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Article

Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp.

by
Michiko Furugaito
1,2,
Yuko Arai
3,
Yutaka Uzawa
3,
Toshinori Kamisako
4,
Kohei Ogura
5,
Shigefumi Okamoto
1 and
Ken Kikuchi
3,*
1
Department of Clinical Laboratory and Biomedical Sciences, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
2
Department of Clinical Laboratory, Kindai University Hospital, Osakasayama, Osaka 589-8511, Japan
3
Department of Infectious Diseases, Tokyo Women’s Medical University, Shinjuku-ku, Tokyo 162-8666, Japan
4
Department of Clinical Laboratory Medicine, Faculty of Medicine, Kindai University, Osakasayama, Osaka 589-8511, Japan
5
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan
*
Author to whom correspondence should be addressed.
Antibiotics 2023, 12(10), 1538; https://doi.org/10.3390/antibiotics12101538
Submission received: 20 September 2023 / Revised: 9 October 2023 / Accepted: 9 October 2023 / Published: 14 October 2023 / Corrected: 31 May 2024
(This article belongs to the Section Mechanism and Evolution of Antibiotic Resistance)
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Abstract

:
Gemella is a catalase-negative, facultative anaerobic, Gram-positive coccus that is commensal in humans but can become opportunistic and cause severe infectious diseases, such as infective endocarditis. Few studies have tested the antimicrobial susceptibility of Gemella. We tested its antimicrobial susceptibility to 27 drugs and defined the resistant genes using PCR in 58 Gemella strains, including 52 clinical isolates and 6 type strains. The type strains and clinical isolates comprised 22 G. morbillorum, 18 G. haemolysans (GH) group (genetically indistinguishable from G. haemolysans and G. parahaemolysans), 13 G. taiwanensis, three G. sanguinis, and two G. bergeri. No strain was resistant to beta-lactams and vancomycin. In total, 6/22 (27.3%) G. morbillorum strains were erythromycin- and clindamycin-resistant ermB-positive, whereas 5/18 (27.8%) in the GH group, 6/13 (46.2%) G. taiwanensis, and 1/3 (33.3%) of the G. sanguinis strains were erythromycin-non-susceptible mefE- or mefA-positive and clindamycin-susceptible. The MIC90 of minocycline and the ratios of tetM-positive strains varied across the different species—G. morbillorum: 2 µg/mL and 27.3% (6/22); GH group: 8 µg/mL and 22.2% (4/18); G. taiwanensis: 8 µg/mL and 53.8% (7/13), respectively. Levofloxacin resistance was significantly higher in G. taiwanensis (8/13, 61.5%) than in G. morbillorum (2/22, 9.1%). Levofloxacin resistance was associated with a substitution at serine 83 for leucine, phenylalanine, or tyrosine in GyrA. The mechanisms of resistance to erythromycin and clindamycin differed across Gemella species. In addition, the rate of susceptibility to levofloxacin differed across Gemella spp., and the quinolone resistance mechanism was caused by mutations in GyrA alone.

1. Introduction

Gemella is a catalase-negative, facultative anaerobic coccus [1]. Gemella is Gram-positive; however, its stain can be easily decolorized [2]. Cells appear as single cells, pairs, tetrads, and sometimes irregular clusters. As of July 2023, 10 Gemella sp. have been identified: G. asaccharolytica [3], G. bergeri [4], G. cuniculi [5], G. haemolysans [6], G. morbillorum [7], G. palaticanis [8], G. parahaemolysans [2], G. sanguinis [9], G. taiwanensis [2], and G. massiliensis [10,11]. G. massiliensis was recently categorized as a new species according to the International Code of Nomenclature of Prokaryotes. These Gemella species are commensals in the oral cavities and guts of humans and are occasionally isolated as pathogens from patients with infective endocarditis [12,13,14,15,16,17,18], cerebral abscesses [19], bacteremia [20,21,22], septic shock [23], meningitis [24], and purpura fulminans with Lemierre’s syndrome [25], spondylodiscitis [26]. Phenotypically, Gemella resembles viridans group streptococci, Abiotrophia, or Granulicatella and has thus been misdiagnosed and underestimated in clinical microbiology laboratories [27]. Some Gemella sp., such as G. haemolysans, G. parahaemolysans, and G. taiwanensis, cannot be distinguished even using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry [12] or 16S rDNA sequencing [2,12,28].
The global spread of antimicrobial resistance in pathogenic bacteria is a growing concern. Some Gemella sp. are reportedly resistant to macrolides [2,29,30] and tetracyclines [29,30]. Studies have shown that in Gemella sp., mefA, mefE, and ermB are involved in macrolide resistance and tetO and tetM are involved in tetracycline resistance [2,29,30,31]. In 2016, document M45-Third Edition of the Clinical and Laboratory Standards Institute (CLSI) described a standardized method for antimicrobial resistance testing for Gemella sp. [32]; however, few studies have used this method [22,25,33]. In many cases, clinicians and laboratory technicians are using methods and setting breakpoints without identical criteria. Additionally, it remains unknown whether Gemella sp. has resistance mechanisms for other antimicrobials, such as quinolones.
In this study, we tested the antimicrobial susceptibility of 52 clinical isolates and six type strains of Gemella according to CLSI M45-Third Edition [32]. Additionally, we analyzed macrolide-, tetracycline-, and quinolone-resistant genes in the Gemella strains.

2. Results

2.1. Identification Using 16S rDNA Sequencing and Multilocus Sequence Analysis

Of the 52 clinical isolates of Gemella, strains 21, 2, and 1 were considered G. morbillorum, G. sanguinis, and G. bergeri, respectively, using 16S rDNA sequencing. The origins of the sources are presented in Table 1. The other 28 isolates were assigned to G. haemolysans, G. parahaemolysans, and G. taiwanensis. Discriminating the three species based on 16S rDNA sequencing alone was challenging, owing to the high homology between the species. Therefore, we conducted multilocus sequence analysis (MLSA) [2] using groEL, recA, and rpoB sequences. In total, isolates 4, 3, and 12 were assigned to G. haemolysans, G. parahaemolysans, and G. taiwanensis, respectively, using both 16S rDNA sequencing and MLSA. Consistently, the 12 isolates typed as G. taiwanensis were identified as G. taiwanensis using MLSA with high homology. However, the 16 isolates belonging to G. haemolysans or G. parahaemolysans could not be distinguished even when using MLSA. These were defined as the G. haemolysans—parahaemolysans (GH) group (Table S1). The MLSA homology of strains TWCC 53044 and TWCC 58771 to the type strains of Gemella is divergent, suggesting that they are new species of Gemella. In this study, these strains were tentatively assigned to the GH group and G. taiwanensis, respectively.

2.2. Antimicrobial Susceptibility

2.2.1. Susceptibility to Penicillin G, Cefotaxime, Ceftriaxone, Meropenem, and Vancomycin

The clinical isolates and five type strains (G. morbillorum ATCC 27824T, G. haemolysans ATCC 10379T, G. parahaemolysans JCM 18067T, G. taiwanensis JCM 18066T, and G. sanguinis CCUG 37820T) were susceptible to penicillin G. Because most of the Gemella strains used in our study were highly sensitive to the β-lactams, the MIC50 and MIC90 of the drugs became close or the same. However, the G. taiwanensis type strain JCM 18066T had intermediate susceptibility. The MIC90 value of the 58 Gemella strains was <0.06 µg/mL. All the isolates and type strains were susceptible to ceftriaxone (MIC90 ≤ 0.06 µg/mL), cefotaxime (MIC90 = 0.12 µg/mL), meropenem (MIC90 ≤ 0.06 µg/mL), and vancomycin (MIC90 = 0.50 µg/mL) (Table 2). Because three strains of G. morbillorum (TWCC 57201, TWCC 57818, and TWCC 71529) grew slower than other strains, the MIC of each drug was determined at 72–96 h (Table S1, Figure 1).

2.2.2. Susceptibility to Erythromycin

In total, 20/58 strains were erythromycin-non-susceptible (intermediate or resistant), with MIC90 > 2 µg/mL. Although the ratios of the erythromycin-non-susceptible isolates varied across species, there was no significant difference among G. morbillorum, the GH group, and G. taiwanensis—G. morbillorum: 27.3% (6/22), GH group: 38.9% (7/18), G. taiwanensis: 46.2% (6/13), G. sanguinis: 33.3% (1/3), and G. bergeri: 0.0% (0/2) (Table 2, Figure 1 and Figure 2).

2.2.3. Susceptibility to Clindamycin

In total, 10/58 strains were clindamycin-non-susceptible, resulting in MIC90 > 2 µg/mL. Clindamycin-resistant G. taiwanensis, G. sanguinis, and G. bergeri isolates were not detected, and differences were not significant—G. morbillorum: 27.3% (6/22), GH group: 22.2% (4/18), G. taiwanensis: 0.0% (0/13), and G. sanguinis: 0.0% (0/3) (Table 2, Figure 1 and Figure 2). Interestingly, all six erythromycin-resistant G. morbillorum strains were clindamycin-resistant. In contrast, 5/7 GH group strains, six strains of G. taiwanensis, and one G. sanguinis strain were erythromycin-non-susceptible and clindamycin-susceptible.

2.2.4. Susceptibility to Levofloxacin

In total, 21/58 strains were levofloxacin-resistant, resulting in MIC90 > 128 µg/mL. Ratios of the levofloxacin strains varied across species—G. morbillorum: 9.1% (2/22), GH group: 50.0% (9/18), G. taiwanensis: 61.5% (8/13), G. sanguinis: 66.7% (2/3), and G. bergeri: 0.0% (0/2). The ratio of the resistant strains was significantly higher in G. taiwanensis than in G. morbillorum (p < 0.05 using chi-squared test), whereas the ratio was higher in the GH group than in G. morbillorum (p = 0.08) (Table 2, Figure 1).

2.2.5. Susceptibility to Minocycline

Although the MIC90 value of the tetracycline antibiotic minocycline was 8 μg/mL, overall, the values were lower in G. morbillorum (2 μg/mL) than in the GH group (8 μg/mL) and G. taiwanensis (8 μg/mL) (Table 2).

2.2.6. Susceptibility to Other Antimicrobial Agents

We tested the 18 antimicrobial agents whose breakpoints are not listed in CLSI M45-third edition. Gemella strains showed low MIC values for all beta-lactams: ampicillin, amoxicillin–clavulanic acid, sulbactam–ampicillin, cefazolin, cefdinir, cefepime, and imipenem (MIC90: ≤0.12, ≤0.25/0.12, ≤0.06/0.12, ≤0.25, ≤0.25, ≤0.06, and ≤0.06 μg/mL, respectively). The MIC90 values of clarithromycin and azithromycin were 8 and >4 µg/mL, respectively, consistent with those of erythromycin. The MIC90 values of clarithromycin varied among G. morbillorum (>16 μg/mL), the GH group (8 μg/mL), and G. taiwanensis (2 μg/mL), indicating the acquisition of high resistance to clarithromycin in G. morbillorum strains. The MIC90 value of moxifloxacin was high (>2 μg/mL) in Gemella strains. The MIC90 values of the aminoglycoside antibiotics gentamicin, gentamicin500 (to confirm tolerance to high concentrations of gentamicin), and arbekacin were 8, ≤500, and >8 μg/mL, respectively; sulfamethoxazole–trimethoprim, fosfomycin, and rifampicin were >38/2, ≤16, and ≤0.5 μg/mL, respectively; and the anti-MRSA agents teicoplanin, linezolid, and daptomycin were ≤0.5, 1, and 2 μg/mL, respectively (Table 2). Typically, streptococci are aminoglycoside-resistant. Therefore, we tested gentamicin500 to identify any Gemella strains that are highly resistant to aminoglycoside.

2.3. Phenotypes and Genotypes of Macrolide-Resistant Strains

The six erythromycin–clindamycin-resistant G. morbillorum strains exhibited constitutive resistance to macrolide, lincosamide, and streptogramin B (cMLSB). Their genotypes—mefA/E-negative, ermB-positive, and msrA-negative—were consistent with their phenotypes. Furthermore, five/seven strains of the GH group, six strains of G. taiwanensis, and one strain of G. sanguinis which were erythromycin-non-susceptible and clindamycin-susceptible, had macrolide-resistant (M) phenotypes and mefE- (four strains) or mefA-positive (one strain), erm-negative, and msrA-negative genotypes. In total, 2/7 GH group strains (TWCC 59567 and TWCC 59795) were erythromycin-resistant and clindamycin-non-susceptible and mefE-positive, but showed M phenotype. These results show that erythromycin-resistant G. morbillorum is associated with ermB, and erythromycin-non-susceptible GH-group, G. taiwanensis, and G. sanguinis are associated with mefE. The MIC values for clarithromycin were higher in the six ermB-positive G. morbillorum strains (8 or >16 µg/mL) (Table 3). All erythromycin-susceptible Gemella strains, except the G. sanguinis strain TWCC 70419, lacked mefA/E, erm, or msrA (Table S1).

2.4. Tetracycline Resistance

Next, we analyzed the possession rates of tet. Overall, 17/58 (29.3%) strains were tetM-positive; none of the other tet genes was detected. The ratios of tetM-positive strains in G. morbillorum, the GH group, G. taiwanensis, G. sanguinis, and G. bergeri were 27.3% (6/22), 33.3% (6/18), 38.5% (5/13), 0/3 (0.0%), and 0.0% (0/2), respectively. Among the 41 tetM-negative strains, one had minocycline (MIC = 2 µg/mL). The minocycline MIC values of the others were ≤1 µg/mL. The minocycline MIC of the 17 tetM-positive strains varied: ≤1 for five, 2 for five, and ≥8 µg/mL for seven strains, respectively (Table 4).

2.5. Mutations in gyrA and gyrB

We analyzed the gyrA and gyrB sequences. The 35 quinolone-susceptible strains possessed gyrA, encoding GyrA with a serine residue at 83 (S83). The serine residue was substituted with leucine (S83L), phenylalanine (S83F), or tyrosine (S83Y) in the 21 quinolone-resistant strains. Specifically, two G. morbillorum strains possessed GyrA/S83L, encoding gyrA. Seven of the GH group, seven G. taiwanensis, and two G. sanguinis strains contained S83F. Two in the GH group and one G. taiwanensis strains contained S84Y. GyrB mutations associated with levofloxacin resistance were not detected (Table 5).

3. Discussion

In this study, we tested the antimicrobial susceptibility of 52 clinical isolates and six type strains of Gemella sp. with 27 drugs in accordance with CLSI M45-Third Edition [32]. Discriminating between G. haemolysans and G. parahaemolysans was difficult using MLSA. Therefore, the strains that could not be differentiated were assigned to the GH group. Garcia Lopez et al. [34] proposed grouping the four strains registered as G. haemolysans as the “Haemolysans group” because their average nucleotide identity ranged from 87.2% to 99.9%.
Although susceptibility was judged after 48 h of incubation for most cases, some strains needed 72–96 h incubation. Some studies have used the E-test, using optimal culture media for Gemella, because bacterial growth is poor with the CLSI method [26,35]. To ensure accurate tests for antimicrobial susceptibility, it might be important to update culture conditions, such as adding supplemental nutrition, to promote better growth of Gemella strains.
All the strains were susceptible to beta-lactams, except the G. taiwanensis type strain JCM 18066T, which had intermediate susceptibility to penicillin G. Because all the Gemella strains used in our study were highly sensitive to β-lactams, the MIC50 and MIC90 values of the drugs were similar or the same. Gemella is usually susceptible to beta-lactams [2,14,20,21,22,23,25,35,36]; however, there are some reports of resistance to penicillin G [2,21,23,24], ceftriaxone [24], and meropenem [33]. Overall susceptibility rates for erythromycin, clindamycin, and levofloxacin were 65.5%, 82.8%, and 63.8%, respectively. Consistently, Baghdadi et al. [33] reported that the susceptibility rates of 14 strains of Gemella (not speciated) were 50% for erythromycin, 86% for clindamycin, and 50% for levofloxacin. For G. morbillorum, our MIC90 value (>2 μg/mL) for clindamycin was different from that reported in another study (MIC90 ≤ 0.06 μg/mL) [36], indicating that trends in antimicrobial susceptibility vary among reports. Therefore, antimicrobial susceptibility must be tested for all Gemella isolates, especially those isolated from sterile sites, such as blood, because the isolate is suspected to be a pathogen. In this study, G. morbillorum strains, including type strains, were frequently isolated from sterile materials, such as blood and ascites, as well as wounds (Table 1 and Table S1). In contrast, the GH group and G. taiwanensis strains were derived from respiratory tissues, such as the pharynx (Table 1 and Table S1). This suggests that the pathogenicity and usual colonization sites of Gemella differ across species.
Drugs with no breakpoints in the CLSI M45-Third Edition had the same trend as the beta-lactams, macrolides, and quinolones of the same family. Rifampicin and anti-MRSA drugs have low MIC90 values and may be therapeutic options.
Resistance of streptococci to MLSB antibiotics occurs through two major mechanisms. The first is mediated by the methylation of ribosomal targets of these antibiotics (MLSB resistance). The methylase responsible for this activity is encoded by erm. MLSB resistance can be constitutive (cMLSB) or inducible (iMLSB). MLSB-mediated resistance by erm confers strong resistance to MLSB [37]. The second involves an active efflux system associated with mef, which exhibits low resistance to 14- and 15-membered macrolides only, and the resulting phenotype is M [38,39].
We found that the erythromycin-resistant G. morbillorum possessed ermB, whereas the erythromycin-resistant GH group, G. taiwanensis, and G. sanguinis had mefE. Consistent with our data, reports show that the MIC values of erythromycin are 2 [2] and 1 μg/mL [31] for mef-positive G. haemolysans and G. taiwanensis, respectively. The G. haemolysans strain possesses mef [2], and the G. taiwanensis strain possesses mef but not ermT, ermTR, or ermB [31]. Conversely, Zolezzi et al. [29] detected G. morbillorum with mefA/E, G. haemolysans with phenotype cMLSB and ermB, and G. morbillorum with iMLSB-resistant phenotype and ermB. Although the relationship between gene acquisition and Gemella sp. Is unknown, Gemella sp. mefE shares 99%–100% homology with Streptococcus pneumoniae (European Nucleotide Archive (ENA) Accession No. U83667.1) and Streptococcus salivarius (ENA Accession No. CAC87432.1) mefE, suggesting a genetic exchange between streptococci. In our collection, the two erythromycin-resistant GH group strains (TWCC 59567 and TWCC 59795) were categorized as mefE-positive clindamycin-resistant and intermediate, respectively. Although one G. sanguinis strain (TWCC 70419) possessed mefE, it was susceptible to erythromycin, with a low MIC value of clarithromycin (≤0.12 μg/mL) and contained no mutation in mefE (Table S3). The MIC of azithromycin for the G. sanguinis strain was relatively high (0.25 μg/mL), indicating that mefE is partially involved in susceptibility to azithromycin. The ermB-positive G. morbillorum strains showed higher MIC values to erythromycin, clarithromycin, and azithromycin than to the mefE-positive GH group, G. taiwanensis, G. sanguinis, and G. bergeri strains (Table 3). Our results suggest that Gemella sp. with erm possess higher macrolide resistance than those harboring mef. Consistently, macrolide resistance was higher after the acquisition of erm than that of mef [38,39,40]. Although we did not find msrA-positive strains in our collection, Zolezzi et al. reported msrA + G. morbillorum [30]. Further analysis must clarify the acquisition of macrolide resistance by Gemella sp.
Because ermB, mefE, and tetM are common to viridans group streptococci, etc., it is assumed that there was horizontal gene transfer between them. Zolezzi et al. performed in vitro mefE transfer from Gemella sp. and viridans group streptococci to S. pneumoniae [29]. Streptococcal ermB and tetM are associated with Tn916- and/or Tn916-like conjugative transposons. In this study, ermB, mefE, and tetM of Gemella sp. showed high homology with those of S. pneumoniae, indicating gene transfer from S. pneumoniae to Gemella sp. via the Tn916 family. In total, 12/13 (92.3%) Gemella strains with a minocycline MIC value ≥ 2 μg/mL harbored tetM. Although our data showed possession of only tetM, Zolezzi et al. reported that G. morbillorum and G. haemolysans possess both tetM and tetO [30]. The oral cavity is a suitable environment for horizontal gene transfer because commensal bacteria exist in close proximity to plaques [41]. In a systematic review, Brooks et al. concluded that tetM and Tn916 were the most prevalent gene and mobile genetic element associated with antibiotic resistance in the oral cavity, respectively, and the most common resistance genes varied in these sites, such as tetM in the root canal and ermB in supragingival plaques [42]. Rossi-Fedele et al. reported that Tn916 is involved in the transfer of tetM from Neisseria niger to Enterococcus faecalis in the root canal [43]. Villedieu et al. showed that tetM and ermB could transfer to Enterococcus faecalis through the Tn916-like conjugative transposon Tn1545 [44]. Zolezzi et al. performed in vitro mefE gene transfer from Gemella species and viridans group streptococci to S. pneumoniae [29].
There are three quinolone resistance mechanisms. The first involves reduced drug binding to the enzyme–DNA complex due to resistance mutations in one or both quinolone target enzymes, DNA gyrase and DNA topoisomerase IV. The second involves a resistance mutation in a regulatory gene that controls the expression of the native efflux pump in the bacterial membrane. The third involves a resistance gene acquired on plasmids [45]. In this study, we focused on mutations in DNA gyrase. Quinolones target two essential bacterial type II topoisomerase enzymes—DNA gyrase and DNA topoisomerase IV. Each enzyme is a heterotetramer: gyrase contains two GyrA and two GyrB subunits, whereas topoisomerase IV contains two ParC and two ParE subunits. GyrA is homologous to ParC, and GyrB is homologous to ParE [45]. In Gram-positive bacteria, the gyrA mutation follows the parC mutation. Quinolone resistance in viridans group streptococci [46] and β-hemolytic Streptococcus spp. [47] is higher for parC + gyrA mutations than for parC mutations. Mutations in streptococcal gyrA alone show high resistance to quinolones [47].
Gemella lacks topoisomerase IV, indicating that the Ser83 mutation in GyrA is only responsible for high resistance to levofloxacin. Similar results were observed for Helicobacter pylori [48] and Mycobacterium tuberculosis [49], which lack topoisomerase IV; however, their quinolone resistance was attributed to mutations in the quinolone resistance-determining regions of gyrA. In Japan, the quinolone resistance rates of Gemella tended to be higher than those for similar Abiotrophia and Granulicatella sp. [50], as well as S. pneumoniae [51]. Because Gemella lacks parC, a single mutation in gyrA can occur easily, resulting in the acquisition of higher quinolone resistance than these streptococci. The consumption of oral quinolones is higher in Japan than in other countries, suggesting that high quinolone exposure [52] resulted in the frequent emergence of quinolone-resistant Gemella. The National Action Plan on Antimicrobial Resistance of Japan recommended that oral prescription of quinolones must be reduced for control of quinolone-resistance.
This study has some limitations. We did not collect patient information, such as clinical history, antibiotics used for treatment, prognosis, isolated hospitals, and time period of collection. To further analyze the characteristics of Gemella species, patient information might be helpful.
In conclusion, the mechanisms of macrolide resistance and occupancy of the levofloxacin resistance gene (gyrA) varied across Gemella sp. Our data suggest that species-level identification is required for further characterization of antimicrobial resistance.

4. Materials and Methods

4.1. Bacterial Strains

We collected 52 Gemella strains isolated from clinical sites, such as blood, wound pus, abdominal drain effluent, ascites, closed wounds, open wounds, catheter urine, sputum, lung biopsy needle wash solution, corneal abrasion, bile, and noses (Table S1 and Table 1). Five type strains, namely G. morbillorum ATCC 27824T, and G. haemolysans ATCC 10379T were obtained from American Type Culture Collection Manassas, VA, USA, G. parahaemolysans JCM 18067T, and G. taiwanensis JCM 18066T, were collected from the Japan Collection of Microorganisms, RIKEN BRC, Ibaraki, Japan. G. sanguinis CCUG 37820T and G. bergeri CCUG 37817T were obtained from the Culture Collection University of Götheborg, Götheborg, Sweden. All the strains were stored in 10% skim milk at −80 °C until use.

4.2. DNA Extraction

Bacteria grown on 5% sheep blood agar EX plates (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) were suspended in McFarland 2.0 standard in 100 μL of 10 mM Tris-HCl and 1 mM EDTA (TE) buffer (pH 8.0) supplemented with 320 U of achromopeptidase (Wako Chemical, Osaka, Japan), followed by incubation at 55 °C for 15 min. After centrifugation at 15,000× g for 5 min, the supernatant was used as a crude DNA template for PCR.

4.3. Identification of Gemella sp.

Gemella species were identified based on 16S rDNA sequencing and MLSA [2], which utilize concatenated sequences of groEL, recA, and rpoB. Primers for MLSA were designed by Hung et al. [28] and our team. The primers used are listed in Table S2. The genes were amplified using TaKaRa Ex Taq polymerase Hot Start Version (TaKaRa Bio, Shiga, Japan) according to the following thermal cycle: initial denaturation at 95 °C for 10 min, 30 cycles of denaturation at 95 °C for 30 s, primer annealing at the indicated temperatures (Table S2) for 1 min, and extension at 72 °C for 1–4 min depending on product size (1 kb/min), and final extension at 72 °C for 5 min. The amplicons were purified and applied to dye terminator cycle sequencing using each of the primers and the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions. After clean-up using the BigDye XTerminator Purification Kit (Thermo Fisher Scientific), the sequences were analyzed using an automatic DNA sequencer (ABI Prism 310 genetic analyzer; Applied Biosystems, Foster City, CA, USA).

4.4. Detection of Macrolide-Resistant Genes

Macrolide resistance-related genes mefA/E [30], ermA [53], ermB [54], ermC [53], ermM [53], ermTR [53], and msrA [53] were detected according to published reports. Sanger sequencing was conducted to confirm mefA or mefE. Amplified PCR fragments were analyzed using sequencing, as described in Section 4.3.

4.5. Detection of Tetracycline-Resistant Genes

Tetracycline-resistant genes tetM, tetO, tetK, tetL, tetT, tetS, and tetW were detected as described, with slight modifications [53,55]. Briefly, the genes were amplified using the TaKaRa Ex Premier DNA Polymerase (TaKaRa Bio) and pairs of primers (Table S2) with the following thermal cycle: initial denaturation at 94 °C for 5 min, 30 cycles of denaturation at 98 °C for 10 s, primer annealing at the indicated temperatures (Table S2) for 15 s, and extension at 68 °C for 70 s, and final extension at 72 °C for 5 min.

4.6. Detection of Quinolone-Resistant Genes gyrA and gyrB

Sequences of gyrA and gyrB were amplified with our designed primers (Table S2) and analyzed using sequencing. Gene sequences were translated into amino acid sequences, followed by alignment and detection of amino acid substitutions.

4.7. Antimicrobial Susceptibility Test

The antimicrobial susceptibility test was conducted in accordance with the CLSI M45-Third Edition for penicillin G, ampicillin, amoxicillin/clavulanic acid, sulbactam/ampicillin, cefazolin, cefdinir, ceftriaxone, cefotaxime, cefepime, imipenem, meropenem, erythromycin, clarithromycin, azithromycin, clindamycin, erythromycin/clindamycin, levofloxacin, moxifloxacin, minocycline, sulfamethoxazole/trimethoprim, gentamicin, gentamicin500, arbekacin, fosfomycin, rifampicin, vancomycin, teicoplanin, linezolid, and daptomycin [32]. Briefly, Gemella strains grown on 5% sheep blood agar EX plates (Shimadzu Diagnostics Corporation, Tokyo, Japan) were suspended in saline and inoculated in Difco cation-adjusted Mueller Hinton Broth (Becton, Dickinson, Sparks, MD, USA) supplemented with lysed 5% horse blood (KOHJIN BIO, Saitama, Japan) at 5 × 105 CFU/mL. Dry plates 34, 42, and 44 were purchased from Eiken Chemical Co., Ltd. (Tokyo, Japan). Dry plates were incubated at 35 °C and 5% CO2 for 48 h. In cases of poor growth until 48 h, the strains were cultured for 72–96 h. MICs were interpreted using the CLSI M45 breakpoints for Gemella spp. when available [32]. For quality control, CLSI-Third Edition-recommended Streptococcus pneumoniae ATCC 49619 was used [32]. Quality control was performed for each change of plates in every lot.

4.8. Statistical Analysis

Ratios of resistant and non-susceptible strains were statistically analyzed using the Chi-square test.

4.9. Ethical Approval

The isolation, storage, and utilization of clinical strains were conducted according to the guidelines of each of the participating hospitals.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/2079-6382/12/10/1538/s1, Table S1: Gemella strains used in this study; Table S2: Primers used in this study; Table S3: mefE sequence of Gemella sanguinis TWCC 70419.

Author Contributions

M.F. collected the clinical isolates, analyzed the genes, and wrote the manuscript; Y.A. and Y.U. conducted the antimicrobial susceptibility test and contributed to the analysis of the genes; T.K. and S.O. supervised M.F. and contributed to the manuscript revision; K.O. revised the manuscript regarding intellectual content; K.K. conceived and designed this study. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author KK upon reasonable request.

Acknowledgments

We thank Norihiko Takemoto (National Center for Global Health and Medicine) for his support with the manuscript preparation.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Antibiotics 12 01538 g001
Figure 1. Ratios of resistant strains. S (blue), I (yellow), and R (Red) indicate sensitive, intermediate, and resistant, respectively.
Figure 1. Ratios of resistant strains. S (blue), I (yellow), and R (Red) indicate sensitive, intermediate, and resistant, respectively.
Antibiotics 12 01538 g001
Antibiotics 12 01538 g002
Figure 2. Distribution of erythromycin/clindamycin resistance in Gemella strains. Blue, yellow, and red boxes indicate sensitive, intermediate, and resistant, respectively.
Figure 2. Distribution of erythromycin/clindamycin resistance in Gemella strains. Blue, yellow, and red boxes indicate sensitive, intermediate, and resistant, respectively.
Antibiotics 12 01538 g002
Table 1. Isolated sites of Gemella species used in this study.
Table 1. Isolated sites of Gemella species used in this study.
SpecimenNumber of Strains
Gemella morbillorumGHgroupGemella taiwanensisGemella sanguinisGemella bergeri
Blood8451
Ascites2
Bile 1
Pleural effusion 1
Wound pus1024 1
Sputum 2
Lung biopsy 1
Pharynx 52
Nose 1
Urine1
Cornea 1
Total21161221
Isolated sites of Gemella type strain. G. morbillorum ATCC 27824T: lung abscess, G. haemolysans ATCC 10379T: unknown, G. parahaemolysans JCM 18067T: blood, G. taiwanensis JCM 18066T: blood, G. sanguinis CCUG 37820T: bood, G. bergeri CCUG 37817T: blood.
Table 2. Susceptibility to antimicrobial agents with breakpoints listed in CLSI M45-third edition.
Table 2. Susceptibility to antimicrobial agents with breakpoints listed in CLSI M45-third edition.
Antimicrobial Agents/
Gemella spp. 		MIC (μg/mL) Interpretive Breakpoint (μg/mL) a
or % of Isolates
RangeMIC50MIC90SusceptibleIntermediateResistant
Penicillin G c≤0.06–>4 ≤0.120.25–2≥4
Gemella morbillorum≤0.06≤0.06≤0.06100.00.00.0
GH group≤0.06≤0.06≤0.06100.00.00.0
Gemella taiwanensis≤0.06–0.25≤0.06≤0.0692.37.70.0
Gemella sanguinis≤0.06––100.00.00.0
Gemella bergeri≤0.06––100.00.00.0
Total≤0.06–0.25≤0.06≤0.0698.31.70.0
Ampicillin≤0.12–>4
Gemella morbillorum≤0.12–0.25≤0.12≤0.12NA bNANA
GH group≤0.12≤0.12≤0.12NANANA
Gemella taiwanensis≤0.12–0.5≤0.12≤0.12NANANA
Gemella sanguinis≤0.12––NANANA
Gemella bergeri≤0.12––NANANA
Total≤0.12–0.5≤0.12≤0.12NANANA
Amoxicillin–clavulanic acid≤0.25/0.12–>4/2
Gemella morbillorum≤0.25/0.12≤0.25/0.12≤0.25/0.12NANANA
GH group≤0.25/0.12≤0.25/0.12≤0.25/0.12NANANA
Gemella taiwanensis≤0.25/0.12≤0.25/0.12≤0.25/0.12NANANA
Gemella sanguinis≤0.25/0.12––NANANA
Gemella bergeri≤0.25/0.12––NANANA
Total≤0.25/0.12≤0.25/0.12≤0.25/0.12NANANA
Sulbactam–ampicillin≤0.06/0.12–>2/4
Gemella morbillorum≤0.06/0.12≤0.06/0.12≤0.06/0.12NANANA
GH group≤0.06/0.12≤0.06/0.12≤0.06/0.12NANANA
Gemella taiwanensis≤0.06/0.12–0.25/0.5≤0.06/0.12≤0.06/0.12NANANA
Gemella sanguinis≤0.06/0.12––NANANA
Gemella bergeri≤0.06/0.12––NANANA
Total≤0.06/0.12–0.25/0.5≤0.06/0.12≤0.06/0.12NANANA
Cefazolin ≤0.25–>2
Gemella morbillorum≤0.25≤0.25≤0.25NANANA
GH group≤0.25–0.5≤0.250.5NANANA
Gemellataiwanensis≤0.25–0.5≤0.250.5NANANA
Gemella sanguinis≤0.25––NANANA
Gemella bergeri≤0.25––NANANA
Total≤0.25–0.5≤0.25≤0.25NANANA
Cefdinir≤0.25–>1
Gemella morbillorum≤0.25≤0.25≤0.25NANANA
GH group≤0.25≤0.25≤0.25NANANA
Gemella taiwanensis≤0.25≤0.25≤0.25NANANA
Gememlla sanguinis≤0.25–0.5––NANANA
Gemella bergeri≤0.25––NANANA
Total≤0.25–0.5≤0.25≤0.25NANANA
Ceftriaxone c≤0.06–>2 ≤12≥4
Gemella morbillorum≤0.06–0.5≤0.06≤0.06100.00.00.0
GH group≤0.06≤0.06≤0.06100.00.00.0
Gemella taiwanensis≤0.06≤0.06≤0.06100.00.00.0
Gemella sanguinis0.25–1––100.00.00.0
Gemella bergeri≤0.06 100.00.00.0
Total≤0.06–1≤0.06≤0.06100.00.00.0
Cefotaxime c≤0.06–>2 ≤12≥4
Gemella morbillorum≤0.06–0.12≤0.06≤0.06100.00.00.0
GH group≤0.06–0.12≤0.06≤0.06100.00.00.0
Gemella taiwanensis≤0.06–0.12≤0.06≤0.06100.00.00.0
Gemella sanguinis0.25–1––100.00.00.0
Gemella bergeri≤0.06––100.00.00.0
Total≤0.06–1≤0.060.12100.00.00.0
Cefepime≤0.06–>2
Gemella morbillorum≤0.06–0.5≤0.06≤0.06NANANA
GH group≤0.06–0.12≤0.060.12NANANA
Gemella taiwanensis≤0.06–0.12≤0.06≤0.06NANANA
Gemella sanguinis0.25–1––NANANA
Gemella bergeri≤0.06––NANANA
Total≤0.06–1≤0.060.12NANANA
Imipenem≤0.06–>4
Gemella morbillorum≤0.06≤0.06≤0.06NANANA
GH group≤0.06≤0.06≤0.06NANANA
Gemella taiwanensis≤0.06≤0.06≤0.06NANANA
Gemella sanguinis≤0.06––NANANA
Gemella bergeri≤0.06––NANANA
Total≤0.06≤0.06≤0.06NANANA
Meropenem c≤0.06–>2 ≤0.51≥2
Gemella morbillorum≤0.06≤0.06≤0.06100.00.00.0
GH group≤0.06≤0.06≤0.06100.00.00.0
Gemella taiwanensis≤0.06≤0.06≤0.06100.00.00.0
Gemella sanguinis≤0.06––100.00.00.0
Gemella bergeri≤0.06––100.00.00.0
Total≤0.06≤0.06≤0.06100.00.00.0
Erythromycin c≤0.25–>2 ≤0.250.5≥1
Gemella morbillorum≤0.25–>2≤0.25>272.70.027.3
GH group≤0.25–>2≤0.25>261.15.633.3
Gemella taiwanensis≤0.25–>2≤0.25>253.80.046.2
Gemella sanguinis≤0.25–1––66.70.033.3
Gemella bergeri≤0.25––100.00.00.0
Total≤0.25–>2≤0.25>265.51.732.8
Clarithromycin≤0.12–>16
Gemella morbillorum≤0.12–>16≤0.12>16NANANA
GH group≤0.12–16≤0.128NANANA
Gemella taiwanensis≤0.12–8≤0.122NANANA
Gemella sanguinis≤0.12–0.25––NANANA
Gemella bergeri≤0.12––NANANA
Total≤0.12–>16≤0.128NANANA
Azithromycin≤0.12–>4
Gemella morbillorum≤0.12–>4≤0.12>4NANANA
GH group≤0.12–>4≤0.12>4NANANA
Gemella taiwanensis≤0.12–>40.25>4NANANA
Gemella sanguinis0.25–4––NANANA
Gemella bergeri0.25––NANANA
Total≤0.12–>4≤0.12>4NANANA
Clindamycin c≤0.25–>2 ≤0.250.5≥1
Gemella morbillorum≤0.25–>2≤0.25>272.70.027.3
GH group≤0.25–1≤0.250.577.816.75.6
Gemella taiwanensis≤0.25≤0.25≤0.25100.00.00.0
Gemella sanguinis≤0.25––100.00.00.0
Gemella bergeri≤0.25––100.00.00.0
Total≤0.25–>2≤0.25>282.85.212.1
Erythromycin/clindamycin ≤1/0.5–>1/0.5
G. morbillorum≤1/0.5–>1/0.5≤1/0.5>1/0.5NANANA
GH group≤1/0.5–>1/0.5≤1/0.5≤1/0.5NANANA
G. taiwanensis≤1/0.5≤1/0.5≤1/0.5NANANA
G. sanguinis≤1/0.5––NANANA
Gemella bergeri≤1/0.5––NANANA
Total≤1/0.5–>1/0.5≤1/0.5>1/0.5NANANA
Levofloxacin c≤0.004–>128 ≤24≥8
Gemella morbillorum0.03–>1280.25190.90.09.1
GH group0.125–>1281>12850.00.050.0
Gemella taiwanensis0.125–>128>128>12838.50.061.5
Gemella sanguinis0.5–>128––33.30.066.7
Gemella bergeri0.5––100.00.00.0
Total0.03–>1280.5>12863.80.036.2
Moxifloxacin≤0.5–>2
Gemella morbillorum≤0.5–>2≤0.5>2 NANANA
GH group≤0.5–>2≤0.5>2NANANA
Gemella taiwanensis≤0.5–>2>2>2NANANA
Gemella sanguinis≤0.5–>2––NANANA
Gemella bergeri≤0.5––NANANA
Total≤0.5–>2≤0.5>2NANANA
Minocycline≤1–>8
Gemella morbillorum≤1–>8≤12NANANA
GH group≤1–8≤18NANANA
Gemella taiwanensis≤1–8≤18NANANA
Gemella sanguinis≤1––NANANA
Gemella bergeri≤1––NANANA
Total≤1≤18NANANA
Sulfamethoxazole–
trimethoprim		≤9.5/0.5–>38/2
Gemella morbillorum≤9.5/0.5–>38/219/1>38/2NANANA
GH group≤9.5/0.5–>38/238/2>38/2NANANA
Gemella taiwanensis≤9.5/0.5–>38/219/119/1NANANA
Gemella sanguinis19/1–>38/2––NANANA
Gemella bergeri≤9.5/0.5––NANANA
Total≤9.5/0.5–>38/219/1>38/2NANANA
Gentamicin≤1–>8
Gemella morbillorum≤1–828NANANA
GH group≤1–2≤12NANANA
Gemella taiwanensis≤1–424NANANA
Gemella sanguinis≤1–8––NANANA
Gemella bergeri2, 4––NANANA
Total≤1–828NANANA
Gentamicin 500≤500–>500
Gemella morbillorum≤500≤500≤500NANANA
GH group≤500≤500≤500NANANA
Gemella taiwanensis≤500≤500≤500NANANA
Gemella sanguinis≤500––NANANA
Gemella bergeri≤500––NANANA
Total≤500≤500≤500NANANA
Arbekacin≤1–>8
Gemella morbillorum≤1–88>8NANANA
GH group≤1–848NANANA
Gemella taiwanensis2–>848NANANA
Gemella sanguinis4–>8––NANANA
Gemella bergeri4, >8––NANANA
Total≤1–84>8NANANA
Fosfomycin≤16–>128
Gemella morbillorum≤16–32≤16≤16NANANA
GH group≤16≤16≤16NANANA
Gemella taiwanensis≤16≤16≤16NANANA
Gemalla sanguinis≤16––NANANA
Gemella bergeri≤16––NANANA
Total≤16–32≤16≤16NANANA
Rifampicin≤0.5–>2
Gemella morbillorum≤0.5≤0.5≤0.5NANANA
GH group≤0.5≤0.5≤0.5NANANA
Gemella taiwanensis≤0.5≤0.5≤0.5NANANA
Gemella sanguinis≤0.5––NANANA
Gemella bergeri≤0.5 NANANA
Total≤0.5≤0.5≤0.5NANANA
Vancomycin c≤0.25–>2 ≤1
Gemella morbillorum≤0.25–0.50.50.5100.00.00.0
GH group≤0.25–0.50.50.5100.00.00.0
Gemella taiwanensis≤0.25–0.50.50.5100.00.00.0
Gemella sanguinis≤0.25–0.5––100.00.00.0
Gemella bergeri0.5––100.00.00.0
Total≤0.25–0.50.50.5100.00.00.0
Teicoplanin≤0.5–>16
Gemella morbillorum≤0.5≤0.5≤0.5NANANA
GH group≤0.5≤0.5≤0.5NANANA
Gemella taiwanensis≤0.5≤0.5≤0.5NANANA
Gemella sanguinis≤0.5––NANANA
Gemella bergeri≤0.5––NANANA
Total≤0.5≤0.5≤0.5NANANA
Linezolid≤0.5–>4
Gemella morbillorum≤0.5–1≤0.51NANANA
GH group≤0.5–1≤0.51NANANA
Gemella taiwanensis≤0.5≤0.5≤0.5NANANA
Gemella sanguinis≤0.5–1––NANANA
Gemella bergeri≤0.5, 2––NANANA
Total≤0.5≤0.51NANANA
Daptomycin≤0.25–>4
Gemella morbillorum≤0.25–422NANANA
GH group0.5–212NANANA
Gemella taiwanensis≤0.25–212NANANA
Gemella sanguinis1–4––NANANA
Gemella bergeri2, 4––NANANA
Total≤0.25–412NANANA
a Interpretive breakpoints are shown in bold for each antibiotic. b NA, not applicable (breakpoints not established). c Antimicrobial agents with breakpoints listed in CLSI M45-third edition.
Table 3. Distribution of macrolides and clindamycin MICs and possession of the mef, erm, and msrA genes in erythromycin-non-susceptible Gemella isolates.
Table 3. Distribution of macrolides and clindamycin MICs and possession of the mef, erm, and msrA genes in erythromycin-non-susceptible Gemella isolates.
Strain No.IdentificationMIC (μg/mL)Macrolide
Phenotype a,b		mefA/EermmsrA
ErythromycinClindamycinErythromycin/ClindamycinClarithromycin Azithromycin
TWCC 57201Gemella morbillorum>2>2>1/0.58>4cMLSB-ermB-
TWCC 57818Gemella morbillorum>2>2>1/0.5>16>4cMLSB-ermB-
TWCC 57944Gemella morbillorum>2>2>1/0.5>16>4cMLSB-ermB-
TWCC 59111Gemella morbillorum>2>2>1/0.58>4cMLSB-ermB-
TWCC 71703Gemella morbillorum>2>2>1/0.5>16>4cMLSB-ermB-
TWCC 72266Gemella morbillorum>2>2>1/0.5>16>4cMLSB-ermB-
TWCC 52027GH group0.5≤0.25≤1/0.582MmefE--
TWCC 59566GH group2≤0.25≤1/0.52>4MmefE--
TWCC 59567GH group>21>1/0.516>4MmefE--
TWCC 59795GH group10.5≤1/0.50.52MmefE--
TWCC 70939GH group>2≤0.25≤1/0.52>4MmefE--
TWCC 71200GH group1≤0.25≤1/0.522MmefA--
TWCC 71814GH group1≤0.25≤1/0.50.51MmefE--
TWCC 55344Gemella taiwanensis>2≤0.25≤1/0.58>4MmefE--
TWCC 58522Gemella taiwanensis>2≤0.25≤1/0.524MmefE--
TWCC 70386Gemella taiwanensis>2≤0.25≤1/0.524MmefE--
TWCC 72085Gemella taiwanensis>2≤0.25≤1/0.52>4MmefE--
TWCC 70387LGemella taiwanensis2≤0.25≤1/0.50.5>4MmefE--
TWCC 70387SGemella taiwanensis>2≤0.25≤1/0.52>4MmefE--
TWCC 54965Gemella sanguinis1≤0.25≤1/0.50.254MmefE--
TWCC 70419Gemella sanguinis≤0.25 c≤0.25≤1/0.5≤0.120.25not MmefE--
a cMLSB: macrolide–lincosamide–streptogramin B-resistant phenotype. b M: macrolide-resistant phenotype. c Erythromycin-susceptible.
Table 4. Distribution of minocycline MIC and ermB in Gemella isolates harboring the tetM gene.
Table 4. Distribution of minocycline MIC and ermB in Gemella isolates harboring the tetM gene.
Strain No.IdentificationtetMMinocycline
MIC (μg/mL)		ermB
TWCC 57944Gemella morbillorum+2+
TWCC 57987Gemella morbillorum+≤1−
TWCC 59111Gemella morbillorum+2+
TWCC 70937Gemella morbillorum+>8−
TWCC 71703Gemella morbillorum+2+
TWCC 72266Gemella morbillorum+≤1+
TWCC 51800GH group+8−
TWCC 59795GH group+≤1−
TWCC 70939GH group+8−
TWCC 71814GH group+2−
TWCC 53044Gemella taiwanensis+8−
TWCC 56546Gemella taiwanensis+2−
TWCC 58522Gemella taiwanensis+8−
TWCC 70386Gemella taiwanensis+8−
TWCC 72085Gemella taiwanensis+8−
TWCC 70387LGemella taiwanensis+≤1−
TWCC 70387SGemella taiwanensis+≤1−
Table 5. Distribution of MIC of tested quinolones and amino acid substitutions in gyrA gene in quinolone-resistant Gemella isolates.
Table 5. Distribution of MIC of tested quinolones and amino acid substitutions in gyrA gene in quinolone-resistant Gemella isolates.
StrainnMIC (μg/mL)GyrA Amino Acid Substitutions a
LevofloxacinMoxifloxacin
Gemella morbillorum2>128>2Ser83 > Leu83 (n = 2)
GH group9128–>128>2Ser83 > Phe83 (n = 7), Ser83 > Tyr83 (n =2)
Gemella taiwanensis8128–>128>2Ser83 > Phe83 (n = 7), Ser83 > Tyr83 (n = 1)
Gemella sanguinis2128–>128>2Ser83 > Phe83 (n = 2)
a gyrA-Ser83 Leu: serine to leucine at codon 83; Ser83 Phe: serine to phenylalanine at codon 83; Ser83 Tyr; serine to tyrosine at codon 83.
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MDPI and ACS Style

Furugaito, M.; Arai, Y.; Uzawa, Y.; Kamisako, T.; Ogura, K.; Okamoto, S.; Kikuchi, K. Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp. Antibiotics 2023, 12, 1538. https://doi.org/10.3390/antibiotics12101538

AMA Style

Furugaito M, Arai Y, Uzawa Y, Kamisako T, Ogura K, Okamoto S, Kikuchi K. Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp. Antibiotics. 2023; 12(10):1538. https://doi.org/10.3390/antibiotics12101538

Chicago/Turabian Style

Furugaito, Michiko, Yuko Arai, Yutaka Uzawa, Toshinori Kamisako, Kohei Ogura, Shigefumi Okamoto, and Ken Kikuchi. 2023. "Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp." Antibiotics 12, no. 10: 1538. https://doi.org/10.3390/antibiotics12101538

APA Style

Furugaito, M., Arai, Y., Uzawa, Y., Kamisako, T., Ogura, K., Okamoto, S., & Kikuchi, K. (2023). Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp. Antibiotics, 12(10), 1538. https://doi.org/10.3390/antibiotics12101538

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