Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of β-lactamase–negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to β-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported.
KEYWORDS: quinolone resistance, Haemophilus influenzae, antimicrobial resistance, fluoroquinolone
ABSTRACT
Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of β-lactamase–negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to β-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported. The current risk of fluoroquinolone resistance in H. influenzae (especially in high-BLNAR) has not yet been evaluated. Here, we examined the development of fluoroquinolone resistance in fluoroquinolone-susceptible clinical H. influenzae isolates in vitro during passaging in the presence of moxifloxacin (from 0.03 to 128 mg/liter). Twenty-nine isolates were examined. Seventeen isolates (58.6%) showed reduced moxifloxacin susceptibility, and 10 of these 17 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute breakpoint for moxifloxacin (MIC of >1 mg/liter) after repeat cultivation on moxifloxacin-containing agar. Seven of these ten isolates were high-BLNAR and represented multiple lineages. We identified 56 novel mutations in 45 genes induced during the development of fluoroquinolone resistance, except the defined quinolone resistance-determining regions (Ser84Leu and Asp88Tyr/Gly/Asn in GyrA and Gly82Asp, Ser84Arg, and Glu88Lys in ParC). Glu153Leu and ΔGlu606 in GyrA, Ser467Tyr and Glu469Asp in GyrB, and ompP2 mutations were novel mutations contributing to fluoroquinolone resistance in H. influenzae. In conclusion, H. influenzae clinical isolates from multiple lineages can acquire fluoroquinolone resistance by multiple novel mutations. The higher rate of derivation of fluoroquinolone-resistant H. influenzae from high-BLNAR than β-lactamase-negative ampicillin-susceptible isolates (P = 0.01) raises the possibility of the emergence and spread of fluoroquinolone-resistant high-BLNAR in the clinical setting.
INTRODUCTION
Haemophilus influenzae is a pathogenic microorganism that mainly causes respiratory and otolaryngological infections (1, 2). β-Lactam antimicrobials, in combination with β-lactamase inhibitors, are the first-choice drugs for treating these infections (3–5). However, β-lactam-resistant strains, termed β-lactamase-positive ampicillin-resistant H. influenzae (BLPAR) (6) and β-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) (7), have emerged. In particular, the prevalence of BLNAR is increasing in various countries (8–13). We recently reported the high prevalence (54.8% of all H. influenzae clinical isolates) of β-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) exhibiting multiclonal expansion in Japan (14). Such an increase has also been reported recently in South Korea (15). These observations are a warning about the ineffectiveness of β-lactam antimicrobials for the treatment of high-BLNAR infections.
Fluoroquinolones are potentially a promising class of antimicrobials for the treatment of β-lactam-resistant H. influenzae infections (3–5). Indeed, a low prevalence of fluoroquinolone-nonsusceptible H. influenzae (approximately 0.1 to 3% in Japan and other countries) has been reported (16–22). However, a few reports describing the increase of prevalence of fluoroquinolone-resistant H. influenzae have been published. In Taiwan, the prevalence of levofloxacin-resistant H. influenzae increased from 2% in 2004 to 24.3% in 2010 (23). In Denmark, clonal spread of high-level ciprofloxacin-resistant H. influenzae was reported in 2016 (24). We recently reported four (3.8% of all H. influenzae clinical isolates) H. influenzae clinical isolates exhibiting reduced fluoroquinolone susceptibility in Japan during 2017 and 2018; three of these isolates are high-BLNAR (14). The occurrence of fluoroquinolone-resistant H. influenzae has a clinical impact, and the increasing occurrence of fluoroquinolone-resistant high-BLNAR is a serious problem in the clinical setting. Therefore, the frequency of occurrence of fluoroquinolone-resistant H. influenzae (especially among high-BLNAR) should be evaluated. However, such investigations have not been performed to date.
The main mechanism of reduced fluoroquinolone susceptibility in H. influenzae involves amino acid substitutions in quinolone resistance-determining regions (QRDRs) in DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) (25). In QRDRs, the amino acid substitutions Ser84 and Asp88 in GyrA and Gly82, Ser84, and Glu88 in ParC (termed “defined QRDR mutations”) have been reported as the main contributors to the development of fluoroquinolone resistance in H. influenzae (26). However, the fluoroquinolone resistance mechanism in clinical isolates of H. influenzae cannot be fully explained by the presence of the defined QRDR mutations, because H. influenzae clinical isolates exhibiting fluoroquinolone resistance or reduced fluoroquinolone susceptibility without any defined QRDR mutations were reported without detailed genetic analysis (19, 21, 27, 28). Therefore, undefined resistance mechanisms should be investigated.
Understanding the detailed mechanism of emergence of fluoroquinolone-resistant H. influenzae, which takes into account the association with high-BLNAR, is required for the appropriate use of β-lactams and fluoroquinolones to treat H. influenzae infections and to prevent further spread of fluoroquinolone-resistant H. influenzae, including high-BLNAR. In the current study, we multiply passaged various fluoroquinolone-susceptible clinical isolates of H. influenzae in the presence of moxifloxacin, a fluoroquinolone, and then examined mutations that occurred in the derived fluoroquinolone-resistant mutants by whole-genome sequencing. We aimed to (i) assess the risk of the emergence of fluoroquinolone-resistant H. influenzae, particularly among high-BLNAR, and (ii) identify novel genetic factors contributing to fluoroquinolone resistance in H. influenzae.
RESULTS
Selection of fluoroquinolone-resistant mutants in vitro.
We selected 29 clinical fluoroquinolone-susceptible isolates of H. influenzae based on the genotypes of the ftsI gene, which encodes penicillin-binding protein 3 (PBP3), the production of β-lactamase (TEM-1), and the sequence type (ST). We confirmed nonbiased distribution of clonality by ST and phylogenetic analysis (Table 1; see also Fig. S1 in the supplemental material). We passaged the isolates on supplemented brain heart infusion (sBHI) agar containing moxifloxacin (from 0.03 to 128 mg/liter). We used moxifloxacin as the selective agent because it is one of the most frequently used fluoroquinolones in respiratory medicine in many countries, including Japan (29, 30).
TABLE 1.
Sequence types, MXF MIC, and AMP resistance genotypes of H. influenzae isolates used in this studya
| Isolate | ST | MXF MIC (mg/liter) | AMP resistance genotype |
|---|---|---|---|
| SMHi6b | 159 | 0.03 | BLNAS |
| SMHi11c | 404 | 0.03 | BLNAS |
| SMHi16 | 1653 | 0.03 | BLNAS |
| SMHi21 | 159 | 0.03 | BLNAS |
| SMHi24 | 1653 | 0.03 | BLNAS |
| SMHi35 | 1878 | 0.03 | BLNAS |
| SMHi37c | 1879 | 0.015 | BLNAS |
| SMHi39c | 147 | 0.03 | BLNAS |
| Hui113 | 18 | 0.03 | BLNAS |
| Hui160c | 2141 | 0.03 | BLNAS |
| SMHi22b | 103 | 0.06 | BLPAR |
| Hui85 | 3 | 0.03 | BLPAR |
| Hui128c | 103 | 0.06 | BLPAR |
| Hui293 | 3 | 0.03 | BLPAR |
| SMHi4b | 142 | 0.03 | Low-BLNAR (II) |
| Hui227c | 33 | 0.03 | BLPACR (II) |
| SMHi2b | 34 | 0.03 | High-BLNAR (III) |
| SMHi3 | 183 | 0.03 | High-BLNAR (III) |
| SMHi5 | ND | 0.03 | High-BLNAR (III) |
| SMHi9b | 156 | 0.25 | High-BLNAR (III) |
| SMHi10 | 155 | 0.03 | High-BLNAR (III) |
| SMHi15b | 834 | 0.03 | High-BLNAR (III) |
| SMHi17b | 422 | 0.06 | High-BLNAR (III) |
| SMHi18b | 1218 | 0.25 | High-BLNAR (III) |
| SMHi23b | 422 | 0.03 | High-BLNAR (III) |
| Hui436b | 1429 | 0.03 | High-BLNAR (III) |
| SMHi25 | 183 | 0.03 | High-BLNAR (III-like) |
| Hui68 | 986 | 0.03 | BLPACR (III) |
| Hui410c | 986 | 0.03 | BLPACR (III) |
MXF, moxifloxacin; AMP, ampicillin; BLNAS, β-lactamase negative, AMP susceptible; BLNAR, β-lactamase negative, AMP resistant; BLPACR, β-lactamase-producing amoxicillin, clavulanate resistant; BLPAR, β-lactamase producing, AMP resistant. Parentheses indicate genogroup of the ftsI gene encoding PBP3 (4, 6, 11). ST, sequence type; ND, not determined because of failed fucK PCR amplification during typing.
Fluoroquinolone-resistant mutants (exceeding breakpoint) obtained.
Mutants with reduced fluoroquinolone susceptibility, but not fluoroquinolone resistant, obtained.
Of the 29 clinical isolates examined, 17 isolates (58.6%) showed reduced moxifloxacin susceptibility after the first passage on moxifloxacin-containing agar (Table 2). Among these 17 isolates, 10 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute (CLSI) breakpoint for moxifloxacin (MIC of >1 mg/liter). At least two passages were required for the 10 isolates to exceed the moxifloxacin breakpoint (Table 2) and exhibit resistance to other fluoroquinolones (levofloxacin and tosufloxacin), but most mutants remained susceptible to sitafloxacin after multiple passages on moxifloxacin-containing agar (Table S1). The appearance frequency of mutants with reduced moxifloxacin susceptibility after various passages varied among the parental isolates, from 3.5 × 10−9 to 8.3 × 10−7 (Tables S1 and S2). The frequency tended to be higher for isolates that gave rise to mutants exceeding the CLSI breakpoint for fluoroquinolones than for those that gave rise to mutants that did not exceed the breakpoint.
TABLE 2.
Fluoroquinolone MIC values and resultant amino acid substitutions in GyrA, GyrB, ParC, and ParE in mutants exhibiting the highest MXF MIC derived from the respective clinical isolates of H. influenzae
| In vitro-derived mutant | No. of passages required to highest MXF MICj | MICk (mg/liter) |
Substitution inl: |
||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| GyrA |
GyrB |
ParC |
ParE | ||||||||||||||||||
| MXF | LVX | TFX | SFX | S84 | D88 | E153 | Q606* | S467 | E469 | T472 | A21* | G82 | D83 | S84 | E88 | R515* | R555* | R618* | L502 | ||
| In vitro-derived mutants exceeding MXF breakpoint | |||||||||||||||||||||
| SMHi4-H | 5 (3) | 128 | 64 | 32 | 4 | L | N | Da ,f | |||||||||||||
| SMHi18-H | 5 (3) | 32 | 32 | 16 | 1 | L | D | Ra ,b ,c ,e ,f | |||||||||||||
| SMHi2-H | 6 (4) | 32 | 8 | 8 | 1 | N | K** | Yc ,f | Ka ,b ,e ,f | ||||||||||||
| SMHi6-H | 4 (2) | 8 | 4 | 4 | 0.5 | L | H | ||||||||||||||
| SMHi17-H | 3 (2) | 8 | 4 | 4 | 0.5 | L | |||||||||||||||
| SMHi23-H | 3 (2) | 8 | 2 | 4 | 0.13 | L | Ib ,h | Nb | |||||||||||||
| SMHi15-H | 3 (3) | 4 | 4 | 2 | 0.5 | La ,b ,c ,e ,f ,g ,i | L | ||||||||||||||
| SMHi9-H | 3 (2) | 4 | 1 | 1 | 0.13 | L | V | ||||||||||||||
| Hui436-H | 4 (4) | 2 | 0.5 | 0.25 | 0.06 | Y | K | F | |||||||||||||
| SMHi22-H | 2 (2) | 2 | 0.5 | 0.25 | 0.06 | Na ,b ,c ,e ,f ,i | Δ | C | |||||||||||||
| In vitro-derived mutants not exceeding MXF breakpoint | |||||||||||||||||||||
| SMHi39-H | 3 | 1 | 1 | 0.5 | 0.03 | D | R | ||||||||||||||
| Hui128-H | 2 | 1 | 0.25 | 0.25 | 0.03 | Y | |||||||||||||||
| SMHi11-H | 2 | 0.5 | 0.25 | 0.25 | 0.03 | Ga ,b ,e ,g | |||||||||||||||
| Hui410-H | 3 | 0.5 | 0.25 | 0.13 | 0.015 | Ya ,d ,e ,f | |||||||||||||||
| Hui160-H | 1 | 0.06 | 0.06 | 0.015 | <0.008 | Y | |||||||||||||||
| Hui227-H | 1 | 0.06 | 0.03 | 0.03 | 0.015 | I | |||||||||||||||
| SMHi37-H | 1 | 0.06 | 0.03 | 0.015 | 0.015 | ||||||||||||||||
Reported in ciprofloxacin (CIP)-resistant H. influenzae, Spain, 2000–2013 (18).
Reported in CIP-resistant H. influenzae, Japan, 2001–2007 (31).
Reported in CIP- and LVX-resistant H. influenzae, Japan, 2002–2004 (17).
Reported in fluoroquinolone-resistant H. influenzae, US, 2002–2004 (32).
Reported in LVX-resistant H. influenzae, Taiwan, 2004–2010 (23).
Reported in LVX- or MXF-resistant H. influenzae, Japan, 2006–2010 (19).
Reported in fluoroquinolone-resistant H. influenzae, Switzerland, 2016–2018 (33).
Reported in CIP-resistant H. influenzae, Spain (year unknown) (27).
Reported in CIP-resistant H. influenzae, Denmark (year unknown) (25).
Parentheses indicate the number of passages required beyond the moxifloxacin MIC breakpoint (>1 mg/liter).
LVX, levofloxacin; TFX, tosufloxacin; SFX, sitafloxacin.
Amino acid substitutions were detected in the in vitro-derived H. influenzae mutants with the highest MFX MIC, detected after sequence comparison with the parental H. influenzae clinical isolates. Boldface, known amino acid substitutions associated with fluoroquinolone resistance (defined QRDR mutations). *, Substitutions outside the QRDR regions, did not identify by PCR using specific primer sets (14, 27); **, substitutions present in SMHi2-1 but not in SMHi2-3.
Based on β-lactam susceptibility, high-BLNAR was the population of strains that most frequently acquired fluoroquinolone resistance (7 of 11 isolates examined) compared with other populations: β-lactamase-negative ampicillin-susceptible (BLNAS) isolates (1 of 10 isolates, P = 0.01 compared with BLNAR) and BLPAR isolates (1 of 4 isolates). No fluoroquinolone-resistant mutants were derived from three β-lactamase-producing amoxicillin-clavulanate-resistant H. influenzae (BLPACR) isolates.
Detection of defined QRDR mutations in the gyrA, gyrB, parC, and parE genes in mutants with reduced fluoroquinolone susceptibility.
We then examined the presence of defined QRDR mutations (resulting in amino acid substitutions at previously identified positions in GyrA and ParC proteins) (26) in 17 mutants that showed the highest moxifloxacin MIC from the respective parental clinical isolates. Fourteen mutants harbored between one and three amino acid substitutions that corresponded with the defined QRDR mutations (Table 2).
For GyrA, the amino acid substitution Ser84Leu was the dominant defined QRDR mutation observed in seven mutants, all of which ultimately exceeded the moxifloxacin breakpoint. The amino acid substitution Asp88 in GyrA was also observed in seven mutants. We detected three types of amino acid substitutions (Asp88Tyr, Asp88Gly, and Asp88Asn). Similarly, in ParC, we detected three types of amino acid substitutions, Gly82Asp, Ser84Arg, and Glu88Lys, in four mutants.
Detection of undefined and non-QRDR mutations by whole-genome sequencing of mutants that exceeded the CLSI breakpoint for moxifloxacin.
We detected 56 novel mutations in 45 genes by using whole-genome sequencing in 10 mutants that exceeded the CLSI breakpoint for moxifloxacin. These mutants harbored 11 types of amino acid substitutions other than those caused by the defined QRDR mutations: Glu153Lys and ΔGln606 in GyrA; Ser467Tyr, Glu469Asp, and Thr472Ile in GyrB; Ala21Val, Asp83Asn, Arg515His, Arg555Leu, and Arg618Cys in ParC; and Leu502Phe in ParE (Table 2). The mutations, in addition to the defined QRDR mutations, accumulated in 10 mutants that exceeded the moxifloxacin breakpoint (Table 2 and Fig. 1). Besides the gyrA, gyrB, parC, and parE genes, 45 mutations occurred in 41 genes upon multiple passages on moxifloxacin-containing agar (Table 3). Specifically, mutations in ompP2 occurred in four mutants: SMHi2 acquired one point mutation, C392A, corresponding to Thr131Lys; and OmpP2 of SMHi15, SMHi17, and SMHi18 was disrupted by frameshifts at Val161 (insertion of AA between nucleotides 480 and 481), Glu278 (insertion of GG between nucleotide 832 and 833), and Val37 (C nucleotide deletion at position 108), respectively.
FIG 1.
Relationship between the development of fluoroquinolone resistance and QRDR mutations. The clinical isolates were grown on sBHI agar containing moxifloxacin (from 0.03 to 128 mg/liter) at 37°C under an atmosphere of 5% CO2 for 48 h. y axis, number of clinical isolates and obtained mutants with reduced fluoroquinolone susceptibility or fluoroquinolone resistance. Defined and undefined/non-QRDR mutations were both considered. Moxifloxacin MIC of >1 mg/liter indicates resistance.
TABLE 3.
Mutations resulting in amino acid substitutions in H. influenzae mutants exceeding the CLSI breakpoint for moxifloxacin
| Strain | Mutated gene(s)a (corresponding amino acid substitution[s]) |
|---|---|
| SMHi4-H | acrB (A813V), asd (A371V), atpB (I255V), ccmH (R20V), fabA (M145I), lexA (P91S), mlaE (G24S), pdxY (T257fs), rfaF (Y324fs), rpoB (L1102P), torY (A197fs), acetyltransferase (S61A), UPF0701 protein (V269A), six hypothetical proteins,b integrase (P63L), lysozyme (E152G), putative acyl-coenzyme A thioester hydrolase (Δ20R), putative glycosyltransferase (Y210fs) |
| SMHi18-H | artM (T59fs), ompP2 (V37fs), putative HTH-type transcriptional regulator (G86S), hypothetical protein (T22fs) |
| SMHi2-H | ompP2 (T132K), fabH (G38S), rpoD (P520S), tadA (R157C), 5′–3′ exoribonuclease (H10fs) |
| SMHi6-H | secD (R599C), udk (L115fs), glycosyl transferase family 1 (P335fs) |
| SMHi17-H | corA (S231L), ompP2 (E278fs) |
| SMHi23-H | fmt (D37_K38insN), putative HTH-type transcriptional regulator (D161fs) |
| SMHi15-H | fabZ (E49G), ompP2 (Val161fs) |
| SMHi9-H | dnaK (G221S), infC (D107E), dusB (I3fs) |
| Hui436-H | adk (G80S) |
| SMHi22-H | None |
Mutations in genes other than gyrA, gyrB, parC, and parE (shown in Table 2) in the in vitro-derived H. influenzae mutants with the highest moxifloxacin MIC determined are shown. The mutations were determined by sequence comparison with the parental H. influenzae clinical isolate. fs, frameshift.
Six hypothetical proteins with one mutation each (Table S3).
We also extensively passaged some H. influenzae isolates on sBHI agar without moxifloxacin as a control experiment. We did not observe mutations in the control experiment that were detected in mutants with reduced moxifloxacin susceptibility in strains (Table S3).
Growth of fluoroquinolone-resistant mutants.
To evaluate the impact of the acquisition of fluoroquinolone resistance on bacterial growth, we next examined the growth of fluoroquinolone-resistant mutants compared with that of the parental isolates (Fig. 2). Among 10 H. influenzae isolates that acquired moxifloxacin resistance during multiple passages on moxifloxacin-containing agar, the growth of mutants derived from SMHi2 and Hui436 was gradually reduced compared with that of the parental isolates. For the mutant series derived from SMHi4, SMHi17, SHMi18, and SMHi9, the growth of mutants exhibiting a moxifloxacin MIC of 2 mg/liter was similar to that of the parental strain, whereas the growth of mutants with the highest moxifloxacin MIC (>4 mg/liter) was reduced. In contrast, the growth of the remaining mutant series (derived from SMHi6, SMHi15, SMHi22, and SMHi23) was similar to that of the parental strain.
FIG 2.
Effect of developing fluoroquinolone resistance on bacterial growth during the development of fluoroquinolone resistance. The growth of the parental H. influenzae clinical isolates (gray lines), mutants exhibiting a moxifloxacin MIC exceeding the breakpoint (>1 mg/liter) (dotted lines), and mutants with the highest moxifloxacin MIC (boldface lines) are shown. H. influenzae isolates or mutants were grown in 0.1 ml of sBHI broth in a 96-well plate at 37°C for 24 h with shaking at 140 rpm. The data are representative of triplicate experiments.
Introduction of specific gyrA, gyrB, parC, parE, and ompP2 mutations into the H. influenzae Rd strain.
To investigate whether the identified mutations contribute to the reduced fluoroquinolone susceptibility of H. influenzae, we next attempted to introduce the gyrA, gyrB, parC, and parE mutations associated with the specific amino acid substitutions identified in the mutants with reduced moxifloxacin susceptibility into H. influenzae wild-type strain Rd (Table 4). We generated the Rd strain transformants by homologous recombination. The gyrA transformants of strain Rd were Rd_GyrASer84Leu, Rd_GyrAAsp88Asn, and Rd_GyrAAsp88Tyr (these mutations were used as control experiments), as well as Rd_GyrAGlu153Lys and Rd_GyrAΔGlu606; the mutations corresponded to those identified in SMHi15-H, SMHi2-H, Hui436-1, SMHi2-1, and SMHi22-1, respectively. In the introduction of each mutation into gyrB, parC, and parE sequences of Rd by using the site-direct mutagenesis method, we obtained gyrB transformants (Rd_GyrBSer467Tyr and Rd_GyrBGlu469Asp). We were unable to obtain Rd transformants harboring the specific mutations in parC and parE. Most of the fluoroquinolone MICs of the Rd gyrA and gyrB mutants were more than two times higher than the MIC of the parental strain (Table 4).
TABLE 4.
Fluoroquinolones and β-lactam MIC values of gyrA and/or ompP2 mutants derived from strain Rd
| Strain | MICa
(mg/liter) |
||||||
|---|---|---|---|---|---|---|---|
| MXF | LVX | TFX | SFX | AMP | CRO | CTX | |
| Rd | 0.015 | 0.015 | 0.008 | 0.002 | 0.25 | 0.004 | 0.015 |
| Rd_GyrASer84Leu | 0.13 | 0.13 | 0.06 | 0.015 | 0.25 | 0.004 | 0.015 |
| Rd_GyrAAsp88Asn | 0.06 | 0.06 | 0.03 | 0.015 | 0.25 | 0.004 | 0.015 |
| Rd_GyrAAsp88Tyr | 0.06 | 0.06 | 0.03 | 0.008 | 0.25 | 0.004 | 0.015 |
| Rd_GyrAGlu153Lys | 0.06 | 0.06 | 0.015 | 0.015 | 0.25 | 0.004 | 0.015 |
| Rd_GyrAΔGlu606 | 0.03 | 0.03 | 0.015 | 0.008 | 0.25 | 0.004 | 0.015 |
| Rd_GyrBser467Tyr | 0.06 | 0.06 | 0.015 | 0.008 | 0.25 | 0.004 | 0.015 |
| Rd_GyrBGlu469Asp | 0.06 | 0.06 | 0.008 | 0.004 | 0.25 | 0.004 | 0.015 |
| Rd_ΔompP2 | 0.06 | 0.06 | 0.015 | 0.008 | 0.25 | 0.06 | 0.03 |
| Rd_ΔompP2/pMC-tandem GFP(−) | 0.03 | 0.06 | 0.015 | 0.008 | 0.25 | 0.06 | 0.03 |
| Rd_ΔompP2/pMCompP2 | 0.015 | 0.015 | 0.008 | 0.002 | 0.25 | 0.004 | 0.015 |
| Rd_GyrASer84Leu + ΔompP2 | 0.25 | 0.5 | 0.06 | 0.06 | 0.25 | 0.06 | 0.03 |
MXF, moxifloxacin; LVX, levofloxacin; TFX, tosufloxacin; SFX, sitafloxacin; AMP, ampicillin; CRO, ceftriaxone; CTX, cefotaxime.
We also attempted to generate an Rd transformant harboring the ompP2 mutation, resulting in the Thr132Lys substitution observed in mutants with reduced moxifloxacin susceptibility derived from SMHi2. However, the attempt was unsuccessful. We then generated an Rd mutant lacking the ompP2 gene, the RdΔompP2 strain, using a kanamycin resistance cassette to disrupt ompP2 by homologous recombination, to represent the disruption of OmpP2 observed in mutants from SMHi15, SMHi17, and SMHi18. The fluoroquinolone MIC of the RdΔompP2 strain was more than two times higher than that of the parental strain (Table 4). The susceptibility of the strain to other antimicrobials was similarly altered, with the ceftriaxone and cefotaxime MICs more than two times higher than those of the parental strain. The complementation of ompP2 into the RdΔompP2 strain recovered the susceptibility to fluoroquinolones and cephalosporins equivalent to that of the wild type Rd strain (Table 4).
Finally, we generated an Rd mutant lacking ompP2 in the Rd_GyrASer84Leu background (Rd_GyrASer84Leu + ΔompP2 strain). The fluoroquinolone MIC of the mutant was even higher than that of the Rd_GyrASer84Leu strain (Table 4).
We then determined the growth of the gyrA and ompP2 Rd transformant series (Fig. 3). The growth of gyrA series transformants was not significantly different from that of the parental strain, Rd. In contrast, the growth of Rd transformants lacking ompP2 (RdΔompP2 and Rd_GyrASer84Leu + ΔompP2 strains) was reduced compared with that of the Rd strain.
FIG 3.
Growth of H. influenzae mutants derived from strain Rd, harboring gyrA mutations and/or lacking ompP2. The data are representative of triplicate experiments.
DISCUSSION
Considering the increasing prevalence of fluoroquinolone resistance in H. influenzae in clinical settings, here we aimed to delineate the mechanism underlying the emergence of fluoroquinolone-resistant H. influenzae, taking into account the association with high-BLNAR. We successfully obtained H. influenzae mutants that acquired fluoroquinolone resistance by exposing susceptible clinical isolates representing multiple lineages to moxifloxacin in vitro. After passaging, 34.5% of the isolates (10 of the 29 isolates) exhibited fluoroquinolone resistance. The growth of 27.6% of the mutants (derived from 8 of 29 isolates examined) was comparable to that of the parental isolates. Notably, 7 of 10 fluoroquinolone-resistant mutants (70%) were derived from high-BLNAR strains. These observations have important bearing on the clinical setting, because they indicate that multiple lineages of high-BLNAR can develop fluoroquinolone resistance while retaining bacterial fitness.
Here, we identified novel mutations associated with fluoroquinolone resistance. Most of the in vitro-derived moxifloxacin-resistant H. influenzae mutants harbored amino acid substitutions in GyrA, ParC, GyrB, and/or ParE proteins (Table 2; see also Table S1 in the supplemental material). Hence, multiple amino acid substitutions in DNA gyrase II and topoisomerase IV are the main mutations required for the acquisition of fluoroquinolone resistance in H. influenzae, and successive passages on moxifloxacin-containing agar were required to obtain mutants that exceeded the moxifloxacin breakpoint. Many of the detected amino acid substitutions in GyrA and ParC proteins resulted from the defined QRDR mutations, previously identified in fluoroquinolone-resistant clinical isolates (17–19, 23, 24, 27, 31–33). The majority of the first-passage mutants shared an amino acid substitution in Ser84 or Asp88 of GyrA, with additional, subsequent amino acid substitutions in QRDRs in ParC (at Gly82, Ser84, or Glu88). This suggests that the development of fluoroquinolone resistance demonstrated in the current study can occur in the clinical setting in the corresponding order of accumulating defined QRDR mutations. Previously, we obtained four H. influenzae clinical isolates (three of which were high-BLNAR) with reduced fluoroquinolone susceptibility (moxifloxacin MICs of 0.25 or 1 mg/liter), which harbored one or two amino acid substitutions in GyrA and/or ParC (14) in a Japanese university hospital. Only one additional mutation can confer fluoroquinolone resistance in these clinical isolates.
In contrast, we also obtained mutants with increased moxifloxacin MIC in the absence of the defined QRDR mutations. This suggested that undefined QRDR and/or non-QRDR mutations were contributing to the development of fluoroquinolone resistance in these mutants. Whole-genome analysis revealed 56 novel mutations in 45 genes arising during the development of fluoroquinolone resistance other than the defined QRDR mutations (Tables S4 and S5). We do not know whether these undefined QRDR and/or non-QRDR mutations also occur in the clinical isolates, because detailed analysis of whole-genome sequences of the fluoroquinolone-resistant clinical isolates has not been performed to date. Such uncovered mutations might explain the unknown mechanism(s) of fluoroquinolone resistance contributing to the observed difference in fluoroquinolone MICs in strains with the same QRDR genotype (14, 18, 19, 27) and in isolates exhibiting fluoroquinolone resistance or reduced fluoroquinolone susceptibility without any defined QRDR mutations (19, 21, 27, 28).
Among the 56 novel mutations, other than the defined QRDR mutations, were those that resulted in 11-amino-acid substitutions in GyrA, GyrB, ParC, and ParE. From these, we showed that mutations resulting in Glu153Lys and ΔGln606 in GyrA as well as Ser467Tyr and Glu469Asp in GyrB, which lie outside QRDRs, are novel contributors to fluoroquinolone resistance in H. influenzae. The increased levels of moxifloxacin MIC observed in Rd isogenic mutants of gyrA tended to be lower than those of clinical isolates. The reason is unclear, but we have considered that differences of growth or other factors between clinical isolates and Rd strain could affect the increased level of fluoroquinolone MICs.
The growth of the respective mutants suggested that these mutations did not affect cellular fitness, as revealed by comparisons with the growth of the parental strains. None of the other detected undefined QRDR or non-QRDR amino acid substitutions (Thr472 in GyrB; Ala21, Asp83Asn, Arg515, Arg555, or Arg618 in ParC; and Leu502 in ParE) have been reported previously as contributors to fluoroquinolone resistance in H. influenzae. Leu502Phe in ParE, together with Ser83Phe in GyrA, was observed in a Salmonella enterica serovar Typhi strain, but these alterations do not significantly contribute to fluoroquinolone resistance of that strain (34). We were unable to introduce the specific parC and parE mutations into the Rd strain. This could be explained by the notion that the amino acid substitutions do not by themselves enhance fluoroquinolone resistance, whereas their combinations with other QRDR mutations confer increased resistance, known as compensatory mutations (31). Otherwise, some of the mutations could occur and be maintained stably only in clinical isolates, which have a different genetic backbone.
We focused on ompP2, which encodes an outer membrane porin (35), because mutations in ompP2 were the most frequent, except for gyrA, gyrB, parC, and parE, in the derived fluoroquinolone-resistant mutants. We demonstrated the novel contribution of ompP2 to decreased fluoroquinolone susceptibility and that combination of ompP2 deletion with Ser84Leu substitution in GyrA further increased (more than 4-fold) the fluoroquinolone MICs of the resultant strain. Various lines of evidence suggest that ompP2 is important for the acquisition of fluoroquinolone resistance (as observed for SMHi2 derivatives in the current study) and for increased fluoroquinolone resistance (as observed for the derivatives of SMHi18, SMHi17, and SMHi15 isolates).
Interestingly, disruption of ompP2 also increased the cephalosporin MICs of the Rd transformants. ompP2 mutations lead to increased MICs for various hydrophilic drugs, such as β-lactams, novobiocin, streptomycin, and chloramphenicol, in H. influenzae (36–38) because of a decreased channel conductance of the OmpP2 porin (37). Further, OmpP2 proteins of various amino acid sequences and molecular masses (from 34 to 42 kDa) were identified in H. influenzae isolates (39, 40). These observations suggest that different fluoroquinolone susceptibilities of strains with the same QRDR genotype (14, 19, 27) and different cephalosporin susceptibilities of strains with the same PBP3 genotype (14, 41) can be explained by the OmpP2 diversity of the clinical isolates of H. influenzae.
In conclusion, in the current study, we identified novel mutations associated with fluoroquinolone resistance in H. influenzae. Surveillance of clinical isolates of H. influenzae that exhibit not only resistance but also reduced susceptibility to fluoroquinolones might be an important measure for the prevention of the emergence of fluoroquinolone-resistant isolates in the clinical setting. We believe that the novel mutations identified in the current study will contribute to the understanding of the emergence of fluoroquinolone-resistant H. influenzae (especially high-BLNAR) in the clinical setting in the future.
MATERIALS AND METHODS
Bacterial isolates.
Twenty-nine fluoroquinolone-susceptible clinical isolates of H. influenzae from bacterial collections in Sapporo, Japan (14, 17), and the Rd strain (42) were used in the current study. All strains were grown on chocolate agar II (Nippon Becton, Dickinson, Tokyo, Japan) at 37°C under an atmosphere of 5% CO2 for 24 to 48 h. The isolates were stored at –80°C using a Microbank system (Pro-Lab Diagnostics, Round Rock, TX) until use. These isolates were confirmed as H. influenzae using a MALDI Biotyper (Bruker Daltonics, Billerica, MA) and also based on the PCR amplification of ompP6, the gene encoding the outer membrane protein P6, as described previously (6). Strain ST was determined by multilocus sequence typing (MLST) based on seven housekeeping genes (adk, atpG, frdB, fucK, mdh, pgi, and recA), as previously described (43). Allele numbers and STs were assigned using the tools available at http://haemophilus.mlst.net. A phylogenetic tree based on the MLST data was constructed using the neighbor-joining method (44) in MEGA7 (45).
Selection of fluoroquinolone-resistant mutants in vitro.
To select fluoroquinolone-resistant mutants, 29 fluoroquinolone-susceptible H. influenzae isolates (moxifloxacin MIC values ranging from 0.015 to 0.25 mg/liter) were used as the parental strains. A single colony of each strain was incubated in 1 ml of sBHI broth (Nippon Becton, Dickinson), supplemented with 15 mg/liter β-NAD (factor V) and 15 mg/liter hemin (factor X), at 37°C under an atmosphere of 5% CO2 for 24 h. The culture was spread on sBHI agar with or without moxifloxacin at a concentration of 2× MIC of the parental strain and incubated at 37°C under an atmosphere of 5% CO2 for 48 h. The frequency of the induction of mutants exhibiting reduced moxifloxacin susceptibility was calculated by dividing the number of colonies obtained on moxifloxacin-containing sBHI agar by the number of colonies obtained on plain sBHI agar. The experiments were performed in triplicate. The moxifloxacin MICs of the obtained mutants were determined, and the mutants were serially passaged on sBHI agar containing moxifloxacin at a concentration equal to 2-fold the MIC of the respective mutants. The resultant mutants were designated 1, 2, … n, and H, in agreement with the respective passage number. Each culturing step was performed in triplicate, and a randomly selected colony was picked from the agar plate for further analysis.
Bacterial growth curve determination.
Bacterial growth curves were determined by measuring values for the optical density at 600 nm (OD600) using an Infinite M200 PRO multimode microplate reader (Tecan, Männedorf, Switzerland). H. influenzae isolates or mutants were grown in 0.5 ml of sBHI broth overnight at 37°C under an atmosphere of 5% CO2, and 1 × 105 CFU/ml bacteria were subsequently cultured in 0.1 ml of sBHI broth in a 96-well plate at 37°C with shaking at 140 rpm. OD600 values were measured every 10 min for 24 h.
Antimicrobial susceptibility testing.
MIC values were determined using the microdilution method according to the CLSI guidelines (46). The following drugs were tested: moxifloxacin (Bayer, Osaka, Japan), levofloxacin (Daiichi-Sankyo, Tokyo, Japan), tosufloxacin (Toyama Chemical, Tokyo, Japan), sitafloxacin (Daiichi-Sankyo), ampicillin (Wako Pure Chemical Industry, Tokyo, Japan), ceftriaxone (Tokyo Chemical Industry, Tokyo, Japan), and cefotaxime (Tokyo Chemical Industry). Strains with MIC values exceeding 2 mg/liter for levofloxacin and 1 mg/liter for moxifloxacin were defined as quinolone nonsusceptible according to CLSI guidelines (46). Because no CLSI breakpoints for tosufloxacin and sitafloxacin have been established, strains with respective MIC values exceeding 1 mg/liter were defined as resistant, as stated by the Japanese Society of Chemotherapy (47).
Identification of mutations in the QRDRs of gyrA, gyrB, parC, and parE.
The nucleotide sequences of QRDRs of the gyrA, gyrB, parC, and parE genes were determined by PCR amplification and DNA sequencing, as previously described (14).
Whole-genome sequencing.
Bacterial genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen, Hilden, Germany). The isolated DNA was sequenced using MiSeq (Illumina, San Diego, CA) and a Nextera XT DNA library prep kit (Illumina). For the sequence assembly, the contigs were mapped using 300-bp paired-end reads to the genome of the parental isolates, which was followed by polishing using the CLC Genomic Workbench (Qiagen). The genomes were annotated using DFAST, based on PROKKA (48). The genomes of fluoroquinolone-resistant mutants and the parental strains were compared, and nonsynonymous mutated genes were detected by CLC Genomic Workbench using the Basic Variant Detection method. The obtained count mutation frequency was greater than 70%. The detected mutations were confirmed by Sanger DNA sequencing using specific primer pairs (see Table S6 in the supplemental material).
Introduction of specific mutations of the gyrA, gyrB, parC, and parE genes into the Rd strain.
The cells were transformed by electroporation. The electrocompetent cells were prepared as described by Ubukata et al. (7). Specific primers (Table S6) were used to PCR amplify amplicons of approximately 3 kb; 200 ng of the DNA was then mixed with electrocompetent cells followed by electroporation. The mixtures were incubated for 24 h at 37°C under an atmosphere of 5% CO2. The transformants were selected by spreading on sBHI agar containing 0.03 and 0.06 mg/liter moxifloxacin. The target genes in the transformants selected on the moxifloxacin-containing agar (at least three transformants for each transformation attempt) were sequenced to confirm the presence of the target mutations, and subsequently MIC values of the transformants were determined as described above. We also confirmed the contribution of the gyrB, parC, and parE mutations by introducing each mutation into gyrB, parC, and parE sequences of Rd by using site-direct mutagenesis methods as described in Table S7 and Fig. S2.
Generation of the H. influenzae mutant lacking the ompP2 gene and ompP2 complementation.
The mutant lacking the ompP2 gene was generated by the homologous recombination method, as previously described (49), with slight modification. Regions 1,100 bp and 1,181 bp upstream and downstream of the ompP2 gene were amplified by PCR using primer sets Rd_OmpP2RedF1 and Rd_OmpP2RedR1 as well as Rd_OmpP2RedF2 and Rd_OmpP2RedR2, respectively, with genomic DNA of the Rd strain as a template (Table S6). A kanamycin resistance cassette was PCR amplified using the primers KM-RedF and KM-RedR (Table S6), with FRT-PGK-gb2-neo-FRT (Gene Bridges, Heidelberg, Germany) as a template. An approximately 3.5-kb fragment of pHSG576 (obtained from the National BioResource Project, Mishima, Japan) was amplified by inverse PCR using primers pHSG576Invert common1 and pHSG576Invert common2, with pHSG576 as a template. The four gene fragments were assembled using NEBuilder HiFi DNA assembly master mix (New England BioLabs Japan, Tokyo, Japan), generating pHSG576_ompP2_KanR. The construct harbors a kanamycin resistance cassette sandwiched by the 5′ and 3′ regions of ompP2. The entire fragment (5′ region of ompP2 kanamycin resistance cassette-3′ region of ompP2) was PCR amplified using the primers RdOmpP2_delF and RdOmpP2_delR. The amplicon (200 ng) was then used to transform the electrocompetent Rd or RdSer84Leu cells by electroporation (1.25 kV/cm, 200 Ω, and 25 μF). The cells were then immediately mixed with 1 ml of sBHI broth and incubated at 37°C, under 5% CO2, for 24 h. After cultivation, 100 μl of the suspension was spread on sBHI agar containing 30 mg/liter kanamycin. The colonies obtained on the kanamycin-containing sBHI agar were picked, and ompP2 deletion was confirmed by PCR using the specific primers OmpP2_NewF2 and OmpP2_NewR2 (Table S6).
Complementation of ompP2 was performed by the transformation of pMC-ompP2 vector into the RdΔompP2 strain. An approximately 6.2-kb fragment of pMC-Tandem (50), kindly provided by P. R. Langford, was amplified by inverse PCR using primers pMCinvert1-2 and pMCinvert2-2, with pMC-Tandem as a template, followed by DpnI digestion. This amplification removes xylE and gfpmut3, present in the pMC-Tandem sequence (50). ompP2 containing the promoter regions (414 bp upstream of ompP2 sequence) (51) was amplified by PCR using primers pMCompP2F2 and pMCompP2R2. The two gene fragments were assembled using NEBuilder HiFi DNA assembly master mix (New England BioLabs Japan), generating pMC-ompP2. pMC-ompP2 and pMC-tandem GFP(−) (as an empty vector) were then used to transform the electrocompetent RdΔompP2 strain by electroporation (1.25 kV/cm, 200 Ω, and 25 μF). The cells were then immediately mixed with 1 ml of sBHI broth and incubated at 37°C, under 5% CO2, for 24 h. After cultivation, 100 μl of the suspension was spread on sBHI agar containing 1 mg/liter chloramphenicol. The colonies obtained on the chloramphenicol-containing sBHI agar were picked, and ompP2 complementation was confirmed by PCR using the specific primers OmpP2_NewF2 and OmpP2_NewR2 (Table S6).
Statistical analysis.
The chi-squared test was used to compare the appearance frequency of fluoroquinolone-resistant H. influenzae mutants in the two cell populations, high-BLNAR and BLNAS. In the analysis, a P value below 0.05 was considered statistically significant.
Ethics statement.
This study was approved by the Institutional Review Board of Sapporo Medical University Hospital (302-3670).
Supplementary Material
ACKNOWLEDGMENTS
pMC-Tandem was kindly provided by P. R. Langford.
This research was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), for the Joint Research Program of the Hokkaido University Research Center for Zoonosis Control, to Y.S., and in part by the Japan Agency for Medical Research and Development (AMED) (grant number JP18fm0108008).
Footnotes
Supplemental material is available online only.
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