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

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.

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.

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.The clinical isolates and five type strains (G.morbillorum ATCC 27824 T , G. haemolysans ATCC 10379 T , G. parahaemolysans JCM 18067 T , G. taiwanensis JCM 18066 T , and G. sanguinis CCUG 37820 T ) were susceptible to penicillin G.Because most of the Gemella strains used in our study were highly sensitive to the β-lactams, the MIC 50 and MIC 90 of the drugs became close or the same.However, the G. taiwanensis type strain JCM 18066 T had intermediate susceptibility.The MIC 90 value of the 58 Gemella strains was <0.06 µg/mL.All the isolates and type strains were susceptible to ceftriaxone (MIC 90 ≤ 0.06 µg/mL), cefotaxime (MIC 90 = 0.12 µg/mL), meropenem (MIC 90 ≤ 0.06 µg/mL), and vancomycin (MIC 90 = 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).Gemella morbillorum a gyrA-Ser83 Leu: serine to leucine at codon 83; Ser83 Phe: serine to phenylalanine at codon 83; S Tyr; serine to tyrosine at codon 83.

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 quinoloneresistant 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).

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 18066 T , which had intermediate susceptibility to penicillin G.Because all the Gemella strains used in our study were highly sensitive to β-lactams, the MIC 50 and MIC 90 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 MIC 90 value (>2 µg/mL) for clindamycin was different from that reported in another study (MIC 90 ≤ 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 (Tables 1 and S1).In contrast, the GH group and G. taiwanensis strains were derived from respiratory tissues, such as the pharynx (Tables 1 and 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 MIC 90 values and may be therapeutic options.
Resistance of streptococci to MLS B antibiotics occurs through two major mechanisms.The first is mediated by the methylation of ribosomal targets of these antibiotics (MLS B resistance).The methylase responsible for this activity is encoded by erm.MLS B resistance can be constitutive (cMLS B ) or inducible (iMLS B ). MLS B -mediated resistance by erm confers strong resistance to MLS B [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 cMLS B and ermB, and G. morbillorum with iMLS B -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] 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.

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

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

Figure 2 .
Figure 2. Distribution of erythromycin/clindamycin resistance in Gemella strains.Blue, yellow, and red boxes indicate sensitive, intermediate, and resistant, respectively.

Figure 2 .
Figure 2. Distribution of erythromycin/clindamycin resistance in Gemella strains.Blue, yellow, and red boxes indicate sensitive, intermediate, and resistant, respectively.

Table 1 .
Isolated sites of Gemella species used in this study.

Table 2 .
Susceptibility to antimicrobial agents with breakpoints listed in CLSI M45-Third Edition.
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 4 .
Distribution of minocycline MIC and ermB in Gemella isolates harboring the tetM gene.

Table 5 .
Distribution of MIC of tested quinolones and amino acid substitutions in gyrA gene in quinolone-resistant Gemella isolates.

µg/mL) GyrA Amino Acid Substitutions a Levofloxacin Moxifloxacin
. 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 Tn916like conjugative transposon Tn1545 [44].Zolezzi et al. performed in vitro mefE gene transfer from Gemella species and viridans group streptococci to S. pneumoniae [29].