ABSTRACT
The in vitro activity of two new fluoroquinolones, delafloxacin and finafloxacin, were evaluated against M. hominis and Ureaplasma spp. The MICs of delafloxacin, finafloxacin, and two classical fluoroquinolones (moxifloxacin and levofloxacin) were tested against 29 M. hominis and 67 Ureaplasma spp. isolates using the broth microdilution method. The molecular mechanisms underlying fluoroquinolone resistance were also investigated. Delafloxacin exhibited low MICs against M. hominis and Ureaplasma spp., including the levofloxacin-resistant isolates. For M. hominis, delafloxacin showed low MIC90 value of 1 μg/mL (MIC range, <0.031 -1 μg/mL) compared to 8 μg/mL for finafloxacin, 16 μg/mL for moxifloxacin, and 32 μg/mL for levofloxacin. For U. parvum and U. urealyticum, delafloxacin had low MIC90 values (U. parvum, 2 μg/mL; U. urealyticum, 4 μg/mL) compared to 16 -32 μg/mL for finafloxacin, 16 μg/mL for moxifloxacin, and 32 - >32 μg/mL for levofloxacin. The two mutations GyrA S153L and ParC S91I were commonly identified in fluoroquinolone-resistant M. hominis, and ParC S83L was the most frequent mutation identified in fluoroquinolone-resistant Ureaplasma spp. Delafloxacin displayed lower MICs against fluoroquinolone-resistant isolates of both M. hominis and Ureaplasma spp. that have mutations in the quinolone resistance determining regions (QRDRs) than the two classical fluoroquinolones. Delafloxacin is a promising fluoroquinolone with low MICs against fluoroquinolone-resistant M. hominis and Ureaplasma spp. Our study confirms the potential clinical use of delafloxacin in treating antimicrobial-resistant M. hominis and Ureaplasma spp. infections.
IMPORTANCE Fluoroquinolone resistance in Mycoplasma hominis and Ureaplasma spp. is on the rise globally, which has compromised the efficacy of the currently available antimicrobial agents. This study evaluated the antimicrobial activity of two new fluoroquinolones, delafloxacin and finafloxacin, for the first time, against M. hominis and Ureaplasma spp. clinical isolates. Delafloxacin and finafloxacin displayed different antimicrobial susceptibility profiles against M. hominis and Ureaplasma spp. in vitro. Delafloxacin was found to be more effective against M. hominis and Ureaplasma spp. than three classical fluoroquinolones (finafloxacin, moxifloxacin, and levofloxacin). Finafloxacin displayed activity similar to moxifloxacin but superior to levofloxacin against M. hominis and Ureaplasma spp. Our findings demonstrate that delafloxacin is a promising fluoroquinolone with outstanding activity against fluoroquinolone-resistant M. hominis and Ureaplasma spp.
KEYWORDS: Mycoplasma hominis, Ureaplasma spp., delafloxacin, finafloxacin, fluoroquinolone-resistant, in vitro activity
INTRODUCTION
Mycoplasma hominis and Ureaplasma species (U. parvum and U. urealyticum) are members of the class of Mollicutes and common commensals colonizing in the urogenital tract of humans. However, they have been associated with chorioamnionitis, adverse pregnancy outcomes, infertility, and neonatal diseases in some instances (1–3). Both M. hominis and Ureaplasma spp. lack cell wall; thus, antimicrobial treatments are limited to those that can inhibit DNA replication (e.g., fluoroquinolones) and protein synthesis (e.g., macrolides and tetracyclines). An increasing number of reports confirmed the presence of antimicrobial resistance in clinical M. hominis and Ureaplasma spp. isolates, especially against fluoroquinolones. Recent surveillance studies reported that fluoroquinolone resistance in M. hominis and Ureaplasma spp. isolates recovered from Chinese patients is on the rise, which has compromised the efficacy of the currently available fluoroquinolones (4–6). In our previous study, > 80% M. hominis and > 50% Ureaplasma spp. isolates were resistant to levofloxacin and moxifloxacin, respectively (4). Therefore, new fluoroquinolones against M. hominis and Ureaplasma spp. are urgently needed to establish effective treatment.
In M. hominis and Ureaplasma spp., amino acid mutations in quinolone resistance determining regions (QRDRs), including DNA gyrase (GyrA and GyrB) and the topoisomerase IV complex (ParC and ParE), are a key mechanism of fluoroquinolone resistance (7). Delafloxacin and finafloxacin are novel fluoroquinolones with improved antibacterial activity against various infections. Delafloxacin is a new dual-targeting anionic fluoroquinolone with modifications at carbon C-7 and C-8 (8), and its unique molecular structure increases its intracellular penetration and potency against multidrug-resistant bacteria - especially under acidic environments (9–11). Unlike other fluoroquinolones, delafloxacin can target DNA gyrase and topoisomerase IV equally, decreasing the selection of resistant mutations. Finafloxacin is another novel fluoroquinolone with 8-cyano and C-7 substituents, which also inhibits topoisomerase IV and DNA gyrase (12, 13). Therefore, both delafloxacin and finafloxacin can be effectively used in treating infections caused by a broad spectrum of organisms, including those with mutations in the QRDRs.
In this study, we evaluated the activity of delafloxacin and finafloxacin in vitro against M. hominis and Ureaplasma spp., compared with two classical fluoroquinolones (moxifloxacin and levofloxacin) using the broth microdilution method. We also identified the mutations in QRDRs and evaluated their associations with the MICs of delafloxacin and finafloxacin.
RESULTS
MICs of the four fluoroquinolones against M. hominis and Ureaplasma spp. isolates.
The MIC distribution, MIC50, and MIC90 values, and the rates of antimicrobial resistance for the four fluoroquinolones (delafloxacin, finafloxacin, moxifloxacin, and levofloxacin) are determined and represented in Table 1.
TABLE 1.
MIC distributions of delafloxacin, finafloxacin, moxifloxacin, and levofloxacin against M. hominis and Ureaplasma speciesa
| Organism and antimicrobials | No. of isolates with the indicated MIC (μg/mL) |
MIC50 (μg/mL) |
MIC90 (μg/mL) | Resistance % | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <0.031 | 0.031 | 0.063 | <0.125 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | ||||
| M. hominis (n = 29) | |||||||||||||||||
| Delafloxacin | 6 | 1 | 3 | 12 | 7 | 0.5 | 1 | NA | |||||||||
| Finafloxacin | 5 | 1 | 1 | 4 | 16 | 2 | 8 | 8 | NA | ||||||||
| Moxifloxacin | 5 | 1 | 3 | 1 | 3 | 15 | 1 | 16 | 16 | 24 (82.76%) | |||||||
| Levofloxacin | 2 | 1 | 2 | 3 | 3 | 17 | 1 | 32 | 32 | 27 (93.10%) | |||||||
| U. parvum (n = 52) | |||||||||||||||||
| Delafloxacin | 5 | 1 | 10 | 2 | 4 | 21 | 6 | 3 | 1 | 2 | NA | ||||||
| Finafloxacin | 1 | 7 | 7 | 6 | 20 | 10 | 1 | 8 | 16 | NA | |||||||
| Moxifloxacin | 3 | 6 | 6 | 19 | 8 | 9 | 1 | 4 | 16 | 37 (71.15%) | |||||||
| Levofloxacin | 5 | 6 | 7 | 11 | 12 | 9 | 2 | 8 | 32 | 41 (78.85%) | |||||||
| U. urealyticum (n = 15) | |||||||||||||||||
| Delafloxacin | 1 | 1 | 1 | 7 | 5 | 2 | 4 | NA | |||||||||
| Finafloxacin | 2 | 1 | 10 | 2 | 16 | 32 | NA | ||||||||||
| Moxifloxacin | 2 | 1 | 4 | 7 | 1 | 16 | 16 | 13 (86.67%) | |||||||||
| Levofloxacin | 2 | 4 | 6 | 3 | 32 | >32 | 13 (86.67%) | ||||||||||
For M. hominis, the breakpoints were ≥ 2 μg/mL and ≥ 0.5 μg/mL for levofloxacin and moxifloxacin, respectively. For Ureaplasma spp., the breakpoints were ≥ 4 μg/mL for levofloxacin and moxifloxacin. NA, not applicable (no CLSI breakpoint).
For the 29 M. hominis isolates tested, delafloxacin showed low MICs (MIC50 = 0.5 μg/mL; MIC90 = 1 μg/mL) compared to finafloxacin, moxifloxacin, and levofloxacin, with MICs ranging from < 0.031 μg/mL to 1 μg/mL. However, finafloxacin, another new fluoroquinolone, showed relatively high MIC50 and MIC90 values (MIC50 = 8 μg/mL; MIC90 = 8 μg/mL), with MICs ranging from < 0.031 μg/mL to 16 μg/mL. According to the CLSI guideline, 82.76% (24/29) and 93.1% (27/29) isolates exhibited resistance to moxifloxacin and levofloxacin, respectively, with MIC90 values of 16 μg/mL for moxifloxacin and of 32 μg/mL for levofloxacin.
Of the 67 Ureaplasma spp. tested, delafloxacin also exhibited low MIC50 (U. parvum, 1 μg/mL; U. urealyticum, 2 μg/mL) and MIC90 (U. parvum, 2 μg/mL; U. urealyticum, 4 μg/mL) values, with MICs ranging from < 0.031 μg/mL to 4 μg/mL for U. parvum and from 0.125 μg/mL to 4 μg/mL for U. urealyticum. In contrast, finafloxacin exhibited high values of MIC50 (U. parvum, 8 μg/mL; U. urealyticum, 16 μg/mL) and MIC90 (U. parvum, 16 μg/mL; U. urealyticum, 32 μg/mL) in both U. parvum and U. urealyticum. The resistance rate of moxifloxacin ranged from 71.15% (37/52) in U. parvum to 86.67% (13/15) in U. urealyticum, with elevated MIC50 values (U. parvum, 4 μg/mL; U. urealyticum, 16 μg/mL) and the same MIC90 values (16 μg/mL). Levofloxacin resistance was found in 78.85% (41/52) U. parvum and 86.67% (13/15) U. urealyticum isolates, with elevated MIC50 (U. parvum, 8 μg/mL; U. urealyticum, 32 μg/mL) and MIC90 values (U. parvum, 32 μg/mL; U. urealyticum, > 32 μg/mL).
Molecular mechanism of fluoroquinolone resistance.
The nucleotide and deduced amino acid sequences of the QRDRs of gyrA, gyrB, parC, and parE for all the 29 clinical M. hominis and 67 clinical Ureaplasma spp. isolates were compared with reference strains of M. hominis and Ureaplasma spp., respectively. In general, levofloxacin-resistant strains demonstrated reduced susceptibilities to delafloxacin, finafloxacin, and moxifloxacin compared to those of levofloxacin-susceptible strains of the same species (Tables 2 and 3).
TABLE 2.
The MICs and genetic alterations of M. hominis
| Delafloxacin MIC (μg/mL) |
Finafloxacin MIC (μg/mL) |
Moxifloxacin MIC (μg/mL) |
Levofloxacin MIC (μg/mL) |
Genetic alterationb |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of isolatesa | Range | MIC50 | Range | MIC50 | Range | MIC50 | Range | MIC50 | GyrA | GyrB | ParC | ParE |
| M. hominis (S = 2) | ||||||||||||
| 2 | <0.031 | <0.063 | <0.125 | 0.5 | N | N | N | N | ||||
| M. hominis (R = 27) | ||||||||||||
| 1 | <0.031 | <0.063 | <0.125 | 2 | N | N | N | N | ||||
| 2 | <0.031-1 | <0.063-8 | <0.125-8 | 4-16 | G272T(S91I) | G1249A(V417I) | ||||||
| 1 | 0.5 | 4 | 2 | 8 | A431G(K144R) | |||||||
| 1 | 1 | 8 | 2 | 8 | A431G(K144R) | G1249A(V417I) | ||||||
| 1 | <0.031 | <0.063 | <0.125 | 8 | G1249A(V417I) & G1276A(D426N) | |||||||
| 1 | 0.0625 | 1 | 2 | 4 | C458T(S153L） | T274C(S92P) & A431G(K144R) | ||||||
| 2 | <0.031-0.5 | 0.5-4 | 1-4 | 16−32 | T457G(S153A) | G272T(S91I) | G1249A(V417I) | |||||
| 1 | 0.5 | 8 | 16 | 32 | C458T(S153L) | G272T(S91I) | ||||||
| 1 | 0.5 | 8 | 16 | 32 | C458T(S153L) | G272T(S91I) & A431G(K144R) | ||||||
| 4 | 0.5-1 | 0.5 | 8-16 | 8 | 16-32 | 16 | 32 | 32 | C458T(S153L) | G272T(S91I) | G1249A(V417I) | |
| 10 | 0.25-1 | 0.5 | 4-8 | 8 | 8-16 | 16 | 32->32 | 32 | C458T(S153L) | G272T(S91I) & A431G(K144R) |
G1249A(V417I) | |
| 1 | 1 | 8 | 8 | 16 | C458T(S153L) | G272T(S91I) & A431G(K144R) & G460A(A154T) |
G1249A(V417I) | |||||
| 1 | 0.5 | 4 | 16 | 32 | C458T(S153L) | C1418T(A473V) | G272T(S91I) | |||||
| ATCC 23114 | <0.031 | 0.5 | 0.25 | 0.5 | ||||||||
S, susceptible; R, resistant.
N, no mutation detected. Numbers and letters in parentheses indicate amino acid substitutions that occurred because of DNA point mutations. Novel mutations are highlighted in bold. Mutations unrelated to fluoroquinolone resistance are identified in italic.
TABLE 3.
The MICs and genetic alterations of U. parvum and U. urealyticum
| Delafloxacin MIC (μg/mL) |
Finafloxacin MIC (μg/mL) |
Moxifloxacin MIC (μg/mL) |
Levofloxacin MIC (μg/mL) |
Genetic alterationb |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of isolatesa | Range | MIC50 | Range | MIC50 | Range | MIC50 | Range | MIC50 | GyrA | GyrB | ParC | ParE |
| U. parvum (S = 11) | ||||||||||||
| 4 | 0.125 | 0.125 | 1-2 | 1 | 0.5-1 | 0.5 | 1-2 | 1 | N | N | N | N |
| 1 | 0.125 | 1 | 0.5 | 1 | T250C(S84P) | |||||||
| 1 | <0.031 | 0.5 | 1 | 1 | G310A(D104N) | |||||||
| 1 | 0.25 | 2 | 2 | 2 | T331A(S111T) | |||||||
| 4 | <0.031-0.125 | 0.125 | 1 | 1 | 1-2 | 1 | 1-2 | 2 | G406T(A136S) | |||
| U. parvum (R = 41) | ||||||||||||
| 2 | 1 | 8 | 4-16 | 8-32 | N | N | N | N | ||||
| 5 | <0.031-0.125 | 0.063 | 2-4 | 2 | 2-4 | 4 | 4 | 4 | G1343A(R448K) | |||
| 1 | <0.031 | 2 | 2 | 4 | G406T(A136S) | G1343A(R448K) | ||||||
| 2 | 0.25-1 | 4-16 | 4-16 | 8-32 | G259A(E87K) | |||||||
| 1 | 0.5 | 4 | 8 | 8 | T247G(S83A) | |||||||
| 24 | 0.5-2 | 1 | 4-16 | 8 | 2-16 | 4 | 4-32 | 16 | C248T(S83L) | |||
| 1 | 2 | 8 | 8 | 16 | A1442G(N481S) | C248T(S83L) | ||||||
| 1 | 0.5 | 8 | 8 | 16 | C1384T(P462S) | C248T(S83L) | ||||||
| 1 | 0.5 | 16 | 16 | 32 | A1446C(E482D) | C248T(S83L) | ||||||
| 1 | 4 | 16 | 16 | 32 | C248T(S83L) | G1441A(A481T) | ||||||
| 1 | 4 | 4 | 16 | >32 | A1328C(D443A) | C248T(S83L) | ||||||
| 1 | 4 | 32 | 32 | >32 | C310A(Q104K) | C248T(S83L) | ||||||
| U. urealyticum (S = 2) | ||||||||||||
| 2 | 0.125-0.25 | 2 | 1 | 2 | N | N | N | N | ||||
| U. urealyticum (R = 13) | ||||||||||||
| 1 | 2 | 16 | 8 | 16 | N | N | N | N | ||||
| 1-4 | 2 | 16-32 | 16 | 4-16 | 16 | 16->32 | 32 | C248T(S83L) | ||||
| 1 | 2 | 8 | 16 | 32 | A1456T(N486Y) | C248T(S83L) | ||||||
| 1 | 4 | 32 | 32 | >32 | A299T(Q100L) | C248T(S83L) | ||||||
| ATCC 27618 | <0.031 | 0.5 | 0.5 | 2 | ||||||||
S, susceptible; R, resistant.
N, no mutation detected. Numbers and letters in parentheses indicate amino acid substitutions that occurred because of DNA point mutations. Mutations unrelated to fluoroquinolone resistance are identified in italic.
Of the 29 M. hominis, the mutations of K144R in ParC and V417I in ParE, unrelated to fluoroquinolone resistance, were observed in 24 resistant isolates. Significant differences in MICs of fluoroquinolones were observed in the two isolates that harbored the ParC S91I mutation and the two isolates that harbored double mutations GyrA S153A/ParC S91I. Sixteen isolates, harbored the double mutations GyrA S153L/ParC S91I, displayed increased MIC50 values for delafloxacin (0.5 μg/mL), finafloxacin (8 μg/mL), moxifloxacin (16 μg/mL), and levofloxacin (32 μg/mL). Both the isolate harbored GyrA S153L/ParC S91I/ParC A154T mutations and the isolate harbored GyrA S153L/GyrB A473V/ParC S91I mutations displayed similar MICs with the above 16 isolates.
Among the 11 levofloxacin-sensitive U. parvum isolates, 7 isolates had a single mutation in ParC, including D104N, S111T, or A136S. For the five isolates that harbored a single mutation of R448K in ParE, the delafloxacin exhibited a low MIC50 value of 0.063 μg/mL compared to 2 μg/mL for finafloxacin, 4 μg/mL for moxifloxacin, and 4 μg/mL for levofloxacin. One isolate harboring the double mutations ParC A136S/ParE R448K showed similar MICs to the isolates harboring ParE R448K. For the 24 isolates harboring a single mutation of S83L in ParC, the delafloxacin also showed a low MIC50 value of 1 μg/mL compared to finafloxacin (8 μg/mL), moxifloxacin (4 μg/mL), and levofloxacin (16 μg/mL). For the six isolates harboring double mutations of ParC S83L along with GyrA Q104K, GyrB (N481S, P462S, E482D, or D443A), or ParE A481T, the MIC50 values of the four fluoroquinolones (delafloxacin, 2 μg/mL; finafloxacin, 8 μg/mL; moxifloxacin, 16 μg/mL; levofloxacin, 32 μg/mL) were comparable to the isolates with a single mutation of ParC S83L.
Of the 13 levofloxacin-resistant U. urealyticum, mutations in the QRDRs were identified in 12 isolates. 10 isolates had a single mutation of S83L in ParC. Compared to U. parvum with the same mutation, the MIC50 values of the four fluoroquinolones (delafloxacin, 2 μg/mL; finafloxacin, 16 μg/mL; moxifloxacin, 16 μg/mL; levofloxacin, 32 μg/mL) against U. urealyticum increased 2- to 4-fold. Two isolates carrying ParC S83L and GyrA Q100L or GyrB N486Y showed similar MICs to the ParC S83L harboring isolates.
DISCUSSION
Increasing fluoroquinolones resistance in M. hominis and Ureaplasma spp. has been well-documented and dramatically limits the clinical treatment options. This study compared the in vitro activity of two new fluoroquinolones, delafloxacin and finafloxacin, against M. hominis and Ureaplasma spp.
In the present study, high levels of moxifloxacin and levofloxacin resistance against M. hominis and Ureaplasma spp. were identified, in which 93.1% M. hominis, 78.85% U. parvum, and 86.67% U. urealyticum showed levofloxacin-resistant, and 82.76% M. hominis, 71.15% U. parvum, and 86.67% U. urealyticum were moxifloxacin-resistant. A high level of fluoroquinolone resistance was discovered in M. hominis and Ureaplasma spp. in different parts of China. Zhang et al. showed that 85.7% of M. hominis isolates were levofloxacin-resistant, and 73.8% were moxifloxacin-resistant (14). Ma et al. showed that 72.87% of Ureaplasma spp. were levofloxacin-resistant, and 89.92% were ciprofloxacin-resistant (5). In our previous study, the resistance rate of moxifloxacin was 25.3% for U. parvum and 50% for U. urealyticum, and that of levofloxacin was 75.9% for U. parvum and 71.4% for U. urealyticum, suggesting an increasing trend of moxifloxacin and levofloxacin resistance against Ureaplasma spp. in recent years (15). It was noteworthy that fluoroquinolones resistance levels varied significantly between countries. In France, the levofloxacin and moxifloxacin resistance rates of Ureaplasma spp. were 1.2% and 0.1%, respectively, and those for M. hominis were 2.7% and 1.6%, respectively (16). In the United States, low levofloxacin-resistant rates (1.6% for U. parvum, 0% for U. urealyticum, and 0% for M. hominis) were observed (17). In contrast, a high fluoroquinolone resistance has been identified in Japan (57.15% levofloxacin resistance rate for Ureaplasma spp.) (18). Fluoroquinolone resistance in M. hominis and Ureaplasma spp. has been extremely high in China, perhaps due to the inappropriate fluoroquinolone agents in both clinical settings and the poultry industry (19, 20).
Delafloxacin and finafloxacin are novel synthetic anionic fluoroquinolones with quinolone structure modifications to improve antibacterial effectiveness, pharmacokinetic profile, and toxicity profile and have been approved by the US Food and Drug Administration (FDA). Both delafloxacin and finafloxacin exhibited a broad spectrum of activity against Gram-positive and Gram-negative bacteria (8–13). However, to our knowledge, until now, no studies have been undertaken in China to determine the in vitro activity of delafloxacin and finafloxacin against M. hominis and Ureaplasma spp. using the current CLSI guidelines. In this study, delafloxacin and finafloxacin displayed different antimicrobial susceptibility profiles against M. hominis and Ureaplasma spp. in vitro. Delafloxacin was found to be more effective against M. hominis and Ureaplasma spp. than the other three fluoroquinolones (finafloxacin, moxifloxacin, and levofloxacin). Finafloxacin displayed activity similar to moxifloxacin but superior to levofloxacin against M. hominis and Ureaplasma spp.
The study by Waites and colleagues tested the antimicrobial activity of multiple fluoroquinolones, including delafloxacin, finafloxacin, moxifloxacin, and levofloxacin, and showed low MIC90 values against 10 clinical M. hominis isolates (delafloxacin, 0.016 μg/mL; finafloxacin, 0.063 μg/mL; moxifloxacin, 0.5 μg/mL; levofloxacin, 0.5 μg/mL) and 22 clinical Ureaplasma spp. isolates (delafloxacin, 0.25 μg/mL; finafloxacin, 0.5 μg/mL; moxifloxacin, 2 μg/mL; levofloxacin, 2 μg/mL) collected from the US in 2003 (21). However, relatively higher MIC90 values of fluoroquinolones against M. hominis (delafloxacin, 1 μg/mL; finafloxacin, 8 μg/mL; moxifloxacin, 16 μg/mL; levofloxacin, 32 μg/mL) and Ureaplasma spp. (delafloxacin, 2 - 4 μg/mL; finafloxacin, 16 - 32 μg/mL; moxifloxacin, 16 μg/mL; levofloxacin, 32 - >32 μg/mL) were observed in this study. Considering the high resistance levels to moxifloxacin and levofloxacin against M. hominis and Ureaplasma spp. in China, delafloxacin may provide an alternative for developing an effective regimen against M. hominis and Ureaplasma spp. infections.
Resistance to fluoroquinolones is associated with genetic mutations in the QRDRs in M. hominis and Ureaplasma spp., including GyrA/B and ParC/E, which serve as the target of most fluoroquinolones. Several investigations have identified the mutations in the ofloxacin/ciprofloxacin/levofloxacin/moxifloxacin-resistant mutants, such as ParC S91I and GyrA S153L in M. hominis, and ParC S83L and ParE R448K in Ureaplasma spp. (4, 14, 15, 18, 22, 23). For M. hominis, mutations were identified in QRDRs of the 26 out of 27 levofloxacin-resistant isolates. These mutations contained S153L and S153A in GyrA; A473V in GyrB; S91I, K144R, S92P, and A154T in ParC; and V417I and D426N in ParE. However, a previous study identified ParC K144R and ParE V417I mutations unrelated to fluoroquinolone resistance (4). For Ureaplasma spp., 51 out of 54 levofloxacin-resistant isolates harbored amino acid changes in QRDRs, including Q100L and Q104K in GyrA; N486Y, N481S, P462S, E482D, and D443A in GyrB; E87K, S83A, and S83L in ParC; and R448K and A481T in ParE. Notably, S84P, D104N, S111T, and A136S mutations in ParC were observed in fluoroquinolone susceptible Ureaplasma spp. isolates, suggesting that these mutations were not involved in fluoroquinolone resistance. The MICs of delafloxacin were lower than finafloxacin and the two classical fluoroquinolones against levofloxacin-resistant isolates of M. hominis and Ureaplasma spp., although all the four fluoroquinolones demonstrated reduced activities in comparison to those of levofloxacin-susceptible isolates of the same species. Although delafloxacin may be affected by mutations in QRDRs, the in vitro antibacterial activity of delafloxacin is still significantly higher than other fluoroquinolones, including finafloxacin, moxifloxacin, and levofloxacin.
In conclusion, this study demonstrates the spectrum of activity of delafloxacin and finafloxacin against fluoroquinolone-resistant M. hominis and Ureaplasma spp. This study suggests that delafloxacin could be a promising therapeutic option for the treatment of M. hominis and Ureaplasma spp. infections, including those caused by fluoroquinolone-resistant isolates. More comprehensive and large-scale surveillance studies are warranted to better understand the antimicrobial susceptibility patterns of new fluoroquinolones.
MATERIALS AND METHODS
Bacterial strains and clinical specimens.
During the period of September 2018 to July 2019, 67 Ureaplasma spp. (52 Ureaplasma parvum and 15 Ureaplasma urealyticum) and 29 M. hominis isolates were cultured from clinical patients at Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China. Among these isolates, 70 isolates were recovered from the urogenital tracts of females aged 21–63 years, and 26 isolates were collected from the urogenital tracts of males aged 22–44 years.
Antimicrobial agents and antimicrobial susceptibility testing.
Delafloxacin was obtained from WuXi AppTec (Wuhan, China). Finafloxacin, moxifloxacin, and levofloxacin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antimicrobial agents were dissolved according to the manufacturers’ instructions. Delafloxacin was dissolved in dimethyl sulfoxide (DMSO), and the other three fluoroquinolones were dissolved in deionized water. The MICs of four fluoroquinolones (delafloxacin, finafloxacin, moxifloxacin, and levofloxacin) were determined in duplicate by the microdilution broth method according to the guidelines of the CLSI. The concentration ranges for moxifloxacin and levofloxacin was from 0.125 μg/mL to 32 μg/mL, while those for finafloxacin and delafloxacin were from 0.063 μg/mL to 32 μg/mL and from 0.031 μg/mL to 16 μg/mL, respectively. The breakpoints were interpreted according to CLSI guideline M43-A. M. hominis ATCC 23114 and U. urealyticum ATCC 27618 were used as reference strains.
Identification of Ureaplasma species and genetic mechanisms of fluoroquinolone resistance.
Zero-point five milliliters (0.5 mL) of a broth culture of M. hominis or Ureaplasma spp. strain was used to isolate genomic DNA. To distinguish U. parvum from U. urealyticum, amplification was conducted with Ureaplasma species, as previously described (15). Amplifications of the QRDRs of M. hominis and Ureaplasma spp. isolates were conducted using previously reported primers (22–24). Purified PCR products were sequenced and then mapped to the corresponding sequences of U. parvum ATCC 700970, U. urealyticum ATCC 33699, or M. hominis ATCC 23114.
ACKNOWLEDGMENT
This study was supported by the National Natural Science Foundation of China (82072342 and 82102429).
We have no conflicts of interest to delcare.
Contributor Information
Xinyou Xie, Email: scottxie@zju.edu.cn.
Jun Zhang, Email: jameszhang2000@zju.edu.cn.
Zhi Ruan, Email: r_z@zju.edu.cn.
Cezar M. Khursigara, University of Guelph
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