Journal of Infection and Chemotherapy 29 (2023) 401–406

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Journal of Infection and Chemotherapy

journal homepage: www.elsevier.com/locate/jic

Original Article

Bactericidal effect of lascufloxacin on HEp-2 cell-internalized group

A Streptococcus
Masamitsu Kono a, Hideki Sakatani a, Tetsuya Kinoshita a, Hisato Sadakata b, Shun Miyazaki b,
Takako Sano b, Muneki Hotomi a, *
a
Department of Otorhinolaryngology-Head and Neck Surgery, Wakayama Medical University, 811-1 Kimiidera, Wakayama-shi, Wakayama, 641-8510, Japan
b
TIMELAPSE VISION Inc., PLAZA TORIYAMA 4F, 5-23-11, Honcho, Shiki-shi, Saitama, 353-0004, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Introduction: Although amoxicillin (AMPC) is recommended as first-line therapy for acute pharyngotonsillitis
Group A streptococci caused by group A streptococci (GAS), it often fails to eradicate infections. Internalization and subsequent
Acute pharyngotonsillitis intracellular survival of GAS are considered major mechanisms for penicillin therapeutic failure. It is, therefore,
Internalization
desirable to administer drugs that exert bactericidal effects on extracellular and intracellular GAS. In this study,
AMPC treatment failure
Lascufloxacin
we aim to investigate the bactericidal effects of lascufloxacin (LSFX) on internalized GAS in HEp-2 cells.
Materials and methods: The GAS strain M1 and clinical isolate strain #2 were used in this study. Following
treatment of GAS-infected human pharyngeal carcinoma epithelial HEp-2 cells with LSFX or AMPC, internalized
GAS cells were recovered. The concentrations of LSFX and AMPC were equivalent to 1 × and 2 × MIC for strain
M1. Culture medium was used as a control. Time-lapse and fluorescence images of GAS invading HEp-2 cell were
obtained. LIVE/DEAD fluorescence staining was used to confirm the viability of internalized GAS.
Results: LSFX significantly reduced the number of cell-internalized M1 and #2 GAS strains compared to the
control (p < 0.01) in a dose-dependent manner. However, AMPC did not reduce this in both strains. Both live and
dead intracellular GAS were confirmed in HEp-2 cells exposed to LSFX. In contrast, intracellular GAS survived in
HEp-2 cells exposed to AMPC and in the control.
Conclusion: LSFX elicits significant bactericidal effects on cell-internalized GAS, hence it may represent a potent
therapeutic option for patients with acute pharyngotonsillitis in whom AMPC treatment has failed.

1. Introduction pharyngotonsillitis caused by GAS [4]. However, S. pyogenes infections

Acute pharyngotonsillitis, primarily characterized by a severe sore
throat, is one of the most common infectious diseases in the field of
otolaryngology. Hence, identification of causative pathogens is critical
for the development of effective therapeutic strategies. The causative
pathogens of acute pharyngotonsillitis reportedly include viruses and
bacteria, with mixed infections reported [1]. Among the bacterial
pathogens, Streptococcus pyogenes (group A streptococci, GAS) can cause
a broad range of illness, from mild pharyngotonsillitis to severe invasive
disease [2]. According to the seventh nationwide surveillance of six
otorhinolaryngological infectious diseases in Japan, the detection rate of
S. pyogenes was 10–30% in acute tonsillitis [1,3].
Amoxicillin (AMPC) is recommended as a first-line therapy for acute
often fail to be eradicated even with optimal treatment [5–7]. Several
mechanisms may lead to treatment failure with penicillin, including
co-infection with β-lactamase-producing bacteria [8], non-susceptibility
[9], biofilm formation [10], as well as internalization and intracellular
survival of GAS [11,12]. Given that penicillin poorly penetrates the
mammalian cell membrane, the intracellular niche is considered to
protect GAS from antimicrobial agents. Consequently, internalized GAS
contributes to recurrent infections and asymptomatic pharyngeal car­
riage, with potential for subsequent dissemination throughout the host
[11,13]. A molecular typing assay for S. pyogenes in patients with
recurrent pharyngotonsillitis after treatment failure reported that more
than 80% had the identical isolates to those obtained in the first episode
[14]. Therefore, in the treatment of GAS infections, it is desirable to use

Abbreviations: GAS, group A streptococci; LSFX, Lascufloxacin; AMPC, Amoxicillin.

* Corresponding author. Department of Otorhinolaryngology-Head and Neck Surgery, Wakayama Medical University, 811-1 Kimiidera, Wakayama-shi, Wakayama,
641-8510, Japan.
E-mail address: mhotomi@wakayama-med.ac.jp (M. Hotomi).

https://doi.org/10.1016/j.jiac.2023.01.008
Received 3 October 2022; Received in revised form 29 December 2022; Accepted 17 January 2023
Available online 18 January 2023
1341-321X/© 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control.
Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Kono et al.
drugs that permeate cell membranes, reach high concentrations, and
have bactericidal effects on both extracellular and intracellular GAS [4,
15].
Lascufloxacin (LSFX) is a novel respiratory quinolone available in
Japan for treating acute pharyngotonsillitis. The minimum inhibitory
concentration 90 (MIC90) of LSFX against S. pyogenes is 0.06 μg/mL,
indicating potent antibacterial activity [16]. It also has high bioavail­
ability [17] and favorable tissue penetration, as indicated by a
tissue-plasma concentration ratio >2 in the sinus mucosa, middle ear
mucosa, and palatine tonsil following oral administration [18].
Furthermore, the frequency of selecting resistant strains when first-step
quinolone resistance-determining region mutants are exposed to anti­
microbial agents is reportedly lower for LSFX than for levofloxacin
(LVFX) and garenoxacin (GRNX) [19].
Respiratory quinolones are considered therapeutic options for
intracellular GAS, however, there are no reports visually confirming that
they kill intracellular GAS. In this study, GAS invasion of human
laryngeal carcinoma HEp-2 cells was observed via inverted microscope
under cell culture conditions and the bactericidal effects of LSFX on
internalized GAS were investigated via intracellular invasion assays, and
the bactericidal effects were visualized by LIVE/DEAD fluorescence
staining.
2. Materials and methods
2.1. Bacterial strains and growth conditions
GAS strain M1 (BAA-947; ATCC, Manassas, VA, USA) and clinical
isolate strain #2 were used in this study. Strain M1 – a widely studied
strain with reported genome sequence [20] – was used as a standard.
Strain #2 was originally isolated from a patient with pharyngotonsillitis.
GAS were grown in Todd-Hewitt broth (Becton, Dickinson and Com­
pany, New Jersey, USA) supplemented with 0.2% yeast extract (THY,
Becton, Dickinson and Company) at 37 ◦ C for 12 h (late-log phase).
2.2. Cell culture
The human pharyngeal carcinoma epithelial HEp-2 cells (ATCC
CCL23) were cultured in Dulbecco’s modified eagle’s medium (DMEM,
Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10%
fetal bovine serum (FBS, Thermo Fisher Scientific) at 37 ◦ C in a 5% CO2
atmosphere unless specifically described. HEp-2 cells were seeded into
24-well plates at a density of 5 × 104 cells/well and grown for 24 h.
Semi-confluent monolayers were used in the following assays.
2.3. Antibacterial agents
Cells were treated with lascufloxacin hydrochloride (MIC of 0.031
μg/mL for M1 and 0.016 μg/mL for strain #2; Kyorin Pharmaceutical
Co., Ltd., Tokyo, Japan) and amoxicillin trihydrate (MIC of 0.016 μg/mL
for both strains; FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan). MICs of each agents were determined according to the standard
microdilution assay [21].
2.4. Time-lapse and fluorescence images of GAS invading HEp-2 cell
HEp-2 cells (2.0 × 105) cultured in a 27-mm glass dish were infected
with 1–3 × 107 colony forming units (CFU) of strain #2. The invading
GAS were observed over time under cell culture conditions using an
inverted microscope (Nikon Eclipse Ti-E, Nikon Corporation, Tokyo,
Japan). Time-lapse images were taken at 10-s intervals with a Canon
EOS5D (Canon Inc., Tokyo, Japan). To corroborate the internalization of
GAS, fluorescence staining was performed to differentiate between
intracellular and extracellular GAS. Suspensions of strain #2 in Hanks’
balanced salt solution (HBSS) were stained with acridine orange (AO,
DOJINDO LABORATORIES, Kumamoto, Japan). After washing with
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Journal of Infection and Chemotherapy 29 (2023) 401–406
HBSS, strain #2 was resuspended in DMEM supplemented with 10%
FBS. The stained bacteria were added to HEp-2 cells at a concentration
of 1–3 × 107 CFU and incubated for 2 h to allow internalization.
Cellstain-DAPI (DOJINDO LABORATORIES) was then added to the
culture medium. Fluorescence images were obtained at 37 ◦ C in a 5%
CO2 atmosphere using a confocal laser scanning microscope and the
attached software (LSM-700, Carl Zeiss AG, Oberkochen, Germany). AO
and DAPI were simultaneously excited by 405 nm laser in order to
capture matched images. Fluorescence images of AO (green) and DAPI
(blue) were obtained with a LP498 and a SP451 filter, respectively.
2.5. Intracellular invasion assays
Assays of GAS invasion into HEp-2 cells and the effects of antibac­
terial agents on internalized GAS were performed as previously
described [22,23] with slight modifications. Briefly, GAS cells (1–3 ×
106 CFU/well) were inoculated onto semi-confluent HEp-2 cell mono­
layers (1.1 × 105 cells/well) grown in 24-well culture plates. The
monolayers were incubated for 2 h and washed with FBS-free DMEM.
After washing, the medium was changed to FBS-free DMEM containing
gentamicin (200 μg/mL; FUJIFILM Wako Pure Chemical Corporation) to
kill extracellular GAS. After 2 h of gentamicin treatment, the monolayers
were washed with FBS-free DMEM and incubated for an additional 2 h in
FBS-free DMEM containing antibacterial agents. Control cells were
treated with FBS-free DMEM without antibacterial agents. The mono­
layers were washed with phosphate buffered saline (− ) and the cells
were detached using 0.05% trypsin/0.0076% EDTA. The removed
HEp-2 cells were resuspended in sterile distilled water and vortexed for
disruption. The cell suspensions were plated on THY agar plates at 37 ◦ C
for 20 h to determine the number of recovered bacteria. The concen­
trations of LSFX were 0.031 μg/mL and 0.062 μg/mL, and those of AMPC
were 0.016 μg/mL and 0.031 μg/mL, respectively, which were 1 × MIC
and 2 × MIC for strain M1, respectively. Prior to this assay, the invasion
rates of both strains into HEp-2 cells were determined. GAS (1–3 × 107
CFU) was added to the HEp-2 cell cultures and incubated for 2 h. The
monolayers were treated with gentamicin (200 μg/mL), after which the
number of internalized GAS was counted. Invasion rates were deter­
mined as recovered CFU/inoculated CFU × 100.
2.6. LIVE/DEAD staining of internalized GAS
HEp-2 cells (2.0 × 105) cultured in a 27-mm glass cover dishes were
infected with strain #2 (1–3 × 106 CFU) for 2 h. HEp-2 cells were
washed with FBS-free DMEM and treated with gentamicin (200 μg/mL).
After washing with FBS-free DMEM, HEp-2 cells were treated with LSFX
and AMPC at 0.031 μg/mL. For controls, only FBS-free DMEM was
added. After 2 h treatment, HEp-2 cells were stained using the “live/
dead” staining kit (LIVE/DEAD BacLight Bacterial Viability Kit, Thermo
Fisher Scientific) for 15 min at 37 ◦ C in 5% CO2. Fluorescence images
were obtained at 37 ◦ C, 5% CO2 using an LSM-700 laser scanning
confocal microscope and the attached software. SYTO9 and propidium
iodide images were obtained using excitation 488 nm and SP555 filters,
and 555 nm and LP566 filter, respectively.
2.7. Statistical analysis
Intracellular invasion assays were independently performed at least
five times; values are presented as the mean ± standard deviation.
Statistical comparisons with control were performed using analysis of
variance (ANOVA) followed by a post-hoc Tukey test. Statistical sig­
nificance was considered when the p-value <0.05. All statistical data
were analyzed using JSTAT (version 6.9).
M. Kono et al.
3. Results
3.1. Time-lapse and fluorescence images of GAS invading HEp-2 cells
Planktonic GAS in the medium moved irregularly, however, stopped
moving following adherence to the cell surface (Fig. 1, Supplementary
Video S1). GAS then invaded HEp-2 cells via endocytosis-like uptake and
migrated intracellular. Fluorescence microscopy images confirmed that
GAS was stained only with AO and with both AO and DAPI. Given that
functional cell membranes separate the inside and outside of the cell, the
presence of GAS stained with only AO demonstrated that GAS was
internalized by HEp-2 cells (Fig. 2).
Supplementary data related to this article can be found at https://do
i.org/10.1016/j.jiac.2023.01.008.
3.2. Effect of antibacterial agents on internalized GAS
Following gentamycin treatment, the invasion rates of GAS into HEp-
2 cells found to be 0.05% for strain M1 and 11.3% for strain #2. The
bactericidal effects of LSFX and AMPC on internalized GAS in both
strains are shown in Fig. 3 (a, b). The number of internalized GAS in the
control was 272-fold higher in strain #2 than in strain M1 (2.4 × 106 vs.
8.8 × 103). LSFX significantly reduced the number of recovered GAS
compared to the control in a dose-dependent manner in both strains.
However, AMPC did not reduce this in both strain M1 and strain #2.
3.3. LIVE/DEAD staining of internalized GAS
Fluorescence microscopy images of GAS strain #2-infected HEp-2
cells were obtained after treatment with LSFX and AMPC (Fig. 4). Both
live and dead intracellular GAS cells were confirmed in HEp-2 cells
exposed to LSFX (Fig. 4a). In contrast, nearly all intracellular GAS cells
survived in HEp-2 cells exposed to AMPC, similar to that observed in
control cells (Fig. 4b and c).
4. Discussion
Since internalized GAS survives intracellularly and is thought to be
associated with intractable or recurrent pharyngotonsillitis and
asymptomatic carriage, it is important to highlight the need for eradi­
cating internalized GAS. Although respiratory quinolones are consid­
ered therapeutic options for intracellular GAS, there are no reports
visually confirming that they kill intracellular GAS. Therefore, this study
was performed to observe the GAS invasion into HEp-2 cells, to evaluate
the bactericidal effects of LSFX on internalized GAS, and to visualize the
intracellular bactericidal effects by using LIVE/DEAD fluorescence
staining. LSFX significantly decreased the number of recovered GAS in a
dose-dependent manner, while AMPC did not decease that. LIVE/DEAD
fluorescence staining confirmed that internalized GAS was dead only in
Fig. 1. Time-lapse images of GAS strain #2 invading HEp-2 cells.
Arrows indicate invading GAS. GAS adheres to the cell surface after 17 min (a). GAS invaded HEp-2 cells via endocytosis-like uptake after 25 min (b). GAS migrated
intracellular at 33 min (C). Scale bar = 10 μm. Supplementary Video S1 provides a time-lapse of images.
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Journal of Infection and Chemotherapy 29 (2023) 401–406
HEp-2 cells treated with LSFX. These results presented herein provide
basic evidence for future treatment.
The GAS strain M1 and #2 were infected to HEp-2 cells to examine
the cell-invasion rate and the effect of the antibacterial agents on
internalized GAS. The multiplicity of infection (MOI) in the intracellular
invasion assay is one of factors which can affect the cell-invasion ability
of GAS. The MOIs in the experiments to investigate the cell-invasion rate
and the bactericidal effects were set to approximately 100 and 10,
respectively. We adjusted MOI in terms of number of recovered GAS and
cytotoxicity due to GAS. Stable data of invasion rate could be obtained
by setting MOI to 100. In the assay to evaluate the bactericidal effects,
the culture term of infected cells becomes longer, so cytotoxicity due to
GAS is likely to occur. From this reason, the MOI was set to approxi­
mately 10, where the experimental system was stable without cytotox­
icity. The influence of MOI to the cell-invasion ability of GAS should be
considered. The cell-invasion rate measured with the MOI = 100 were
0.05% for M1 strain and 11.3% for #2 strain, showing the ability to
invade is 226-fold higher for #2 strain than M1 strain. The number of
recovered GAS of controls in the intracellular invasion assays with the
MOI = 10 were approximately 8.8 × 103 for M1 and 2.4 × 106 for #2
strain. The ratio of recovered GAS was 272, which was almost accorded
with the ratio of cell-invasion rate. Therefore, if the MOI is in the range
of 10–100 in this experimental system, the relative relationship of cell-
invasion abilities between both strains is thought constant, and the in­
fluence of the MOI is thought little in our experiment system.
We observed that strain #2 invaded HEp-2 cells under cell culture
conditions. Indeed, Passali et al. reported that invasion is achieved via
two steps, namely, adhesion and up-take [13]. The invasion process is
regarded as a complicated system due to a number of factors, including
the host extracellular matrix and fibronectin-binding proteins [11].
Moreover, the ability to enter cells differs depending on the genotype
[24]. Hence the difference in invasion rate between strain #2 and M1
may be associated with proteins expressed on the GAS surface and/or
related genes.
Investigation of the effects of LSFX and AMPC on internalized GAS
revealed that LSFX significantly reduced the number of recovered GAS
in both strains compared to the control. In contrast, AMPC did not
predominantly reduce intracellular GAS in neither strain M1 and strain
#2. Nakagawa et al. reported that GAS invades epithelial cells via
endocytosis, which damages the endosome membrane and escapes the
endosome-lysosomal degradation pathway [25]. Some escaped GAS are
killed via autophagy [26], while surviving GAS induce epithelial cell
apoptosis [23]. In the current study, LIVE/DEAD staining confirmed the
presence of dead intracellular GAS only in LSFX-treated cells, suggesting
that dead GAS was due to the bactericidal effect of LSFX rather than cell
defense mechanisms.
Iuchi et al. reported that GRNX and LVFX significantly reduced
intracellular GAS [27]. Antibacterial agents must penetrate cells to exert
bactericidal effects on intracellular GAS. The ratio of intracellular to
M. Kono et al. Journal of Infection and Chemotherapy 29 (2023) 401–406

Fig. 2. Fluorescence microscopic images of intracellular and extracellular GAS.

Phase-contrast images (a). GAS cells stained green with acridine orange (b). Extracellular GAS cells stained blue with DAPI (c). Superimposed images from (b) and
(c). Triangular arrowhead indicates intracellular GAS (green). Arrows indicate extracellular GAS (blue) (d). Scale bar = 10 μm.

Fig. 3. Effects of antibacterial agents on internalized GAS.

Number of recovered GAS following treatment with LSFX and AMPC are shown as mean ± standard deviation from at least five independent experiments. Statistical
comparisons with control were performed ANOVA followed by a post-hoc Tukey test. **p < 0.01. LSFX: lascufloxacin, AMPC: amoxicillin.

extracellular concentration (I/E ratio) of quinolone antibiotics is
approximately eight times higher than that of β-lactam antibiotics,
indicating that quinolone has higher intracellular penetration [28]. It
has also been reported that LSFX has higher I/E ratio in poly­
morphonuclear leukocytes than GRNX and LVFX [29]. Hence, the
difference in bactericidal effects between LSFX and AMPC may be due to
differences in intracellular penetration. It has been reported that the
tissue/plasma concentration ratio of LSFX is 2.76 for the palatine tonsil,
indicating favorable tissue distribution [18], and that the tissue con­
centration in the palatine tonsil when orally administered clinical dose

404
M. Kono et al.
Fig. 4. Fluorescence microscopic images of LIVE/DEAD staining of internalized GAS.
GAS strain #2-infected HEp-2 cells after treatment with (a) LSFX, (b) AMPC, and (c) DMEM control. Green: live GAS; Red: dead GAS. Arrows indicate intracellular
GAS. Scale bar = 10 μm. LSFX: Lascufloxacin, AMPC: amoxicillin.
of LSFX 75 mg is 609 ng/mL [18]. This tissue concentration is 10-fold
higher than MIC90 for S. pyogenes, and higher than the concentration
examined in the present study [16,18].
Although AMPC-resistant GAS does not currently represent a sig­
nificant clinical issue, GAS with penicillin-binding protein PBP2x gene
mutations tends to be non-susceptible to β-lactam antibiotics [9]. At­
tempts to reduce AMPC usage have also been made as a challenge to
antimicrobial resistance (AMR) [30]. On the contrary, a 2021 Cochrane
Review reevaluated antibiotics in patients with sore throat and reported
that antibiotics reduced the incidence of acute otitis media within 14
days and quinsy within two months [31]. Harabuchi et al. proposed that
respiratory quinolones can be administered to patients with severe
pharyngotonsillitis as a first-line treatment [32]. Hence, although a
therapeutic strategy for GAS infections must be carefully developed
considering AMR countermeasures, the appropriate use of antimicro­
bials should be actively promoted.
Certain limitations were noted in the current study. First, in this
experimental system, the externalization of GAS following escape from
the intracellular space to the extracellular space, was not considered.
GAS induces apoptosis in invading cells and is excreted extracellularly.
However, given that the incubation time of HEp-2 cells with GAS was
relatively short, and we had confirmed that cytotoxicity was not
exhibited in all experiments, we believe that the influence of external­
ization of GAS was negligible. Second, since GAS has extraordinary
biological diversity, as shown by the wide range of diseases and their
antigenic heterogeneity, interpretations of in vitro experimental results
using GAS have always been controversial. Further studies with clinical
isolates including non-susceptible strains are warranted. Third, the re­
sults of this in vitro study suggest that LSFX may be a potent therapeutic
option against internalized GAS. However, to prove its clinical useful­
ness, it is necessary to examine the eradication of internalized GAS in
clinical studies of AMPC treatment failure cases and GAS carriers.
In conclusion, this study demonstrates that LSFX, but not AMPC,
significantly reduces the number of internalized GAS in a dose-
dependent manner and these results provided basic evidence for
future studies and practice. Together with its beneficial properties, such
as favorable tissue permeability and low selectivity of resistant strains,
these results suggest that LSFX may be a potent therapeutic option for
patients with acute pharyngotonsillitis caused by GAS who have expe­
rienced failed AMPC treatment.
Funding
Funding for this research and technical support were provided by
Kyorin Pharmaceutical Co., Ltd.
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Journal of Infection and Chemotherapy 29 (2023) 401–406
Authorship statement
All authors met ICMJE criteria for authorship. MHa was the chief
investigator and responsible for the data analysis. MHa conceived of, and
designed, the study. MKa, HSa, and TKa contributed data interpretation.
HSb, SMb, and TSb contributed to data collection and analysis. MHa
wrote the final manuscript and submitted the manuscript. All authors
have read and approved the final manuscript.
Declaration of competing interest
MH has received lecture fees from Kyorin Pharmaceutical Co., Ltd..
This does not alter the authors’ adherence to any publication policies.
All of other authors have no conflicts of interest to declare.
Acknowledgments
We thank Yuki Endo (Wakayama Medical University) for her tech­
nical support and Yukari Yoshino (Kyorin Pharmaceutical Co., Ltd.) for
her coordination of this study. A portion of this work was orally pre­
sented at the 70th Annual Meeting of the Western Japan Branch of the
Japanese Society of Chemotherapy on November 2022, in Nagasaki,
Japan.
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