolone target sites, which gradually results in quinolone resistance in a stepwise manner, as it has been shown that the substitutions occur in GyrA first and then in ParC (15). Furthermore, in recent years, the number of H. influenzae strains with amino acid substitutions in GyrA has been increasing (4, 6, 22, 23), and it has been reported that the emergence of quinolone resistance is related to the increase in quinolone usage (24); nevertheless, the mechanism of emergence and dissemination of isolates less susceptible to quinolone remains unclear. In the current study, the phylogenetic analysis of gyrA sequences from isolates with amino acid substitutions showed that they were highly diverse despite having the same molecular epidemiological backgrounds, strongly suggesting that quinolone resistance in H. influenzae emerged not only by point mutations but also by horizontal gene transfer. Therefore, we hypothesized that the regions of gyrA and parC conferring quinolone resistance to H. influenzae strains can be transferred and recombined. The in vitro horizontal gene transfer assays confirmed the transfer of mutated gyrA from the quinolone-resistant isolates to the quinolone-susceptible strains. In addition, the extracellular DNA in the culture supernatant played an important role in the transfer of quinolone resistance. Thus, our findings suggest that horizontal gene transfer could contribute to the recent increase in quinolone-low-susceptible strains. Furthermore, in the culture supernatant, the amount of DNA responsible for conferring low susceptibility to quinolone was below the quantification limit, indicating that a low DNA concentration is required for the recognition of the uptake signal and recombination. Additionally, our results showed that mutated parC is transferred only to those strains harboring a gyrA with amino acid substitutions, conferring quinolone low susceptibility. These findings further support the development of quinolone resistance in clinical settings. However, the reason the recombinants harboring only ParC substitutions could not be obtained remains unclear. The more likely explanation is DNA was taken up and recombined but did not confer the selected level of resistance against the quinolones commonly used in clinical settings. Without an increased MIC, possible transformants could not be selected for confirmation using the selection strategy of the experiment. Overall, these findings revealed that ParC substitutions may contribute to increased quinolone-resistance, but they are not essential for triggering the resistance in H. influenzae.
Haemophilus spp. are known to have natural transformation ability via recognition of the uptake signal sequence (5′-AAGTGCGGT-3′), a short repetitive sequence associated with gene uptake (17, 18). Our study indicated the presence of an uptake signal sequence in gyrA, and demonstrated its active role in gyrA transfer using a horizontal gene transfer assay. Furthermore, although an intact uptake signal was not found in parC, an uptake signal-like sequence (5′-AAGAGCGGT-3′) was found between the 1,337 and 1,345 bp, which played an important role in the transfer of parC. It has been reported that of the nine bases present in the uptake signal, four (5′-GCGG-3′) are critical for gene transfer (19), and the presence of these four bases in the uptake signal-like sequence further supports its role as an uptake signal. Thus, our study revealed that gyrA and parC transfer across H. influenzae isolates via uptake signal and uptake signal-like sequences, respectively.
The transfer of ftsI, a gene encoding penicillin-binding protein 3, has been previously reported (2527). Accordingly, our study demonstrated the horizontal transfer of quinolone resistance, which may be a factor contributing to the recently observed increased number of strains with low susceptibility to quinolones. As β-lactamase nonproducing ampicillin-resistant H. influenzae strains emerge because of cephem use, the use of quinolones may affect the emergence of strains with low susceptibility to quinolones.
Nonetheless, the current study has several limitations, in particular the small number of quinolone-low-susceptible strains used and the fact that all the strains were isolated from Japan. Hence, our findings may not accurately reflect the global situation. In Japan, quinolone has been used for pediatric patients since 2010, which may have contributed to such resistance acquisition. However, due to the increased emergence of antimicrobial resistant strains, quinolones are likely to be used in other countries, promoting the emergence of quinolone-resistant strains.
Herein, we provide in vitro evidence of the mechanisms underlying the development of quinolone-low-susceptible or resistant H. influenzae strains. Furthermore, the number of strains with amino acid substitutions in gyrA has been increasing in recent years (4, 6, 22, 23), and these strains may harbor mutated parC in the future, resulting in the emergence of quinolone-resistant strains.

MATERIALS AND METHODS

Phylogenetic analysis of gyrA sequence.

The sequence data of the strains with amino acid substitutions in the QRDRs of gyrA were obtained from previous studies (Table S1) (3, 4, 8). Multiple alignment was generated by GENETYX v.10 (GENETYX, Tokyo, Japan), and the sequences of the strains were compared to that of H. influenzae Rd strain (ST47). A phylogenetic dendrogram was constructed using gyrA sequences from 14 H. influenzae strains using the Clustal Omega multiple sequence alignment tool and neighbor-joining method of Tamura-Nei, in the Geneious Prime 2019 software platform (Biomatters, Auckland, NZ).

In vitro horizontal gene transfer assays.

Haemophilus influenzae clinical isolates 2018-Y40 (ST422) and 2018-Y38 (ST107) were used as donor strains, while H. influenzae Rd and the clinical isolates 2019-6 (ST422), 2017-18B (ST57), 2017-22B (ST143), and 2018-Y17 (ST107) were used as recipient strains. The strains were cultured overnight on chocolate agar at 37°C under a 5% CO2 atmosphere.
The recipient strain was cultured until logarithmic growth phase (OD = ca. 0.25) in brain heart infusion (BHI) broth supplemented with 15 μg/mL each of hemin and NAD (sBHI, supplemented brain heart infusion), and 50 μL of the culture was spotted on sBHI agar supplemented. After drying, 50 μL of the concentrated supernatant or PCR products of the donor strain were overlaid, and the plates were then incubated overnight at 37°C. The grown bacteria were scraped into 1 mL sBHI and spread on chocolate agar supplemented with 30 μg/mL of nalidixic acid (Wako, Osaka, Japan) or pipemidic acid (LKT Laboratories, MN, USA). The recombination was confirmed by comparing the strains with the control without DNA (mock control), and by DNA sequencing of QRDRs, as previously reported (10). The concentrated supernatant of the donor strain was prepared from 10 mL of the supernatant. Briefly, the supernatant was passed through a 0.20 μm filter to remove the viable donor strain cells, and the fine particles in the supernatant were then precipitated with PEG8000/NaCl solution to a 10-fold concentration, as previously described (28). The PCR products of gyrA and parC were amplified from the donor strain 2018-Y40. The primers used for PCR amplification are listed in Table S3. Furthermore, to investigate the recombination of determinants, the precipitated supernatant was pretreated with 0.1% Triton X-100 and 1U DNase I (TaKaRa-Bio, Shiga, Japan).

Construction of prophage knockout strains.

Both upstream and downstream (1.5-kb) regions of each prophage from H. influenzae 2018-Y40 strain were amplified by PCR (primer sequences are listed in Table S3). Additionally, the β-lactam-resistant gene, blaTEM-1, was amplified using forward and reverse primers (Table S3) containing 20-bp sequences that were homologous to the upstream and downstream regions of the prophage, respectively. The resulting PCR fragments were then mixed and amplified using the forward and reverse primers against the upstream and downstream regions, respectively, to obtain the resistance cassette for each prophage deletion. These fragments were introduced into H. influenzae 2018-Y40 cells by electroporation following the method described by Ubukata et al. (29). The transformants were selected by spreading the cells on chocolate agar containing 50 μg/mL of ampicillin. Finally, the resulting mutants were confirmed by PCR and DNA sequencing.

Antimicrobial susceptibility test.

MICs were determined by the broth microdilution method, according to the procedure described by the CLSI (14), using levofloxacin (Tokyo Chemical Industry, Tokyo, Japan) and tosufloxacin (FUJIFILM-Wako, Osaka, Japan). In addition, H. influenzae ATCC 49247 and Rd were used as quality control strains.

Statistics.

The statistical differences when comparing the number of resistant colonies with the control without DNA were evaluated using the Welch’s t test. P < 0.05 was considered statistically significant.

ACKNOWLEDGMENT

This study was partially supported by the Nagai Memorial Research Scholarship (No. N-202604) from the Pharmaceutical Society of Japan (to E.T.).

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cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 66Number 215 February 2022
eLocator: e01967-21
PubMed: 34930025

History

Received: 4 October 2021
Returned for modification: 27 October 2021
Accepted: 15 December 2021
Accepted manuscript posted online: 20 December 2021
Published online: 15 February 2022

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Keywords

  1. Haemophilus influenzae
  2. drug resistance mechanisms
  3. quinolones

Contributors

Authors

Department of Clinical Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan
Department of Clinical Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan
Kei-ichi Uchiya
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan
Department of Clinical Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

Notes

The authors declare no conflict of interest.

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American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
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