Serogroup Y Clonal Complex 23 Meningococcus in China Acquiring Penicillin Resistance from Commensal Neisseria lactamica Species

ABSTRACT

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis (NmY) is rare in China; recently, an invasive NmY isolate, Nm512, was discovered in Shanghai with decreased susceptibility to penicillin (Pen^NS). Here, we investigated the epidemiology of NmY isolates in Shanghai and explored the potential commensal Neisseria lactamica donor of the Pen^NS NmY isolate. A total of 491 N. meningitidis and 724 commensal Neisseria spp. isolates were collected. Eleven NmY isolates were discovered from IMD (n = 1) and carriers (n = 10), including two Pen^NS isolates with five-key-mutation-harboring (F504L-A510V-I515V-H541N-I566V) penA genes. Five of the eight ST-175 complex (CC175) isolates had a genotype [Y:P1.5-1,2-2:F5-8:ST-175(CC175)] identical to that of the predominant invasive clone found in South Africa. Only one invasive NmY CC23 isolate (Nm512) was discovered; this isolate carried a novel Pen^NS penA832 allele, which was identified in commensal N. lactamica isolates locally. Recombination analysis and transformation of the penA allele highlighted that N. meningitidis Nm512 may acquire resistance from its commensal donor; this was supported by the similar distribution of transformation-required DNA uptake sequence variants and the highly cognate receptor ComP between N. meningitidis and N. lactamica. In 2,309 NmY CC23 genomes from the PubMLST database, isolates with key-mutation-harboring penA genes comprised 12% and have been increasing since the 1990s, accompanied by recruitment of the bla_ROB-1 and/or quinolone resistance allele. Moreover, penA22 was predominant among genomes without key mutations in penA. These results strongly suggest that Nm512 is a descendant of the penA22-harboring CC23 isolate from Europe and acquired its penicillin resistance locally from commensal N. lactamica species by natural transformation.

INTRODUCTION

Neisseria meningitidis is responsible for invasive meningococcal disease (IMD) globally, presenting mainly as septicemia and meningitis (1). Twelve serogroups can be identified on the basis of capsular polysaccharides, of which six serogroups (A, B, C, W, X, and Y) are responsible for the majority of IMD cases worldwide (2). Recent IMD cases in China have been caused by mainly serogroup C, A, B, and W N. meningitidis isolates (3). Reports of IMD cases due to serogroup Y N. meningitidis (NmY) are rare in China. This is in contrast to the increasing NmY cases in Europe in the 2000s and in the USA since the 1980s; these increases were both attributable to NmY ST-23 complex (CC23) strains (4, 5). So far, there have only been three culture-confirmed NmY IMD cases reported in China. The first case was caused by an NmY ST-175 isolate, Nm-TJ, in December 2015 in Tianjin (6). In March 2019, two quinolone-resistant NmY CC23 isolates were discovered in Guangdong Province from an IMD patient (N19-2-Y) and his asymptomatic contact (N19-3-Y) (7). In December 2019, a 2-year-old boy in Shanghai was diagnosed with fulminant meningococcemia (8), which was caused by a penicillin-nonsusceptible (Pen^NS) NmY CC23 isolate (Nm512).

The β-lactam antibiotics penicillin and third-generation cephalosporins are recommended as lead candidates for treating IMD (9). However, Pen^NS meningococci have been increasing globally and reported in North America (USA, 2015) (10), South America (Brazil, 2009 to 2016) (11), Europe (France, 1999 to 2002; Italy, 2006 to 2016) (12, 13), Oceania (Australia, 2017) (14), Africa (South Africa, 2001 to 2005) (15), and Asia (Iran, 2019) (16). Pen^NS meningococci are mainly associated with five key alterations (F504L, A510V, I515V, H541N, and I566V) in penicillin binding protein 2 (PBP2) encoded by the penA gene (17, 18). Taha et al. elucidated that Pen^NS penA alleles originated from commensal Neisseria species through horizontal gene transfer (HGT) (17), commonly via natural transformation (19). Efficient natural transformation in Neisseria spp. requires the presence of genus-specific DNA uptake sequence (DUS) (20, 21). Eight distinct DUS variants, termed DUS dialects, have been identified in Neisseria spp. (22), constituting up to 1% of the entire neisserial genomes. The pilin protein ComP was identified as the cognate receptor to bind specific DUS and plays an important role for DNA uptake in natural transformation (23).

This study aimed to gain more data on the molecular epidemiology of NmY isolates in Shanghai and to investigate the potential donor of the Pen^NS penA allele of Nm512. The characteristics and genetic relationships of domestic and foreign prevalent NmY isolates were also explored.

RESULTS

Epidemiological and molecular characterizations of the NmY isolates in Shanghai from 1965 to 2020.

A total of 491 N. meningitidis isolates were collected in Shanghai during 1965 to 1985 (n = 303) and 2005 to 2020 (n = 188) from IMD cases (n = 171) and carriage (n = 320) (see Table S1 in the supplemental material). Eleven NmY isolates were discovered among the 491 N. meningitidis isolates, including 1 (Nm512) from the IMD case in 2019 and 10 (7.9%, 10/126) from carriers during the period 1965 to 1976 (see Table S2). Among the 10 NmY carriage isolates, 8 belonged to CC175 and 8 possessed susceptible penA4 alleles. Five of eight CC175 isolates had an identical molecular characterization of Y:P1.5-1,2-2:F5-8:ST-175(CC175). Penicillin susceptibility was investigated among the NmY isolates: 2 (Nm512 and Nm156) of the 11 were Pen^NS, carrying penA genes encoding PBP2 with five mutations (F504L, A510V, I515V, H541N, and I566V). Nm512 carried Pen^NS penA832 belonging to ST-1655 (Y:P1.5-1,10-1:F4-1:ST-1655 [CC23]) (8), while Nm156 was isolated from a carrier in 1967 with Pen^NS penA379 belonging to ST-9532 (Y:P1.12-18,3:F5-135:ST-9532 [singleton]). In addition, there were 31 (6.3%; 31/491) nongroupable isolates (see Table S1).

Potential donor of the penA832 allele from commensal Neisseria isolates.

Among 724 commensal Neisseria isolates collected during 2013 to 2019, eight N. lactamica isolates (Nei341, Nei145, Nei152, Nei282, Nei386, Nei422, Nei432, and Nei439) were discovered to share identical penA832 alleles with Nm512. The N. lactamica isolate Nei341 showed a penicillin MIC of 0.25 μg/mL. To explore potential recombination events between N. meningitidis and N. lactamica isolates, meningococcal CC23 isolates 2960 and N19-2-Y, both closely related to Nm512 and carrying the susceptible penA22 allele (8), and CC4821 isolates Nm040 and 053442, both carrying the susceptible penA1 allele, were used as reference N. meningitidis strains. The nucleotide sequences of the penA gene and its flanking sequences were extracted from the genomes of Nm512, eight N. lactamica isolates, and reference N. meningitidis strains. The genomes of Nm512 and eight commensal N. lactamica isolates shared a 1,377-bp region with 99.5% to 100% nucleotide sequence identity, including a 1,028-bp region of the penA gene and a 349-bp fragment of its downstream gene. The 1,377-bp region exhibited high variation (183 bp differences) compared to the two penA22- and penA1-harboring reference sequences (see Fig. S1 and S2 in the supplemental material). The sequence of Nei282 contained some fragments similar to those in N. meningitidis genomes and showed discrete variations compared to the other seven N. lactamica isolates (Fig. S2). The first nucleotide of the initial codon of penA was recorded as +1. We observed that the upstream recombination breakage point was located at position +718 within penA, the downstream breakage point was located at position +2094, and the recombinant fragment included the 402-bp penA allele of penA832 (+1321 to +1722). The flanking sequences of the recombinant common region exhibited a significant difference between Nm512 and N. lactamica isolates, while they were identical between Nm512 and the penA22-harboring CC23 reference genomes from Italy and Guangdong, China (see Fig. S2). Thus, it was indicated that the commensal N. lactamica isolates were the potential donors of the nonsusceptible penA allele of Nm512, and the flanking sequences shared the same origin with those closely related penA22-harboring NmY CC23 isolates.

Natural transformation of the penA832 allele.

A natural transformation experiment was performed to investigate whether penicillin-susceptible meningococci could acquire nonsusceptible penA alleles from commensal Neisseria species. Recipient CC4821 meningococcal strain Nm040 was transformed with the chromosomal DNA of the commensal N. lactamica strain Nei341, which carried penA832 encoding PBP2 with the five mutations. Four transformants (Nm040Nei341T1-1 to Nm040Nei341T1-4) exhibiting intermediate resistance to penicillin were selected, elevating the MIC values from 0.032 μg/mL (recipient) to 0.19 μg/mL (transformant). Each transformant acquired the penA832 allele from Nei341 but showed a diverse mosaic penA variant. Genomic sequence comparison of the corresponding donor, recipient, and the four transformants illustrated various breakage points at upstream and downstream positions, and the sizes of the recombinant fragments ranged from 1,292 to 7,805 bp (Table 1; see also Fig. S3 in the supplemental material).

TABLE 1.

Characteristics of Nm512 and genetic transformation from Nei341 into Nm040

Strain	Attribute of strain	Penicillin MIC (μg/mL)	penA allele (mutations)	Breakage point positiona		Recombinant size (bp)	PubMLST ID	Reference
				Upstream	Downstream
Nm512	Clinical isolate	0.19	penA832 (F504L, A510V, I515V, H541N, I566V)	+718	+2094	1,377	71401	8
Nei341	Donor	0.25	penA832 (F504L, A510V, I515V, H541N, I566V)				83066	This study
Nm040	Recipient	0.032	penA1 (none)				58130	41
Nm040Nei341T1-1	Transformant	0.19	penA832 (F504L, A510V, I515V, H541N, I566V)	+757	+2048	1,292	111265	This study
Nm040Nei341T1-2	Transformant	0.19	penA832 (F504L, A510V, I515V, H541N, I566V)	+328	+8664	7,805	111266	This study
Nm040Nei341T1-3	Transformant	0.19	penA832 (F504L, A510V, I515V, H541N, I566V)	+328	+5534	4,554	111267	This study
Nm040Nei341T1-4	Transformant	0.19	penA832 (F504L, A510V, I515V, H541N, I566V)	+328	+5534	4,554	111268	This study

^aThe first nucleotide of the initial codon of penA of Nm040 was recorded as +1.

Characterization of DUS variants and cognate ComP in neisserial genomes.

Eight DUS variants (termed DUS dialects) were investigated in five N. meningitidis and eight commensal N. lactamica isolates (Table 2). The AT-DUS duplications were predominantly found in both N. meningitidis and N. lactamica genomes (1,426 to 1,742 copies), accounting for over 80% of all available DUS variants, followed by AG-DUS duplications (187 to 274 copies; approximately 10% to 13%). A further six DUS variants were identified, except for TG-wadDUS (Table 2). The proportion of dominant DUS variants was similar in N. meningitidis and N. lactamica isolates. Duplications of AT-DUS and/or AG-DUS were identified within or adjacent to penA genes of N. meningitidis and N. lactamica isolates (see Table S3). The cognate ComP, a receptor of DUS, was identical in N. meningitidis CC4821 (Nm040 and 053442) and CC23 (Nm512, 2960, and N19-2-Y) isolates, which showed 97% to 98% amino acid identity (2 to 4 residues variation) with those of N. lactamica isolates (Fig. 1).

TABLE 2.

Identification of DUS variants in neisserial genomes

Isolate	DUS variant counta	AT-DUS count (% of content)b	AG-DUS count (% of content)b	Counts for other 6 DUSs						% of content for other DUSsb	PubMLST ID
				AG- mucDUS	AG- simDUS	AG- kingDUS	AA- king3DUS	AG- eikDUS	TG- wadDUS
Nm512	1,797	1,456 (81.0)	193 (10.7)	102	10	10	20	6	0	8.3%	71401
N19-2-Y	1,778	1,445 (81.3)	189 (10.6)	101	10	10	17	6	0	8.1%	46123
2960	1,812	1,465 (80.8)	190 (10.5)	110	10	10	21	6	0	8.7%	84080
Nm040	1,770	1,429 (80.7)	201 (11.4)	90	14	15	17	4	0	7.9%	58130
053442	1,740	1,426 (82.0)	187 (10.7)	86	11	10	16	4	0	7.3%	12672
Nei145	2,123	1,738 (81.9)	252 (11.9)	75	22	20	13	3	0	6.2%	84478
Nei152	2,125	1,739 (81.8)	253 (11.9)	75	22	20	13	3	0	6.3%	111269
Nei282	2,065	1,718 (83.2)	236 (11.4)	46	24	28	10	3	0	5.4%	111270
Nei341	2,039	1,712 (84.0)	234 (11.4)	27	24	28	10	4	0	4.6%	83066
Nei386	2,119	1,725 (81.4)	268 (12.6)	55	26	29	13	3	0	6.0%	108166
Nei422	2,115	1,715 (81.0)	274 (13.0)	55	26	29	13	3	0	6.0%	84261
Nei432	2,122	1,742 (82.1)	248 (11.7)	73	23	20	13	3	0	6.2%	111271
Nei439	2,063	1,711 (82.9)	243 (11.8)	44	24	28	10	3	0	5.3%	111272

^aDUS variants: AT-DUS (5′-ATGCCGTCTGAA-3′), AG-DUS (5′-AGGCCGTCTGAA-3′), AG-mucDUS (5′-AGGTCGTCTGAA-3′), AG-simDUS (5′-AGGCTGCCTGAA-3′), AG-kingDUS (5′-AGGCAGCCTGAA-3′), AA-king3DUS (5′-AAGCAGCCTGCA-3′), AG-eikDUS (5′-AGGCTACCTGAA-3′), TG-wadDUS (5′-TGCCTGTCTGAA-3′).

^bProportion of the DUS(s) among the total for all eight DUS variants; the percent content data shown for the six “other” DUSs represent the group of six combined.

FIG 1.

Sequence alignment of ComP in N. meningitidis and N. lactamica isolates, produced using UGENE. ComP is a protein of 149 amino acids, is virtually identical among N. meningitidis isolates, and shows 97% to 98% identity (2 to 4 diverse residues) between N. meningitidis and N. lactamica isolates. Each horizontal bar represents one ComP sequence; the black arrow corresponds to the diverse residue position.

Distribution of penA alleles in NmY CC23 strains.

There were 2,392 NmY CC23 genomes from at least 29 different countries deposited in the PubMLST Neisseria Database by 29 November 2021. A total of 2,309 genomes with penA allele data represented 46 penA alleles (see Table S4). Only 12.0% (278/2,309) contained PBP2 with key mutations (F504-A510-I515-H541-I566 [n = 277] and F504-I515-H541-I566 [n = 1]), representing 29 penA alleles. Seventeen penA alleles without key mutations represented 88.0% (2,031/2,309) of the isolates. Among the CC23 genomes without key mutations in penA, penA22 was predominant (94.9%; 1,928/2,031), followed by penA1 (3.5%; 71/2,031). Two of the previously reported penicillin-susceptible NmY CC23 isolates, N19-2-Y and N19-3-Y (Y:P1.5-1:10–1:F4-1:ST-1655[CC23]), from China also carried the penA22 allele (7). Among the key-mutation-harboring isolates, penA9 was most prevalent (30.2%; 84/278), followed by penA20 (25.9%; 72/278). No penA832-harboring Neisseria isolates were found except for those in this study. There were 2,386 genomes with gyrA allele data, representing 15 gyrA alleles in the database (Table S4). The susceptible gyrA1 (60.9%; 1,452/2,386) and gyrA2 (22.3%; 532/2,386) alleles were preponderant and accounted for 82% (1,984/2,386) of CC23 isolates. The ROB-1-type beta-lactamase gene (bla_ROB-1) was investigated in 2,392 NmY CC23 genomes, and 43 bla_ROB-1-positive NmY CC23 isolates were found, including 40 from North America (mainly in the USA; 33/40) and 3 from Europe during 2013 to 2020. Almost all of the bla_ROB-1-positive isolates harbored key-mutation-harboring penA9 (97.5%; 42/43), and 25.6% (11/43) of isolates carried mutation-harboring gyrA242 (T91I), which was associated with quinolone resistance (see Table S5).

Since the 1990s, CC23 isolates have increased, including key-mutation-harboring penA NmY CC23 isolates; the proportion of key-mutation-harboring penA NmY CC23 isolates ranged from 5% to 7% in 1993 to 2010, except for in 2007 (18.7% proportion, mainly in USA), and then increased to 8% to 20% in 2011 to 2020 (Fig. 2). The bla_ROB-1-positive NmY CC23 isolates emerged in 2013 and increased to more than 40 during 2016 to 2020 (Fig. 2).

FIG 2.

Period distribution of NmY CC23 isolates since the 1990s. NmY CC23 isolates, including key-mutation-harboring penA isolates, have increased since the 1990s; bla_ROB-1-positive isolates emerged in 2013 and increased above 40 during 2016 to 2020. The number of isolates is indicated for each corresponding category of isolates.

DISCUSSION

Although serogroup Y meningococcal diseases are still rare in China, they have increased in the United States, South Africa, and Europe since the 1990s (24–27). In this study, we investigated the epidemiology of serogroup Y N. meningitidis in IMD cases and healthy populations in Shanghai based on available isolates from 1965 to 2020. We found that the NmY carriage isolates were isolated during the period 1965 to 1976, accounting for 8.7% of carriage isolates at that time. Most of the NmY carriage isolates have a molecular characterization of Y:P1.5-1,2-2:F5-8:ST-175(CC175), which is identical to the predominant invasive clone in South Africa from 1999 to 2002 (24). So far, only one case caused by the NmY ST-175 isolate (Nm-TJ) was reported, in 2015 in Tianjin, China, although the genetic relationships of this isolate are uncertain as the genome of Nm-TJ was unavailable. Here, only one NmY invasive isolate, Nm512, was discovered in Shanghai in 2019 that was reported to be genetically closely related to the CC23 isolates (Y:P1.5-1,10-1:F4-1:ST-1655[CC23]) from Europe and South Africa during 2012 to 2019 and from Guangdong, China in 2019 (8). Unlike other NmY CC23 strains worldwide, Nm512 carried a novel Pen^NS penA allele, penA832, which was discovered only in N. meningitidis and commensal N. lactamica isolates in this study.

Penicillin resistance of N. meningitidis can be caused by two mechanisms. The main mechanism is due to mutations in the penA gene encoding PBP2, which is the main target of β-lactam antibiotics (17, 18). In 2007, the European Monitoring Group on Meningococci (EMGM) analyzed 652 Pen^NS isolates from Europe and the USA and found five key mutations in PBP2 (F504L, A510V, I515V, H541N, and I566V) that lead to penicillin resistance (MIC, 0.094 to 1 μg/mL; intermediate or resistant) (17). The other mechanism is the acquisition of β-lactamase-encoding genes from other species. Indeed, a CC23 isolate has acquired the bla_ROB-1 gene from Haemophilus influenzae, leading to high-level penicillin resistance (MIC, 3 μg/mL) (28).

In the last decade, almost all of the reported β-lactamase-positive meningococcal isolates worldwide were found to belong to NmY CC23 (25–27). CC23 is a hypervirulent lineage associated with most NmY isolates globally (25). Here, we investigated 2,309 NmY CC23 genomes with penA allele data in the PubMLST Database, among which approximately 12% possessed the five key mutations in PBP2. The proportion of penA key-mutation-harboring CC23 isolates has increased from a level of 5% to 7% in the 1990s to 8% to 20% more recently, which is consistent with reports of a global rising trend of Pen^NS isolates (10–16). Recently (after 2016), more than 40 CC23 isolates were found to carry both bla_ROB-1 and the nonsusceptible penA9 allele, while 11 of them also carried the quinolone-resistant gyrA242 allele, suggesting that multidrug-resistant N. meningitidis isolates represent a significant public health threat.

Wang et al. analyzed 288 representative CC23 genomes and identified three sublineages (8), each of which has a global dissemination. In sublineage L23.1, three NmY CC23 isolates (Nm512, N19-2-Y, and N19-3-Y) from China and nine from Europe, Japan, and South Africa were closely related (8). However, all nine isolates from countries outside China were β-lactamase negative, carrying quinolone-susceptible gyrA1 and penicillin-susceptible penA22, while Nm512 from Shanghai harbored Pen^NS penA832 and isolates N19-2-Y and N19-3-Y from Guangdong possessed the quinolone-resistant gyrA9 allele. These two resistant alleles are different from those (penA9 and gyrA242) from European or North American CC23 isolates, indicating different sources of these alleles. Commensal N. lactamica strains are a great pool of resistance genes, especially against penicillin and quinolones in China (29). NmY CC23 isolates in China may be descendants of those from Europe and have acquired penicillin or quinolone resistance genes locally.

A previous study verified that quinolone-susceptible N. meningitidis isolates could capture quinolone-resistant gyrA or parC alleles from commensal Neisseria strains through HGT (30). Indeed, Neisseria gonorrhoeae is also able to acquire the penA gene of penicillin resistance from commensal Neisseria species (31). Nm512 was presumed to have acquired Pen^NS penA allele in a similar way.

Penicillin and fluoroquinolone antibiotics have been widely used in hospitals in China (32, 33). In 2010, penicillin antibiotics consumption reached 1 × 10⁹ standard units, accounting for the highest antimicrobial agent consumption in that period (32). Increased antibiotic consumption was observed globally between 2000 and 2015, especially in low- and middle-income countries, including China (34). For instance, the consumption of broad-spectrum penicillins and of fluoroquinolones increased from 2.8 to 3.9 defined daily doses (DDDs) and 0.7 to 2.4 DDDs per 1,000 inhabitants per day, respectively (34). Increasing international travel and high population density in Shanghai also promote the risk of colonization of Pen^NS/quinolone^NS meningococci and commensal Neisseria strains in the nasopharynx. Commensal Neisseria could provide an ideal gene pool to allow meningococci to acquire antimicrobial resistance traits (29, 35). In this study, we discovered Pen^NS commensal N. lactamica isolates harboring the penA832 allele in healthy populations in Shanghai. Under penicillin selective pressure, meningococci can obtain mutation-harboring penA832 through natural transformation. Unlike the mosaic structure of penA14, which may originate from four diverse commensal Neisseria species (17), penA832 was recovered from a single donor of N. lactamica. Recombination analysis indicated that the mosaic penA in Nm512 is a hybrid of DNA segments from penA832-harboring N. lactamica and penA22-harboring CC23 strains. These results suggest that closely related penA22-harboring NmY CC23 isolates, such as 2960 from Italy, are most likely the predecessors of Nm512.

Neisseria spp. are constantly in a state of competence, which facilitates their taking up exogenous DNA fragments and undergoing frequent natural transformation (36). Natural transformation in Neisseria spp. requires the presence of a genus-specific DUS and depends on the type IV pilus (Tfp) and RecA-mediated homologous recombination (37, 38). A bioinformatic analysis of the Neisseriaceae family genomes revealed eight distinct variants of DUS, among which AT-DUS was the most dominant, showing highest transformation efficacy among several human Neisseria species, such as N. meningitidis, N. gonorrhoeae, and N. lactamica. AG-DUS showed 90% efficacy compared to AT-DUS in N. meningitidis. A similar degree of DUS variant distribution in Neisseria spp. (such as N. meningitidis and N. lactamica) correlated with the level of transformation (22). Most Neisseria spp. genomes contained one dominant DUS variant (22). Here, we found a similar degree of efficient DUS distribution between N. meningitidis and commensal N. lactamica isolates, suggesting the potential for a high level of interchange with each other. ComP, the minor pili protein, had been considered a definite DUS receptor and showed a high affinity with its cognate DUS (23, 39, 40). Identification of extensive genus-specific DUS variants and cognate ComP in N. meningitidis and N. lactamica isolates provides us with more intrinsic factors for the capability of Nm512 to recruit the nonsusceptible penA allele from commensal N. lactamica through HGT.

In summary, NmY cases are still rare in China. However, the invasive NmY CC23, showing a global dissemination, has been detected in China and recruited a novel nonsusceptible penA allele or quinolone resistance genes, which pose some challenges to local IMD prevention and control. Efficient HGT by natural transformation may promote N. meningitidis to accumulate more resistance genes, and commensal N. lactamica plays an important role as a pool of resistance genes. More attention should be paid to the prevalence of Pen^NS isolates and the globally disseminated CC23 clones in China.

MATERIALS AND METHODS

Isolate collection.

A collection of 491 N. meningitidis isolates was obtained in Shanghai during 1965 to 1985 and 2005 to 2020 (see Appendix 1 in the supplemental material). Meningococcal surveillance and carriage survey were implemented by the Shanghai Center for Disease Control and Prevention (CDC) as previously described (33). Since 1950, the Shanghai CDC has collected N. meningitidis isolates from patients and close contacts, with an interruption during 1986 to 2004 (33). Surveys of meningococcal carriage were performed by the Shanghai CDC annually and also conducted prior to the epidemic seasons of meningococcal disease. The carriage survey of commensal Neisseria isolates was performed as previously described (29). A total of 724 commensal Neisseria isolates were collected from posterior oropharyngeal swabs of over 2,000 children (aged <15 years) during 2013 and 2019, including 405 Neisseria subflava, 238 N. lactamica, and 81 other commensal Neisseria spp. (see Appendix 1 in the supplemental material). The Neisseria spp. were identified as previously described (30). The transformation recipient strain Nm040 (N. meningitidis CC4821) was from a patient with meningitis in 2007 in Shanghai (41).

Antimicrobial susceptibility testing.

The MICs of penicillin for Neisseria isolates were determined by the agar dilution method and confirmed by Etest (bioMérieux). The interpretation of breakpoints was a MIC of ≤ 0.06 μg/mL as susceptible and ≥0.5 μg/mL as resistant, according to the 2020 guidelines of the Clinical and Laboratory Standards Institute methods (42).

Serogrouping and penA genotyping.

The serogroups of N. meningitidis were determined by a slide agglutination assay using monoclonal antiserum (Remel Europe Ltd., Dartford, Kent, United Kingdom). The penA gene was amplified and sequenced using previously described primers (17). The sequences of penA were queried in the Neisseria PubMLST database for assigning the penA alleles (hypervariable region), which were defined based on the sequence between nucleotides 1321 and 1722 (402 bp).

Genetic transformation of penA and identification of the recombination breakage point of penA.

The natural transformation experiment was performed as described previously (43). The genomes of Neisseria isolates were sequenced on an Illumina HiSeq platform, including NmY isolates, commensal N. lactamica isolates that shared the identical penA allele with NmY isolates, and transformants of Nm040. The genomes of four N. meningitidis strains were used for the penA reference sequence, including CC4821 isolates Nm040 (PubMLST ID 58130) and 053442 (12672) carrying penA1 (44) and NmY CC23 isolates 2960 (84080) and N19-2-Y (46123) carrying penA22 (8). The recombination breakage points of penA were determined as previously described (30).

Identification of DUS variants and ComP alignment.

The DUS was identified as eight 12-mer distinct DUS variants, termed DUS dialects, including the AT-DUS (5′-ATGCCGTCTGAA-3′), the AG-DUS (5′-AGGCCGTCTGAA-3′), the AG-muDUS (5′-AGGTCGTCTGAA-3′), the AG-simDUS (5′-AGGCTGCCTGAA-3′), the AG-kingDUS (5′-AGGCAGCCTGAA-3′), the AA-king3DUS (5′-AAGCAGCCTGCA-3′), the AG-eikDUS (5′-AGGCTACCTGAA-3′), and the TG-wadDUS (5′-TGCCTGTCTGAA-3′) (22). UGENE was used for searching DUS variants in neisserial genomes, as well as for sequence alignment of ComP in N. meningitidis and N. lactamica isolates.

Data availability.

The genomes of all Neisseria isolates and transformants that were sequenced in this study were submitted to the PubMLST Neisseria Database (see Table S6).