ABSTRACT
Quinolone resistance is increasing in Neisseria meningitidis, with its prevalence in China being high (>70%), but its origin remains unknown. The aim of this study was to investigate the donors of mutation-harboring gyrA alleles in N. meningitidis. A total of 198 N. meningitidis isolates and 293 commensal Neisseria isolates were collected between 2005 and 2018 in Shanghai, China. The MICs of ciprofloxacin were determined using the agar dilution method. The resistance-associated genes gyrA and parC were sequenced for all isolates, while a few isolates were sequenced on the Illumina platform. The prevalences of quinolone resistance in the N. meningitidis and commensal Neisseria isolates were 67.7% (134/198) and 99.3% (291/293), respectively. All 134 quinolone-resistant N. meningitidis isolates possessed mutations in T91 (n = 123) and/or D95 (n = 12) of GyrA, with 7 isolates also harboring ParC mutations and exhibiting higher MICs. Phylogenetic analysis of the gyrA sequence identified six clusters. Among the 71 mutation-harboring gyrA alleles found in 221 N. meningitidis isolates and genomes (n = 221), 12 alleles (n = 103, 46.6%) were included in the N. meningitidis cluster, while 20 alleles (n = 56) were included in the N. lactamica cluster, 27 alleles (n = 49) were included in the N. cinerea cluster, and 9 alleles (n = 10) were included in the N. subflava cluster. Genomic analyses identified the exact N. lactamica donors of seven mutation-harboring gyrA alleles (gyrA92, gyrA97, gyrA98, gyrA114, gyrA116, gyrA151, and gyrA230) and the N. subflava donor isolate of gyrA171, with the sizes of the recombinant fragments ranging from 634 to 7,499 bp. Transformation of gyrA fragments from these donor strains into a meningococcal isolate increased its ciprofloxacin MIC from 0.004 μg/ml to 0.125 or 0.19 μg/ml and to 0.5 μg/ml with further transformation of an additional ParC mutation. Over half of the quinolone-resistant N. meningitidis isolates acquired resistance by horizontal gene transfer from three commensal Neisseria species. Quinolone resistance in N. meningitidis increases in a stepwise manner.
INTRODUCTION
Neisseria meningitidis is a major causative agent of life-threatening septicemia and meningitis. Globally, N. meningitidis is responsible for over 1.2 million cases of invasive meningococcal disease (IMD) every year, with a case-fatality rate of approximately 11% (1). N. meningitidis strains can be divided into 12 serogroups on the basis of their capsular polysaccharides, among which serogroups A, B, C, W, X, and Y are responsible for the majority of IMD cases (2). The prevalence of the various serogroups changes with geographic location and time. Variations within the same serogroup can be further evaluated by multilocus sequence typing (MLST), which establishes sequence types (STs) based on the sequences of the alleles for seven housekeeping genes and clusters close STs into the same clonal complex (CC) (3).
Ciprofloxacin, rifampin, and ceftriaxone are recommended for the chemoprophylaxis of close contacts of IMD cases worldwide to effectively and quickly control the spread of IMD (4). However, reports of ciprofloxacin resistance in meningococcal isolates are increasing. This resistance has already been observed in Europe (Greece, 1992) (5), Oceania (Australia, 1998) (6), South America (Argentina, 2002) (7), Asia (India, 2005) (8), North America (the United States, 2007), and Africa (South Africa, 2009) (9). During the last 3 years, the emergence of ciprofloxacin-resistant N. meningitidis has been reported in several countries (10–12). Except for an outbreak that occurred in India (8), ciprofloxacin resistance has been sporadic in most countries (frequency, <10%) (13, 14), but it is highly prevalent in China (>70%) (15, 16), where ciprofloxacin was recommended as a prophylactic agent for IMD in 2005 (16). The MICs for ciprofloxacin-resistant N. meningitidis isolates were reported to range from 0.06 to 0.25 μg/ml, with mutations in the quinolone resistance-determining region (QRDR) of the gyrase-encoding gene gyrA being responsible for the majority of this resistance (5, 9, 11, 13–15, 17). A study in the United States reported that a gyrA mutation in four ciprofloxacin-resistant meningococci was introduced from a commensal Neisseria donor through horizontal gene transfer (14). In our previous study, among 16 mutation-harboring gyrA alleles from N. meningitidis isolates in Shanghai, China, only 2 ciprofloxacin resistance-conferring alleles were shown to have occurred via point mutation, while the other 14 ciprofloxacin resistance-conferring alleles appeared to have resulted from horizontal gene transfer (15). However, the exact donors of these alleles remain unknown.
It is convenient and helpful to analyze whole-genome sequence (WGS) data on the platform of the Neisseria Bacterial Isolate Genome Sequence Database (BIGSdb) for the purpose of surveillance and genetic characterization of antimicrobial resistance in Neisseria species (18). The database platform automatically performs locus tagging and allele assignment, and the corresponding sequence can easily be extracted and is highly useful for searching for the donor of ciprofloxacin resistance among all the genomes deposited in this database.
To identify the donors of mutation-harboring gyrA alleles in N. meningitidis isolates, we began to collect commensal Neisseria isolates in Shanghai in 2013, including isolates of the species N. lactamica, N. polysaccharea, N. subflava, N. cinerea, N. mucosa, and N. oralis. In this study, phenotypic and genotypic characterization of the quinolone resistance of N. meningitidis and commensal Neisseria isolates was performed; moreover, genomic evidence and data from transformation experiments provide support for the hypothesis that meningococcal quinolone resistance mainly originates from several commensal Neisseria species.
RESULTS
Susceptibility of Neisseria isolates to ciprofloxacin.The MICs of ciprofloxacin for the 198 N. meningitidis isolates ranged from ≤0.015 to 1 μg/ml, with the MIC50 and MIC90 values being 0.125 and 0.25 μg/ml, respectively. For the 293 commensal Neisseria isolates, the MICs ranged from ≤0.015 to 16 μg/ml, with the MIC50 and MIC90 values being 0.25 and 0.5 μg/ml, respectively. The prevalences of quinolone resistance among the N. meningitidis and commensal isolates Neisseria were 67.7% (134/198) and 99.3% (291/293), respectively.
Mutations in QRDRs of quinolone resistance-associated genes.Among the N. meningitidis isolates, all 134 quinolone-resistant isolates possessed mutations in T91 (n = 123) and/or D95 (n = 12) of GyrA, and 7 isolates also harbored mutations in D86 (n = 2), S87 (n = 4), or E91 (n = 1) of ParC. Of the seven isolates (3.5%, 7/198) harboring mutations in ParC, two isolates harbored D86N mutations, one had an S87I mutation, three had S87R mutations, and one had an E91G mutation, with each isolate also harboring a T91I mutation in GyrA (Table 1). These seven isolates with GyrA and ParC mutations belonged to ST-5542 (not assigned to any clonal complex, singleton, n = 3), ST-3200 (CC4821, n = 1), ST-7962 (singleton, n = 1), ST-12869 (singleton, n = 1), and ST-13501 (singleton, n = 1). Almost none of the 64 quinolone-susceptible isolates possessed resistance-associated mutations in the QRDR sequences of GyrA or ParC, with the exception being one serogroup B isolate (Nm403) that harbored a T91A mutation in GyrA but that lacked ciprofloxacin resistance (MIC ≤ 0.015 μg/ml). Nm403 (PubMLST identifier [ID] 34465) was isolated from an 87-year-old female carrier in 2014 and was assigned to ST-11046 (singleton).
Correlation of ciprofloxacin MICs with QRDR mutations in N. meningitidis
Of the 293 commensal Neisseria isolates, except for the 2 quinolone-susceptible isolates, the remaining 291 quinolone-resistant isolates possessed mutations in T91 (n = 271) and/or D95 (n = 38) of GyrA, while 29 isolates (9.9%) also harbored mutations in S87 (n = 19) and/or S88 (n = 11) of ParC (1 isolate harbored two ParC mutations [S87R and S88P] and its parC allele was assigned to SH062) (see Table S3 in the supplemental material).
Correlation of ciprofloxacin MICs with QRDR mutations in N. meningitidis.Meningococci harboring no resistance-associated mutations in GyrA or ParC showed MICs of ciprofloxacin that were lower than 0.015 μg/ml. Isolates harboring single GyrA mutation in D95 showed a ciprofloxacin MIC of 0.06 μg/ml, while those with mutations in T91 (T91I and T91F) showed MICs ranging from 0.06 to 0.5 μg/ml. When isolates possessed one mutation in GyrA combined with another mutation in ParC, the MICs ranged from 0.5 to 1 μg/ml (Table 1).
Phylogenetic analysis of gyrA alleles.The QRDR sequences of the gyrA genes from the 198 N. meningitidis isolates were assigned to 60 different alleles. Fifty-four gyrA alleles were identified from the 134 quinolone-resistant N. meningitidis isolates, with the allele gyrA71 being predominant (25.4%, 34/134), followed by gyrA13 (7.7%, 10/134), with both of these harboring a T91I mutation. The gyrA gene sequences from the 293 commensal Neisseria isolates were assigned to 76 different alleles. Among the 75 alleles found among the 291 quinolone-resistant commensal Neisseria isolates, the gyrA242 allele predominated (14.4%, 42/291), followed by gyrA238 (14.1%, 41/291), and both of these possessed a T91I mutation.
The Neisseria PubMLST database contained 17,096 Neisseria genomes representing 150 gyrA alleles, and of the 12,573 N. meningitidis genomes, only 87 (0.7%) possessed resistance-associated mutations in GyrA (T91 and D95). These gyrA alleles and the alleles identified in this study were used for phylogenetic analysis, which resulted in the identification of six clusters, corresponding to N. meningitidis, N. lactamica, N. gonorrhoeae, N. cinerea, N. mucosa, and N. subflava (Fig. 1). Among the N. meningitidis isolates and genomes, a total of 71 gyrA alleles harbored resistance-associated mutations (represented by 221 isolates and genomes; n = 221) (Table S4). Twelve of these alleles (n = 103 isolates and genomes, 46.6%) were included in the N. meningitidis cluster, 20 alleles (n = 56) were present in the N. lactamica cluster, 27 alleles (n = 49) were present in the N. cinerea cluster, 9 alleles (n = 10) were present in the N. subflava cluster, and 3 alleles (n = 3) were outside all the clusters (Fig. 1).
Phylogenetic analysis of the gyrA quinolone resistance-determining region of the Neisseria isolates and genomes. Phylogenetic analysis of the nucleotide sequences of 263 gyrA alleles (nucleotides 115 to 639) from N. meningitidis (n = 12,771), N. gonorrhoeae (n = 4,118), N. lactamica (n = 562), N. subflava (n = 30), N. cinerea (n = 21), N. mucosa (n = 22), and other commensal Neisseria (n = 63) isolates and genomes collected in this study and from the Neisseria PubMLST database was conducted with the MEGA (version 5) program using the unweighted pair group method with arithmetic mean averages (UPGMA). Clusters were identified if bootstrap values were >70% in the bootstrap test with 1,000 replicates.
Determination of breakage point of recombination events in gyrA.Among the isolates in the N. lactamica cluster, seven mutation-harboring gyrA alleles (gyrA92, gyrA97, gyrA98, gyrA114, gyrA116, gyrA151, and gyrA230) were shared by N. meningitidis and N. lactamica isolates or genomes, suggesting that N. lactamica was likely their donor. Twenty Neisseria genomes harboring these gyrA alleles were used to identify the exact donors (Table S5). First, the donor of the gyrA92 allele was investigated. The allele was shared by the genomes of N. meningitidis CR24 (a strain isolated from the cerebrospinal fluid of a 6-year-old boy in Croatia in 2009) and N. lactamica isolate Nei343 (isolated from a throat swab specimen from a 3-year-old boy in Shanghai in 2016). The nucleotide sequences, including those of the gyrA gene and its flanking sequences, were extracted from the genomes of CR24, Nei343, and three meningococcal reference isolates (isolates 053442, MC58, and Z2491). The genome of N. meningitidis CR24 was found to share a 7.499-kb region exhibiting 100% nucleotide sequence identity with the genome of N. lactamica Nei343, including a 2.459-kb region of the gyrA gene and a 5.04-kb fragment of its upstream DNA. The upstream breakage point was observed to correspond to position 1130 of locus NEIS1312, while the downstream breakage point was located within the gyrA gene at position 2459 (Fig. 2A and B). In contrast to the high sequence similarity of this 7.499-kb region, the sequences flanking this region showed a significant divergence between CR24 and Nei343, while a higher similarity was observed among the meningococcal genomes of CR24 and the three reference genomes (Fig. 2C). N. lactamica isolate Nei343 was identified to be the donor of the gyrA allele of the genome of N. meningitidis CR24, with a recombinant region being observed across the loci NEIS1312, NEIS1314, NEIS1315, NEIS2531, NEIS1317, NEIS1318, NEIS2445, NEIS1319, and gyrA. Using the same strategy, the donor of each of these seven meningococcal gyrA alleles was identified and included recombinant fragments of different sizes ranging from 835 to 7,499 bp. The sequences of all the recombinant fragments shared a high level of similarity with those of their potential donors (≤1 nucleotide difference), whereas they showed a high level of variation (63 to 517 nucleotide differences) when their sequences were compared to those of the three meningococcal reference genomes (Table 2).
Recombination breakage points within gyrA92-harboring N. meningitidis isolate CR24. (A) Sequence alignment of the upstream sequences of the gyrA gene showing the recombination breakage point at position 5040 upstream of the gyrA gene of serogroup C strain 053442. (B) Sequence alignment of gyrA genes showing the recombination breakage point within the gene at position 2459 relative to the sequence of strain 053442. (C) Recombination breakage points detected by at least one of seven methods included in RDP (version 4.97). The topmost horizontal bar represents the CR24 sequence; the blue and red arrows correspond to the locations of the breakage points identified in panels A and B.
Breakage points of recombination events in gyrA alleles shared by N. meningitidis and commensal Neisseria species
Among the isolates in the N. subflava cluster, one allele (gyrA171) was shared by the N. meningitidis and N. subflava isolates, indicating that N. subflava was the donor. Three Neisseria genomes with the gyrA171 allele were used to identify the exact donor. A 634-bp recombinant fragment whose sequence was identical to that of its potential donor and different from the sequences of the three meningococcal reference genomes by at least 130 nucleotides was identified (Table 2).
Phylogenetic analysis of parC alleles.Among the N. meningitidis isolates collected in this study, the QRDR sequences of the parC gene were assigned to 35 different alleles (SH071 to SH105), 5 of which possessed resistance-associated mutations in ParC (2 alleles with D86N, 1 with S87I, 1 with S87R, and 1 with E91G) and were found in seven isolates. Among the 293 commensal Neisseria isolates, 70 parC QRDR alleles (SH001 to SH070) were identified. These included 19 alleles harboring resistance-associated mutations (8 alleles with S87I, 4 with S87R, 6 with S88P, and 1 with double mutations of S87R and S88P) that were found in 29 isolates.
Among the 17,922 Neisseria genomes representing 892 alleles based on the full length of the parC gene in the Neisseria PubMLST database, 568 alleles were found in 13,130 N. meningitidis genomes, and only 2 alleles possessed resistance-associated mutations in ParC (D86N or S87N). These two alleles (parC74 and parC191) were present in 11 genomes (0.008%). Among the genomes deposited in the Neisseria PubMLST database, the first N. meningitidis genome harboring a mutation in ParC was that of a serogroup B isolate, M05 240897 (PubMLST ID 20945), which was isolated from a throat swab specimen from a patient in the United Kingdom in 2005. Among the meningococcal isolates collected in this study, the first isolate harboring a mutation in ParC was also a serogroup B isolate, Nm376 (PubMLST ID 52365), which was isolated from a throat swab specimen from an 83-year-old male carrier in 2013 in Shanghai. The 402-bp parC fragments from these genomes were used in a phylogenetic analysis, the results of which yielded clusters that were not clear, except for the N. gonorrhoeae cluster (Fig. S1). Seven meningococcal parC alleles harbored mutations in ParC and were dispersed.
Genetic transformation of gyrA and parC from commensal Neisseria strains.N. meningitidis strain Nm040, expressing GyrA and ParC without mutations, was transformed with the chromosomal DNA of eight commensal Neisseria isolates, each of which was considered to be one potential donor of the eight meningococcal gyrA alleles based on sequence analysis (Table 3). One donor isolate (Nei139) also possessed a ParC mutation (S87I). After the first round of transformation, eight transformants (gyrA transformants) each showed a gyrA allele different from that of Nm040 and acquired the T91I mutation from the corresponding donor DNA, leading to increases in the ciprofloxacin MIC from 0.004 μg/ml to 0.125 or 0.19 μg/ml. In a comparison of the genome sequences of the corresponding donor and transformant, the length of the recombinant fragment carrying the partial gyrA gene ranged from 608 to 8,188 bp (Table 3), and no recombination involved the parC, gyrB, parE, or mtrR gene. In the second round of transformation, the recipient isolate Nm040_Nei139_T3 acquired an additional mutation (ParC S87I) from the donor strain Nei139 and became transformant Nm040_Nei139_T7, for which the ciprofloxacin MIC elevated from 0.19 μg/ml to 0.5 μg/ml. In addition to the fragment of 8,188 bp carrying the partial gyrA sequence that was acquired in the first round of transformation, another 2,808-bp fragment carrying a partial parC gene was found to be transferred from the same donor DNA, and no recombination occurred in the gyrB, parE, or mtrR gene (Table 3).
Characteristics of strains in the genetic transformation from commensal Neisseria bacteria into N. meningitidis isolates
To investigate the possibility that Nm040 can acquire a single ParC QRDR mutation via natural transformation to become resistant to quinolones, a mixture of parC QRDR allele fragments (SH087, SH095, SH099, SH100, and SH103) from five quinolone-resistant meningococcal isolates (nucleotides 1 to 822, 100 ng each) that harbored ParC mutation D86N, S87I, S87R, or E91G served as the donor DNA. Despite the use of several experiments, no donor DNA could be transformed into Nm040; in contrast, the donor DNA fragments were transformed into three gyrA transformants (Nm040_Nei018_T3, Nm040_Nei082_T3, and Nm040_Nei110_T3) at the first attempt, and for each isolate, the ciprofloxacin MICs were elevated from 0.125 or 0.19 μg/ml to 0.5 μg/ml.
DISCUSSION
In this study, we present the findings of a detailed investigation of the origin of quinolone resistance in N. meningitidis by sequence analysis and a genetic transformation test. Our data suggest that over half of the quinolone-resistant meningococci acquired resistance by horizontal gene transfer from three commensal Neisseria species, N. lactamica, N. cinerea, and N. subflava. Based on genomic evidence, the exact donor isolates of eight mutation-harboring gyrA alleles were identified. These included seven N. lactamica isolates and one N. subflava isolate. It was interesting to observe that an N. lactamica isolate in Shanghai provided a nearly 7.5-kb fragment to an N. meningitidis isolate in Croatia (Fig. 2). The recombinant fragments involved in the transfer of the gyrA sequence were shown to be diverse in size, ranging from hundreds to thousands of base pairs.
Under quinolone selective pressure, we identified that N. meningitidis is able to acquire fragments of up to 8 kb from commensal Neisseria isolates by natural transformation to obtain quinolone resistance. Our findings add more evidence to the theory that commensal Neisseria species have provided a large gene pool that allows N. meningitidis to acquire antimicrobial resistance, including resistance against sulfonamides, β-lactams, and quinolones (14, 19, 20). Due to frequent and extensive genetic exchange via natural transformation, Neisseria species possess an extraordinary ability to rapidly spread new traits, including antimicrobial resistance markers. Neisseria species take up only DNA containing the genus-specific DNA uptake sequence (DUS); therefore, natural transformation almost exclusively occurs using DNA from the same genus (21). Previous studies reported that penA alleles conferring penicillin resistance in N. gonorrhoeae and N. meningitidis resulted from horizontal gene transfer from commensal Neisseria species (20, 22). Unlike the mosaic structure of mutation-harboring penA alleles of penicillin-resistant N. meningitidis isolates, in which a 402-bp fragment of a single penA allele might have originated from four different Neisseria species (20), the 525-bp QRDR fragment of each recombinant gyrA allele originated from a single donor. A similar inference was made in another study from the United States, which identified that an N. lactamica donor provided a 1,265-bp fragment of the gyrA gene to N. meningitidis, based on sequence analysis (14). Besides the genomic evidence, a more important value of the present study is the provision of data from a transformation experiment to prove this inference.
The results of this study show that the prevalence of quinolone resistance in N. meningitidis has been sustained at high levels in China since 2005 and has been observed in almost all commensal Neisseria isolates. In contrast, quinolone-resistant N. meningitidis and commensal Neisseria isolates have been reported only sporadically in most other countries (9–11, 14, 23–26). In the United Kingdom in 2019, three nongroupable ciprofloxacin-resistant meningococci were isolated from patients connected with recent travel to Mecca, where over 2.2 million pilgrims from over 180 countries attended the 2019 Hajj pilgrimage (27). This result highlights the potential for the global spread of ciprofloxacin resistance, which will jeopardize the use of ciprofloxacin for meningococcal chemoprophylaxis. Since the 1980s, fluoroquinolones, such as norfloxacin and ofloxacin, have been widely used in hospitals in China and have become the third most consumed antibiotics since 2010 (28). Thus, the high prevalence of quinolone resistance in Neisseria species can be attributed to antibiotic selective pressure. Interestingly, the frequency of ciprofloxacin resistance observed among commensal Neisseria isolates (99.3%) was much higher than that observed among N. meningitidis isolates (67.7%), allowing N. meningitidis to acquire sequences encoding resistance from commensal Neisseria bacteria, which cocolonize the same nasopharyngeal niche as N. meningitidis (29).
The GyrA mutation T91I was highly prevalent in the ciprofloxacin-resistant isolates. Among the N. meningitidis isolates, over 90% of the ciprofloxacin-resistant isolates harbored T91I, while among the commensal Neisseria isolates, over 85% of the ciprofloxacin-resistant isolates harbored this mutation. For position 95D, the D95N mutation is more common in N. meningitidis (8.2%) than in commensal Neisseria bacteria, whereas the D95Y mutation is more common in commensal Neisseria bacteria (8.9%). Based on the nucleotide sequences observed, the gyrA gene was more divergent in the ciprofloxacin-resistant N. meningitidis isolates (54 alleles in 134 isolates [0.40]) than in the commensal Neisseria isolates (75 alleles in 291 isolates [0.26]). This result can be attributed to the various origins of the resistance-harboring gyrA alleles in meningococci, including point mutations and horizontal gene transfer from several different species of commensal Neisseria bacteria. Although biological costs may be incurred (13), which was also suggested in the reduction in the rate of susceptibility to penicillin G in meningococci (30), mutations in GyrA facilitate the spread of N. meningitidis isolates under the selective pressure of quinolones.
In previous studies, almost all the ciprofloxacin-resistant meningococci harbored mutations only in GyrA and showed ciprofloxacin MICs of no more than 0.25 μg/ml (8, 13, 14). In contrast, in the present study, we identified seven isolates that harbored both GyrA and ParC mutations and that showed correspondingly higher MICs that ranged from 0.5 to 1 μg/ml. Although none of the meningococcal isolates, including the transformants tested in the present study, with only a mutation(s) in ParC showed quinolone resistance, which might be related to the fitness cost, the correlation between ciprofloxacin MIC values and QRDR mutations suggests that ParC mutations contribute to meningococcal quinolone resistance and that resistance in N. meningitidis increases in a stepwise manner: a single amino acid mutation in GyrA confers low-level resistance, and a second mutation in ParC increases the ciprofloxacin MIC, which was observed in the genetic transformation test performed in this study. This phenomenon was also observed by Shultz et al., based on in vitro-derived quinolone-resistant mutants (31). In their study, quinolone resistance in N. meningitidis was shown to increase to higher MICs (from 3 to 16 μg/ml) with the addition of a third mutation in GyrA (31). These results suggest that the development of quinolone resistance in N. meningitidis may be similar to that observed in N. gonorrhoeae, in which quinolone resistance is common and the choice of antibiotics that may be used for treatment is quickly narrowing (32). Although the role of ParC mutations may need further investigation, a database of parC QRDR sequences is needed for global surveillance of the potential appearance of novel ParC-related quinolone resistance mutations in N. meningitidis isolates.
In summary, the results of this study highlight the important role that horizontal gene transfer may play in the emergence of quinolone resistance in N. meningitidis and provide evidence that antibiotic selective pressure is an important factor driving the development of quinolone resistance. The IMD surveillance results showed that antimicrobial susceptibility testing and sequence analysis of resistance-associated genes are necessary to monitor the appearance of novel resistance capabilities and higher MIC values. This report also illustrates how to use WGS data to identify the donors of antimicrobial resistance.
MATERIALS AND METHODS
Isolate collection.A total of 198 N. meningitidis and 293 commensal Neisseria isolates were collected between 2005 and 2018 in Shanghai, China (see Tables S1 and S2 in the supplemental material). The N. meningitidis isolates were obtained from IMD patients (n = 46) and asymptomatic carriers (n = 152). The transformation recipient strain Nm040 (33) was isolated from a 3-month-old male patient with meningitis in 2007, and its genome was sequenced using single-molecule real-time sequencing (Pacific Biosciences, Menlo Park, CA, USA) and is available in the Neisseria PubMLST Database (PubMLST ID 58130), showing molecular characterization as B:P1.20,13-1:ST-5798 (CC4821). All commensal Neisseria isolates, including N. lactamica (n = 252), N. polysaccharea (n = 15), N. subflava (n = 11), N. cinerea (n = 8), N. mucosa (n = 6), and N. oralis (n = 1) were obtained from carriers. The Neisseria spp. were primarily identified by Gram staining, the oxidase reaction, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS; bioMérieux, France), with subsequent confirmation being obtained by analysis of the sequence of the rplF gene, which encodes the 50S ribosomal protein L6 (34). When genomes were available, the species of the isolates were checked using ribosomal multilocus sequence typing (rMLST) (35). All the isolates were stored in brain heart infusion broth supplemented with 20% glycerol at −80°C.
Antimicrobial susceptibility testing.The MICs of ciprofloxacin for all the Neisseria isolates were determined by the agar dilution method and interpreted using the breakpoints for N. meningitidis in the 2019 guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Resistance to ciprofloxacin was defined as an MIC of ≥0.06 μg/ml (13).
Typing of N. meningitidis isolates.The serogroup of the N. meningitidis isolates was determined by slide agglutination using monoclonal antiserum (Remel Europe, Kent, United Kingdom). MLST was performed for all 491 isolates according to previously described protocols (36, 37).
Analysis of QRDRs of quinolone resistance-associated genes.The QRDRs of the quinolone resistance-associated genes gyrA (encoding gyrase) and parC (encoding topoisomerase IV) were amplified and sequenced using previously described primers (13, 14). The gyrA and parC sequences of strain MC58 (N. meningitidis) were used as a reference (38). The sequences of gyrA were queried against the sequences in the Neisseria PubMLST database for assigning alleles of the gyrA QRDR (nucleotides 115 to 639), while novel alleles discovered in this study were submitted and assigned a new allele number in the database. In this study, parC QRDR alleles were defined based on the sequence between nucleotides 100 and 501. Phylogenetic analyses were performed via the unweighted pair group method with arithmetic mean averages (UPGMA) with the MEGA program, using the sequences for gyrA (nucleotides 115 to 639) and parC (nucleotides 100 to 501) collected in this study and those present in the Neisseria PubMLST database from different Neisseria species and different countries deposited prior to 7 March 2019. Visualization was performed using the Interactive Tree of Life (iTOL) (39). Clusters were determined using bootstrap values of >70% from bootstrap tests with 1,000 replicates (40).
Genome sequencing, assembly, and analysis.To identify potential donors of the recombined N. meningitidis gyrA alleles, the genomes of 19 Neisseria isolates harboring gyrA alleles shared by two Neisseria species were sequenced. Purified genomic DNA samples were used to generate multiplexed libraries with a TruSeq Nano DNA sample preparation kit (Illumina, San Diego, CA, USA), and paired-end 150-bp indexed reads were generated on an Illumina HiSeq platform. The average coverage depth of the genomes was approximately 400-fold. The genome sequence data were assembled using the SPAdes (version 3.13.1) algorithm and filtered with a coverage of >10 and a scaffold length of >200 bp (41). The assembled genomes were uploaded to the PubMLST Neisseria database (https://pubmlst.org/neisseria/), which supplements the BIGSdb platform (18).
Identification of recombination donors and positions of breakage points of the meningococcal gyrA alleles.A commensal Neisseria strain was considered to be a potential donor for a mutation-harboring gyrA allele in N. meningitidis if (i) the gyrA allele was assigned outside the N. meningitidis cluster in the phylogenetic analysis of the gyrA QRDR nucleotides, (ii) the allele was shared by N. meningitidis and the commensal Neisseria strain in question, and (iii) less than 1 nucleotide difference per 1,000 bp was observed between the donor and recombinant fragment sequences (42). The genomes of representative N. meningitidis and commensal Neisseria isolates that shared the potential recombinant gyrA allele were sequenced on an Illumina platform to identify the recombination donors and the positions of the breakage points. The full-length gyrA gene sequence, including the flanking sequences, when necessary, was extracted from the genome for sequence alignment to identify the positions of the recombination breakage points, which were identified as points of an abrupt change in sequence similarity between the recombinant and a potential donor strain (43). The recombination breakage points identified by visual inspection were checked, using the Recombination Detection Program (RDP; version 4.97), by at least 1 of 7 algorithms (RDP, Chimera, Bootscan, 3Seq, GENECONV, MaxChi, and SiScan) (44). The corresponding sequences of three meningococcal strains were used as the gyrA reference sequence, including those of strain 053442 (C:P1.7-2,4:ST-4821 [CC4821]; gyrA71; GenBank accession number CP000381), strain MC58 (B:P1.7,16-2:ST-74 [CC32]; gyrA2; GenBank accession number AE002098), and strain Z2491 (A:P1.7,13-1:ST-4 [CC4]; gyrA3; GenBank accession number AL157959) (38, 45, 46). The gyrA QRDR sequences of all three of these strains have been shown to group within the N. meningitidis cluster (14).
Genetic transformation.The transformation of chromosomal fragments from commensal Neisseria strains into N. meningitidis was performed according to a previously described protocol (47). In brief, the chromosomal DNAs of the potential commensal Neisseria donor strains (donor chromosomal DNA), which were identified by genomic analysis and which harbored the GyrA mutation T91I, were purified using a Qiagen DNA minikit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. After overnight growth on Columbia agar (Oxoid, Basingstoke, United Kingdom) supplemented with 5% sheep blood, the quinolone-susceptible recipient strain Nm040 (ciprofloxacin MIC, 0.004 μg/ml) was incubated with each donor chromosomal DNA (500 ng) in proteose peptone medium (Oxoid) supplemented with BBL IsoVitaleX (BD, Sparks, MD, USA) for 5 to 6 h at 37°C. Then, the mixture was plated on Columbia sheep blood agar supplemented with ciprofloxacin (2-fold dilutions; range, 0.03 to 0.5 μg/ml) at 37°C with 5% carbon dioxide for 24 to 48 h. Transformations were repeated once using transformants from plates supplemented with the highest ciprofloxacin concentration as the recipient. The ciprofloxacin MICs and the genomes of the final transformants were determined using Etest (bioMérieux, Marcy l’Etoile, France) and sequencing by the Beijing Genomics Institution (Shenzhen, China), respectively.
Ethical considerations.This study was approved by the Shanghai Municipal Center for Disease Control and Prevention Ethical Review Committee (no. 2016-4). Before sample and data collection, informed consent was obtained from all study participants (or their guardians).
Accession number(s).The sequences of the 105 parC alleles defined in this study were submitted to GenBank under accession numbers MK728699 to MK728731 and MN018761 to MN018832. The 28 genomes of the N. meningitidis isolates, transformants, and commensal Neisseria isolates that were sequenced in this study were submitted to the PubMLST Neisseria Database (PubMLST IDs, 52298, 52302, 52365, 52374, 52400, 84198, 84206, 84263, 84265, 84474, 84477, 89163 to 89165, 92872 to 92876, and 94049 to 94057). All the short reads of the genomes sequenced in this study were submitted to the European Nucleotide Archive (ENA) and assigned accession number PRJEB35093.
ACKNOWLEDGMENTS
We thank Heike Claus of the University of Würzburg for sharing with us the experimental details of the genetic transformation of N. meningitidis. We thank Eva Hong of the Pasteur Institute for her work as the curator of the gyrA database and for assigning the numerous new alleles identified in this study. We thank Bin Liu of Nankai University for helping us polish the language in the manuscript. This study made use of Neisseria genomic data deposited in the Neisseria MLST Database (https://pubmlst.org/neisseria/), which was developed by Keith Jolley and which is located at the University of Oxford.
The development of the Neisseria MLST Database is funded by the Wellcome Trust and European Union. This work was supported by the National Natural Science Foundation of China under grant 81601801, the Shanghai Rising-Star Program of the Shanghai Municipal Science and Technology Commission under grant 17QA1403100, a Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai under grant 2017YQ039, and the 13th Five-Year Project of the National Health and Family Planning Commission of the People’s Republic of China under grants 2017ZX10303405004 and 2017ZX10103009-003.
The funders had no role in the study design, data collection and interpretation, or decision to submit the work for publication.
We have no reported conflicts of interest.
FOOTNOTES
- Received 23 July 2019.
- Returned for modification 10 September 2019.
- Accepted 11 November 2019.
- Accepted manuscript posted online 18 November 2019.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
