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. 2019 Mar 27;63(4):e02158-18. doi: 10.1128/AAC.02158-18

Low Prevalence of Gram-Positive Isolates Showing Elevated Lefamulin MIC Results during the SENTRY Surveillance Program for 2015–2016 and Characterization of Resistance Mechanisms

Rodrigo E Mendes a,✉, Susanne Paukner b, Timothy B Doyle a, Steven P Gelone c, Robert K Flamm a, Helio S Sader a
PMCID: PMC6437497  PMID: 30670418

This study investigated the molecular mechanisms possibly associated with non-wild-type MICs for lefamulin among staphylococci and streptococci included in the lefamulin surveillance program from 2015 to 2016. A total of 2,919 Staphylococcus aureus, 276 coagulase-negative staphylococci (CoNS), 3,923 Streptococcus pneumoniae, 389 β-hemolytic, and 178 viridans group streptococci isolates were included in the surveillance studies.

KEYWORDS: BC-3781, pleuromutilins, vga(A), lsa(E)

ABSTRACT

This study investigated the molecular mechanisms possibly associated with non-wild-type MICs for lefamulin among staphylococci and streptococci included in the lefamulin surveillance program from 2015 to 2016. A total of 2,919 Staphylococcus aureus, 276 coagulase-negative staphylococci (CoNS), 3,923 Streptococcus pneumoniae, 389 β-hemolytic, and 178 viridans group streptococci isolates were included in the surveillance studies. Eleven (0.3% of all S. aureus) S. aureus isolates with lefamulin MICs above the staphylococcal epidemiological cutoff (ECOFF) value (>0.25 μg/ml) were selected for this study. Eight (72.7%) S. aureus (lefamulin MIC, 0.5 to 4 μg/ml) isolates carried vga(A or E), one isolate (MIC, 32 μg/ml) carried lsa(E), one isolate (MIC, 16 μg/ml) had an alteration in L4, and one strain (MIC, 0.5 μg/ml) did not carry any of the investigated resistance mechanisms. A total of 14 (5.1% of all CoNS) CoNS isolates had lefamulin MICs (0.5 to >32 μg/ml) above the ECOFF. Similar to S. aureus, 8 (57.1%) CoNS (lefamulin MIC, 1 to 8 μg/ml) isolates carried vga(A or B), while 2 isolates (MIC, 4 to 32 μg/ml) carried cfr. High genetic diversity was observed among staphylococci, although 3 S. aureus isolates belonged to sequence type 398 (ST398). Among the 3 Streptococcus agalactiae and 3 viridans group streptococci (0.1% of all streptococci surveyed) isolates selected for additional characterization, all but 1 isolate carried lsa(E). This study documents a low occurrence of surveillance isolates exhibiting a non-wild-type MIC for lefamulin, and among these isolates, vga and lsa(E) prevailed in staphylococci and streptococci, respectively.

INTRODUCTION

Lefamulin belongs to the pleuromutilin class of antibiotics, and its antibacterial profile covers the most relevant organisms causing community-acquired bacterial pneumonia (CABP), including Gram-positive, fastidious Gram-negative, and atypical respiratory pathogens (1–3). Lefamulin also shows in vitro activity against multidrug-resistant Neisseria gonorrhoeae and Mycoplasma genitalium (4, 5). Thus, in addition to the clinical utility for treating CABP, the characteristic lefamulin antibacterial profile fits treatment for acute bacterial skin and skin structure infections (ABSSSIs) and sexually transmitted diseases (6).

Lefamulin inhibits bacterial protein synthesis by binding the 23S ribosomal subunit at the A and P sites in the peptidyl transferase center (PTC) via 4 hydrogen bonds and other interactions. An “induced-fit” mechanism, which is characteristic for pleuromutilin antibiotics and causes the tight fit of these molecules to the target site, hinders the correct positioning of the tRNA and thereby prohibits peptide bond formation (7, 8).

Mechanisms mediating resistance to pleuromutilins include mutations within the domain V of the 23S rRNA, including methylation of the nucleotide A2503 by the methyl transferase Cfr (9). Mutations in the 23S rRNA at positions 2032, 2055, 2447, 2499, 2504, and 2572 were previously described to confer resistance to tiamulin in Brachyspira spp. (10), while alterations at positions 2055, 2447, 2504, and 2572 were associated with valnemulin resistance in Mycobacterium smegmatis (11). Ribosomal proteins L3 and L4 are not primarily pleuromutilin targets, but mutations within these molecules may alter the PTC structure and affect binding. L3 (rplC) at the amino acid positions 145, 148, 149, 152, 155, 157, 158, and 159 and L4 (rplD) at position 68 were associated with resistance (7, 10, 12–14). Moreover, ATP-binding cassette F (ABC-F) proteins, such as vga(A–E) and lsa(E), initially described as putative efflux pumps can cause pleuromutilin resistance by ribosomal protection (15, 16).

As part of the clinical development, the in vitro activity of lefamulin and comparator agents have been monitored against a global collection of Gram-positive and fastidious Gram-negative organisms causing CABP and ABSSSI through the SENTRY Antimicrobial Surveillance Program. This study evaluated the occurrence of staphylococci and streptococci displaying elevated lefamulin MICs or above the epidemiological cutoff (ECOFF) during the SENTRY Program from 2015 to 2016 and characterized the possible associated resistance mechanisms among non-wild-type surveillance isolates.

RESULTS

Lefamulin had MIC50 and MIC90 results of 0.06 and 0.12 μg/ml, respectively, with the majority (99.6%) of isolates displaying MICs of ≤0.008 to 0.25 μg/ml (Table 1). Eleven Staphylococcus aureus isolates showed lefamulin MICs above the ECOFF value (i.e., >0.25 μg/ml), and these isolates represented 0.3% of all S. aureus included in the 2015 and 2016 lefamulin surveillance programs (Table 1). When lefamulin was tested against coagulase-negative staphylococci (CoNS), a total of 14 isolates (4 species) had lefamulin MICs (0.5 to >32 μg/ml) above the ECOFF value (Tables 1, 2). The lefamulin MIC50 results obtained against streptococci varied depending on species or group of species (Table 1), and 3 Streptococcus agalactiae, 2 Streptococcus lutetiensis, and 1 Streptococcus gallolyticus isolates showed lefamulin MICs outside the wild-type distribution for the respective species and were further investigated.

TABLE 1.

Lefamulin MICs obtained during surveillance programs for 2015 and 2016a

graphic file with name AAC.02158-18-t0001.jpg

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a

Clinical isolates selected for further analysis with respective lefamulin MICs are highlighted.

bNC, not calculated.

TABLE 2.

MICs obtained for lefamulin and comparator agents tested against isolates included in the studya

Collection no. Species MIC (µg/ml) by agent
Erythromycin Clindamycin Q-D Linezolid Lefamulin Retapamulin Chloramphenicol
975498 S. aureus >8 >64 0.5 1 0.5 (0.5) ≤0.06 16
981256 S. aureus 0.12 ≤0.5 0.5 1 0.5 (0.5) 0.5 4
924825 S. aureus >8 ≤0.5 0.5 1 1 (1) 2 8
953474 S. aureus >8 ≤0.5 0.5 0.5 1 (1) 1 4
879822 S. aureus 0.12 ≤0.5 0.5 1 2 (>1) 4 8
913640 S. aureus >8 >64 0.5 1 2 (>1) 2 8
934242 S. aureus 0.12 ≤0.5 0.5 0.5 2 (1) 2 8
950457 S. aureus 0.12 8 1 1 4 (2) >8 4
916083 S. aureus >8 >64 1 0.25 16 (>1) 8 8
976441 S. aureus >8 >64 4 0.5 32 (16) >8 64
972481 S. aureus 4 4 1 1 >32 (>16) >8 4
939671 S. cohnii >8 ≤0.5 1 1 0.5 (2) 1 4
939504 S. epidermidis >8 ≤0.5 ≤0.25 16 0.5 (1) 0.25 16
947675 S. epidermidis >8 16 ≤0.25 0.5 1 (0.5) >8 4
951555 S. epidermidis >8 >64 4 0.5 1 (0.5) 1 4
955639 S. epidermidis 0.12 ≤0.5 0.5 0.5 1 (1) 1 4
956923 S. epidermidis >8 16 ≤0.25 0.25 2 (0.5) >8 2
949426 S. epidermidis ≤0.06 1 ≤0.25 0.5 2 (2) >8 4
938399 S. epidermidis >8 ≤0.5 ≤0.25 0.5 8 (4) 8 2
952506 S. epidermidis >8 1 ≤0.25 0.5 8 (4) 8 4
958510 S. epidermidis ≤0.06 2 ≤0.25 1 8 (2) >8 4
934123 S. epidermidis 0.5 >64 1 128 32 (8) >8 64
939969 S. haemolyticus >8 >64 4 2 4 (4) 4 32
944662 S. sciuri >8 >64 0.5 1 16 (8) 8 32
941213 S. sciuri 0.25 ≤0.5 1 1 32 (>16) >8 4
960742 S. lutetiensis 0.03 ≤0.5 1 1 0.5 (0.5) 0.5 2
982012 S. lutetiensis ≤0.015 1 0.5 1 2 (1) 2 2
971459 S. agalactiae >32 >64 1 1 8 (8) 8 2
935557 S. agalactiae 0.03 4 0.5 1 8 (8) 4 2
935554 S. agalactiae 0.03 4 0.5 0.5 16 (16) 4 2
965031 S. gallolyticus >32 4 1 2 32 (>16) >8 4
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a

Values within parentheses are the initial lefamulin MICs obtained during the surveillance studies. Q-D, quinupristin-dalfopristin.

Most S. aureus (7/11; 63.6%) isolates displaying lefamulin MICs of >0.25 μg/ml harbored vga(A) (lefamulin MIC, 0.5 to 4 μg/ml), while 2 strains carried either vga(E) (lefamulin MIC, >32 μg/ml) or the lsa(E) gene (lefamulin MIC, 32 μg/ml) (Table 3). Very little variability was observed in the S. aureus 23S rRNA nucleotide and ribosomal sequences. Overall, each isolate contained the same polymorphisms in the 23S rRNA (A21G, A1557T, and/or A2234G), while ribosomal proteins had wild-type sequences. The only exception was observed for isolate 916083, which had a V118A and an E147K in L4 (lefamulin MIC, 16 μg/ml). One S. aureus (975498) isolate with a lefamulin MIC of 0.5 μg/ml did not show any known resistance mechanisms associated with pleuromutilins. High genetic diversity was observed among staphylococci, although 3 S. aureus strains belonged to sequence type 398 (ST398).

TABLE 3.

Molecular epidemiology and resistance mechanism results for isolates included in this study

Species Isolate no. Yr MLSTa Country Lefamulin MIC (μg/ml) Resistance determinantsb
 Ribosomal mutationsc
Pleuromutilins Other 23S rRNA L3 L4 L22
S. aureus 975498 2016 5 United States 0.5 erm(A) A21G, A1557T WT WT WT
S. aureus 981256 2016 4335 New Zealand 0.5 vga(A) A21G, A1557T, A2234G WT WT WT
S. aureus 924825 2015 88 Australia 1 vga(A) erm(C) A21G, A1557T, A2234G WT WT WT
S. aureus 953474 2016 398 France 1 vga(A) erm(T) A21G, A1557T, A2234G WT WT WT
S. aureus 879822 2015 1 Slovenia 2 vga(A) A21G, A2234G WT WT WT
S. aureus 913640 2015 1148 United States 2 vga(A) erm(C) A21G, A2234G WT WT WT
S. aureus 934242 2016 1148 United States 2 vga(A) A21G, A2234G WT WT WT
S. aureus 950457 2016 97 United States 4 vga(A) A21G, A2234G WT WT WT
S. aureus 916083 2015 5 Korea 16 erm(A) A21G, A1557T, A2234G WT V118A, E147K WT
S. aureus 976441 2016 398 Brazil 32 lsa(E) erm(T), lnu(B) A21G, A1557T, A2234G WT WT WT
S. aureus 972481 2016 398 Germany >32 vga(E) erm(A) A21G, A1526G, A1557T WT WT WT
S. cohnii 939671 2016 N/A United States 0.5 msr(A) A107G, A124G, T266G, C450T, T623C, A816G, T1261C, T1448A, T1549A D108E, T190A, N193K, Y208F N20S, A128T, A133T, V155I WT
S. epidermidis 939504 2016 2 Italy 0.5 msr(A) G105A, G241T, T669C, T1236C, G2576T WT WT WT
S. epidermidis 947675 2016 57 United States 1 vga(A) mph(C), msr(A) G241T, T669C, T1236C WT WT WT
S. epidermidis 951555 2016 87 Czech Republic 1 vga(A), vga(B) erm(A) C139T, G241T, T669C, T1236C WT WT WT
S. epidermidis 955639 2016 87 Italy 1 vga(A), vga(B) vat(B) C139T, T669C, T1236C, C2809T WT WT WT
S. epidermidis 956923 2016 679 Brazil 2 vga(A) msr(A) T669C, T1236C V188I WT WT
S. epidermidis 949426 2016 255 United States 2 vga(A) T669C, T1236C WT WT WT
S. epidermidis 938399 2016 5 United States 8 vga(A) mph(C), msr(A) C139T, T669C, T1236C WT WT WT
S. epidermidis 952506 2016 20 Argentina 8 vga(A) mph(C), msr(A) C139T, T669C, T1236C, C1638T WT WT WT
S. epidermidis 958510 2016 487 United States 8 vga(A) G241T, T669C, T1236C, C1638T WT WT WT
S. epidermidis 934123 2016 5 United States 32 cfr T669C, T1236C, C2534T H146Q, V154L, A157R G71_R72insG WT
S. haemolyticus 939969 2016 3 Mexico 4 cfr mph(C), msr(A), erm(C) C1486T, A2235G, T2882C WT WT A29T
S. sciuri 944662 2016 N/A Mexico 16 sal(A) erm(C) WT WT WT A112D
S. sciuri 941213 2016 N/A Australia 32 sal(A) WT WT WT WT
S. lutetiensis 960742 2016 N/A Belgium 0.5 lnu(C) T225C, A2360G WT WT WT
S. lutetiensis 982012 2016 N/A Argentina 2 lsa(E) lnu(B), lnu(C) WT WT WT WT
S. agalactiae 971459 2016 19 Korea 8 lsa(E) lnu(B), erm(B) WT WT R1del, R2K WT
S. agalactiae 935557 2016 19 Mexico 8 lsa(E) lnu(B), erm(B) WT WT R1del, R2K WT
S. agalactiae 935554 2016 19 Mexico 16 lsa(E) lnu(B), erm(B) WT WT R1del, R2K WT
S. gallolyticus 965031 2016 N/A Spain 32 lsa(E) lnu(B), erm(B) C696T WT WT WT
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a

MLST, multilocus sequence typing; N/A, not available.

b

MLSB (macrolide-lincosamide-streptogramin B) and pleuromutilin resistance genes screened as available at https://faculty.washington.edu/marilynr/ermwebA.pdf.

c

23S rRNA mutational analysis performed on nucleotide sequences (Escherichia coli numbering). Protein sequences analyzed for annotating L3, L4, and L22.

Both CoNS with a lefamulin MIC of 0.5 μg/ml, 1 Staphylococcus cohnii and 1 Staphylococcus epidermidis isolate, did not contain any known pleuromutilin resistance genes; however, both isolates had multiple alterations in the 23S rRNA or ribosomal proteins. Eight (57.1%) CoNS isolates contained acquired vga variants (lefamulin MIC, 1 to 8 μg/ml) (Table 3). The cfr gene was detected in 1 Staphylococcus haemolyticus (lefamulin MIC, 4 μg/ml) isolate and 1 S. epidermidis (lefamulin MIC, 32 μg/ml) isolate. The latter also showed multiple mutations in 23S rRNA, L3, and L4 (Table 3). Two Staphylococcus sciuri (lefamulin MIC, 16 to 32 μg/ml) isolates carried the intrinsic putative sal(A) gene (Table 3). In general, S. epidermidis isolates showed alterations in the 23S rRNA, such as G241T, T669C, and T1236C, that could be considered polymorphisms.

Among all streptococci surveyed, including 3,923 S. pneumoniae, 3 S. agalactiae, 2 S. lutetiensis, and 1 S. gallolyticus isolates, those with elevated lefamulin MICs (0.5 to 32 μg/ml) were selected for further evaluation (Tables 1 to 3). All but 1 of the selected streptococci carried lsa(E) (lefamulin MIC, 2 to 32 μg/ml) (Table 3). The S. lutetiensis isolate with a lefamulin MIC of 0.5 μg/ml carried lnu(C) and had a T225C and an A2360G in the 23S rRNA, while alterations within the ribosomal proteins evaluated were not detected (Table 3).

DISCUSSION

This 2-year (2015 to 2016) global surveillance program documents a small number of isolates showing a non-wild-type phenotype for lefamulin. Variants of the vga gene (8/11; 72.7%) were observed among most S. aureus isolates with lefamulin MICs above the ECOFF value (>0.25 μg/ml), while 2 isolates carried lsa(E) or L4 mutations (V118A and E147K). Two S. aureus (975498 and 981256) isolates displayed a lefamulin MIC of 0.5 μg/ml, but only the latter carried a vga(A) gene. Isolate 975498 only showed alterations in the 23S rRNA that was also observed in other S. aureus isolates included in the study, which are likely polymorphisms and not associated with pleuromutilin-resistance phenotypes. Furthermore, these locations are not associated with drug binding (7).

Staphylococci exhibiting elevated MICs to pleuromutilins, lincosamides, and streptogramin A (PLSA) usually carry the ATP-binding cassette F (ABC-F) proteins, such as those belonging to Vga, Lsa, or Sal families (15–17). In fact, similar to S. aureus, vga gene variants were also observed among most CoNS (8/14; 57.1%) or in 64.0% (16/25) of all staphylococci selected herein for further investigation. However, studies have demonstrated that alterations in the 23S rRNA and L3 ribosomal protein can also be responsible for decreased susceptibility to pleuromutilins (10–13, 18, 19), but in general, except for some polymorphisms observed in 23S rRNA, the S. aureus isolates included here showed 23S rRNA and ribosomal protein sequences equivalent to the respective reference strain.

Isolate 916083 displaying a lefamulin MIC of 16 μg/ml was the only S. aureus isolate with alterations in L4 (V118A and E147K). This isolate also exhibited elevated MICs for clindamycin, retapamulin, and erythromycin (Table 2). Previous studies linked L4 alterations with decreased susceptibility to tiamulin, chloramphenicol, and oxazolidinones (14, 20, 21). However, these previously reported alterations surrounded position K68, which is relatively close to the PTC and is responsible for stabilizing this region. V118 and E147 at L4 are located far from the PTC, but a hypothesis would be that L4 mutations may perturb the 3-dimensional structure of the 23S rRNA and minimize drug interaction (22). In fact, non-wild-type lefamulin MICs were obtained against 3 S. aureus surveillance isolates included in the SENTRY Program for 2010, and further investigations detected only the presence of L4 alterations in these older isolates. These isolates belonged to ST59 (lefamulin MIC, >16 μg/ml), carried A50G and V118A at L4 or ST398 (lefamulin MIC, 16 μg/ml), and had V118A and V142I at L4 (unpublished data). These data suggest that V118A, common to these S. aureus isolates from 2010, 2015, and 2016, may be associated with a decreased susceptibility to this agent. However, additional studies are needed to truly link this L4 alteration with the MICs presented here.

Two S. sciuri isolates showed lefamulin MICs of 16 to 32 μg/ml and did not carry any acquired resistance genes associated with the pleuromutilin phenotype. However, the sal(A) gene was detected in both isolates, and this gene was previously determined to be ubiquitous in this species and to cause decreased susceptibility to pleuromutilins and other agents (15, 23, 24). In addition, this gene has been detected in several staphylococcal species other than S. sciuri from animal and human origins, indicating that it has been mobilized to other bacterial species (15).

Both CoNS (939504 and 939671) isolates with a lefamulin MIC of 0.5 μg/ml had multiple alterations in the 23S rRNA. A G2576T was noted in isolate 939504, which is a well-known oxazolidinone resistance mechanism (14, 25) and known to affect tiamulin and valnemulin binding (11, 26). The binding effect for lefamulin appears to be less pronounced, likely because lefamulin appears to have more hydrogen bonds formed at the binding site than tiamulin and valnemulin. It also does not interact directly with G2576, although an alteration at this position causes a shift at the backbone of nucleotides from positions 2504 to 2507 (11), and these nucleotides interact with lefamulin (7). Importantly, isolate 939504 displayed a linezolid MIC of 16 μg/ml (Table 2), indicating the presence of G2576T in several 23S rRNA alleles (14); therefore, the lower lefamulin MIC was likely caused by a minimal effect of G2576T on drug binding rather than a low number of mutated ribosomes. As additional evidence, several staphylococci isolates included in past years of the SENTRY Program were characterized because of elevated linezolid MICs (≥8 μg/ml). Those isolates showing only G2576T had lefamulin MICs of 0.12 to 0.5 μg/ml (unpublished data), which are at the right side of the modal MIC (0.06 μg/ml) for S. aureus (Table 1). All 23S rRNA alterations observed in isolate 939671 are located outside the lefamulin binding site (13), and the L3 and L4 alterations detected have not been previously associated with resistance (19).

One S. epidermidis (lefamulin MIC, 32 μg/ml) and 1 S. haemolyticus (lefamulin MIC, 4 μg/ml) isolate carried cfr. This transferable gene confers a resistance phenotype to several classes of drugs (9), and its dissemination could jeopardize the clinical utility of several agents used in humans and animals. These study results corroborate those from large surveillance investigations that documented a low prevalence of cfr among Gram-positive isolates (25, 27, 28). Several studies reported sporadic outbreaks of cfr-carrying staphylococci; however, it was documented that the dissemination of such isolates are usually controlled by a combination of antibiotic stewardship and infection control measures (29–32).

All but 1 of the 6 streptococcal isolates selected for this study carried lsa(E). This gene has been reported among several Gram-positive isolates recovered from human and animal specimens (15, 24, 33). lsa(E) is usually part of a gene island that includes several resistance genes, including lnu(B) upstream (24, 34), which confers resistance to lincosamides. Among selected streptococcal isolates, pleuromutilin resistance mechanisms were not detected in S. lutetiensis 960742, except for 2 alterations in 23S rRNA (T225C and A2360G) located outside the lefamulin binding site (13). Isolate 960742 displayed a lefamulin MIC of 0.5 μg/ml, which is 32-fold higher than the modal MIC (0.015 μg/ml) shown for this species (Tables 1, 2).

In summary, this study showed a low prevalence of isolates exhibiting a non-wild-type MIC for lefamulin among Gram-positive isolates included in a 2-year global surveillance program. The non-wild-type phenotypes observed here could be generally explained by the presence of vga in staphylococci and lsa(E) in streptococci, which are more often detected among isolates collected from animals (15, 33–37). The association of acquired genes detected here with isolates from animal origins is further evidenced by the presence of 3 S. aureus isolates belonging to ST398, a lineage commonly responsible for infections in animals (16, 36–39). This study benchmarks the lefamulin activity against a global contemporary collection of Gram-positive surveillance isolates, as well as the rare instance of resistance genes associated with decreased susceptibility before clinical approval and use of this unique agent of the pleuromutilin class. Although the prevalence of surveillance isolates exhibiting non-wild-type MICs for lefamulin are rare, continued surveillance to monitor transferable genes and changes in MIC over time will provide valuable information for this new class of antibacterial agents for humans.

MATERIALS AND METHODS

Clinical isolates.

A total of 3,195 staphylococci and 4,489 streptococci isolates were included as part of the lefamulin surveillance program from 2015 to 2016. Based on the MIC distributions shown in Table 1, ECOFF values (≤0.25 μg/ml for S. aureus and CoNS) were calculated to define the lefamulin wild-type population of S. aureus and CoNS that included 99.9% of isolates within each group (40). Thus, staphylococcal isolates exhibiting lefamulin MICs of >0.25 μg/ml were selected for further molecular characterization (Table 1). Streptococci were selected based on species, and those isolates displaying elevated lefamulin MICs within a given species were selected for further molecular characterization (Table 1). Bacterial isolate identification was confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (Bruker Daltonics, Bremen, Germany) and genome sequencing.

Antimicrobial susceptibility testing.

Isolates were tested for susceptibility by broth microdilution methods, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (41). Frozen-form broth microdilution 96-well plates were manufactured by JMI Laboratories and contained cation-adjusted Mueller-Hinton broth (2.5% to 5% lysed horse blood added for testing streptococci). Isolates that met the inclusion criteria were retested for susceptibility in frozen-form panels containing extended ranges for lefamulin among other agents (Table 2). Bacterial inoculum density was monitored by colony counts to ensure an adequate number of cells for each testing event. MICs were validated by concurrently testing CLSI-recommended quality-control strains (42).

Characterization of resistance mechanisms by next-generation genome sequencing and analysis.

Selected isolates had total genomic DNA extracted with the fully automated Thermo Scientific KingFisher Flex magnetic particle processor (Cleveland, OH, USA), which was used as input material for library construction. DNA libraries were prepared using the Nextera library construction protocol (Illumina, San Diego, CA, USA) following the manufacturer’s instructions and were sequenced on a MiSeq sequencer (JMI Laboratories, North Liberty, IA, USA). FASTQ format sequencing files for each sample set were assembled independently using the de novo assembler SPAdes 3.9.0 (43), and an in-house-designed software program was applied to the assembled sequences to align against known macrolide-lincosamide-streptogramin B (MLSB) and pleuromutilin resistance genes, including tva(A) (44–46).

Sequences of 23S rRNA (PTC), rplC (L3), rplD (L4), and rplV (L22) were extracted from the assembled sequences and evaluated against corresponding sequences of susceptible wild-type reference strains. The analysis of 23S rRNA was performed based on nucleotide sequences (Escherichia coli numbering), while those from rRNA proteins were based on amino acid sequences. All intrinsic 23S rRNA target genes or ribosomal protein amino acid sequences were considered wild type if 100% identity was observed with the respective reference sequences. Nucleotide and amino acid differences were annotated when an identity of <100% was observed. Reference sequences were extracted from the following strains: S. aureus (NCTC 8325), S. epidermidis (ATCC 12228), S. cohnii (ATCC 29974), S. haemolyticus (JCSC1435), S. sciuri (ATCC 29062), S. lutetiensis (NCTC 13774), S. agalactiae (NEM316), and S. gallolyticus (ATCC 43143).

Multilocus sequence typing.

Multilocus sequence typing (MLST) was performed by extracting previously defined sets of 7 housekeeping gene fragments (approximately 500 bp) from each assembled sequence. Each fragment was compared with known allelic variants for each locus (housekeeping gene) on the MLST website (PubMLST, https://pubmlst.org). An allele sharing 100% genetic identity with a known variant received a numeric designation, and a 7-number sequence (1 for each housekeeping gene) formed an allelic profile, defined as STs.

Data availability.

This is an original work and the data set repository and published article in which the data set and/or code was originally described and have not been published previously. Upon request, and subject to certain criteria, conditions and exceptions, JMI Laboratories and Nabriva Therapeutics will provide access to the code and databases utilized here. This information may be requested 24 months after study completion and will be made available to researchers whose proposals meet the research criteria and other conditions and for which an exception does not apply, via a secure portal. To gain access, requestors must enter into an information access agreement with JMI Laboratories and/or Nabriva Therapeutics.

ACKNOWLEDGMENTS

The surveillance study and experiments performed in this study were supported by Nabriva Therapeutics.

The authors express appreciation to the following JMI employees for technical support or manuscript assistance: L. Flanigan, J. Oberholser, and C. Smith. Editorial support was provided by Lycely del C. Sepulveda-Torres at C4 MedSolutions, LLC (Yardley, PA), a CHC Group company, and funded by Nabriva Therapeutics.

JMI Laboratories was contracted to perform services in 2017 for Achaogen, Allecra Therapeutics, Allergan, Amplyx Pharmaceuticals, Antabio, API, Astellas Pharma, AstraZeneca, Athelas, Basilea Pharmaceutica, Bayer AG, Becton, Dickinson and Co., Boston Pharmaceuticals, CEM-102 Pharma, Cempra, Cidara Therapeutics, Inc., CorMedix, CSA Biotech, Cutanea Life Sciences, Inc., Entasis Therapeutics, Inc., Geom Therapeutics, Inc., GSK, Iterum Pharma, Medpace, Melinta Therapeutics, Inc., Merck & Co., Inc., MicuRx Pharmaceuticals, Inc., N8 Medical, Inc., Nabriva Therapeutics, Inc., NAEJA-RGM, Novartis, Paratek Pharmaceuticals, Inc., Pfizer, Polyphor, Ra Pharma, Rempex, Riptide Bioscience, Inc., Roche, Scynexis, Shionogi, Sinsa Labs, Inc., Skyline Antiinfectives, Sonoran Biosciences, Spero Therapeutics, Symbiotica, Synlogic, Synthes Biomaterials, TenNor Therapeutics, Tetraphase, The Medicines Company, Theravance Biopharma, VenatoRx Pharmaceuticals, Inc., Wockhardt, Yukon Pharma, Zai Laboratory, and Zavante Therapeutics, Inc. There are no speakers bureaus or stock options to declare for R.E.M., T.B.D., R.K.F. or H.S.S. S.P. and S.P.G. are employees of and hold stock in Nabriva Therapeutics plc.

Footnotes

For a companion article on this topic, see https://doi.org/10.1128/AAC.02161-18.

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