This study investigated the effects of GRNX, CAM, AMPC, CDTR-PI, and LVFX on the invasion of Detroit 562 cells by NTHi and S. pyogenes. The results showed that NTHi and S. pyogenes invaded Detroit 562 cells, AMPC and CDTR-PI did not affect the invasion of Detroit 562 cells by NTHi and S. pyogenes, and GRNX, CAM, and LVFX reduced the invasion of Detroit 562 cells by NTHi and S. pyogenes.
Fibronectin-binding protein (F1 protein) is mentioned as a mechanism by which S. pyogenes invades the cells . In Japan, Ma et al.  reported that 77.3% of S. pyogenes strains possessed F1 protein. More interestingly, many biofilm-producing strains are F1 protein-negative strains . Intracellular invasion ability and biofilm formation ability are negatively correlated, and it is considered that S. pyogenes avoids the attack of antibacterial drugs . In addition, phosphorylcholine is mentioned as a mechanism of intracellular invasion by NTHi, and the higher the expression level of phosphorylcholine, the more it penetrates into cells . The present study showed that it takes a certain period of time for bacteria to adhere to cells and enter the cells, which becomes constant within 6 h. Yamanaka’s report that H. influenzae invades Detroit 562 cells supports the results of the present study .
Since the late 1990s, respiratory tract infections caused by resistant strains of S. pneumoniae and H. influenzae have been rapidly increasing worldwide. Penicillin-resistant S. pneumoniae, such as penicillin intermediately-resistant S. pneumoniae (PISP), penicillin-resistant S. pneumoniae (PRSP), and BLNAR, are particularly common in Japan . In this study, no bacteria resistant to β-lactam were found, but decreased sensitivity to LVFX by S. pyogenes and to CAM by NTHi and S. pyogenes was observed. Quinolone inhibits bacterial growth by disrupting the DNA replication of type II topoisomerase . Type II topoisomerases are currently recognized to include DNA gyrase, which is responsible for the formation and elimination of supercoiled structures in DNA strands, and topoisomerase IV, which cuts and re-ligates tangled DNA during DNA replication . Both of these enzymes are composed of two dimers of subunit types A and B, which together form a tetramer. Amino acid substitutions in either enzyme may lead to the inhibition of such complex formations, and in particular, mutations in the quinolone resistance-determining regions (QRDRs) within subunits A and B are closely related to resistance . Shoji et al.  reported that of the 14 S. pyogenes strains, 12 (85.7%) had two or more mutations in QRDRs. This is considered one of the reasons that streptococcal susceptibility to LVFX was reduced.
Invasion of cells by bacteria has been cited as a cause of repeated tonsillitis. In this study, neither AMPC nor CDTR-PI was found to have a bactericidal effect on bacteria invading the cells. It is known that β-lactam antibacterial drugs have low intracellular transmissibility, and their antibacterial action is reduced against H. influenzae that has entered the cells . Therefore, it is suggested that another antimicrobial treatment is necessary for recurrent tonsillitis.
GRNX shows a favorable pharmacokinetic profile, with good penetration into sputum and otorhinolaryngological tissues, and it is highly effective in the treatment of patients with upper and lower respiratory tract infections . Recent studies have established that the AUC0-24/MIC ratio is an important pharmacodynamic parameter influencing quinolone efficacy. Lister demonstrated that garenoxacin exhibits pharmacodynamics similar to those of clinically available quinolones . In addition, Takagi et al.  reported that GRNX concentrations in plasma and tissues of subjects receiving GRNX 400 mg once a day were higher than the MIC90 of major causative pathogens. The trough concentration (Cmin) in plasma was 1.92 g/mL, a level that was higher than the mutant prevention concentration, suggesting that GRNX is unlikely to induce the selection of resistant strains during treatment. The efficacy rates of GRNX in otorhinolaryngological infections were 91.3% for sinusitis, 81.8% for otitis media, 89.5% for pharyngolaryngitis, and 95.0% for tonsillitis . A double-blind study was conducted comparing GRNX 400 mg once a day with LVFX 100 mg three times a day for 10 days in patients with bacterial pneumonia. The efficacy rate was 94.9% (94/99) in the GRNX group and 92.8% (77/83) in the LVFX group at the 7th day after completion of treatment . No significant difference in efficacy rates was found between GRNX and LVFX, with a 95% CI of – 4.9% to 9.2%, indicating that GRNX is not inferior to LVFX. The bacterial eradication rate was 100% (53/53) in the GRNX group and 87.8% (36/41) in the LVFX group. This difference in the eradication rate was statistically significant, with a 95% CI of 2.4% to 23.9% . In the present study, GRNX was also effective against bacteria invading cells. Since LVFX showed decreased sensitivity, GRNX is effective for recurrent tonsillitis.
The present study showed that CAM was effective against bacteria invading cells. Patel et al. reported that the concentration of CAM in alveolar macrophages of healthy subjects reached a maximum of 1996 μg/mL at 4 h after administration of 500 mg of CAM . Chou et al.  reported that cultured human gingival fibroblasts and SCC-25 cells took up CAM via a concentrative active transport system. However, the concentration of CAM used in this study is far beyond the amount used in actual clinical practice and therefore could not be used in actual clinical practice.
Our study has some limitations. First, BLNAS and other resistant strains were not investigated. Since the number of strains of resistant bacteria is increasing, more resistant strains should be included in future studies. The second limitation pertains to the epithelial cells used. Although the use of normal human epithelial cells may be more clinically relevant, we used a pharyngeal cancer-derived cell line. Because these cells were of human origin, we consider that the results of this study were not different from those that would have been obtained with the use of normal cells.
In conclusion, GRNX was the most effective agent against bacteria invading cells. Administration of GRNX should be considered when the efficacy of penicillin and cephem antibiotics and of β-lactam is insufficient in daily medical practice.