Genetic Diversity and Carriage Dynamics of Neisseria lactamica in Infants
http://www.100md.com
感染与免疫杂志 2005年第4期
The Peter Medawar Building for Pathogen Research and Department of Zoology, Oxford University
Nuffield Department of Clinical Microbiology
Oxford Vaccine Group, Department of Paediatrics, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom
ABSTRACT
Neisseria lactamica, a harmless human commensal found predominantly in the upper respiratory tracts of infants, is closely related to Neisseria meningitidis, a pathogen of global significance. Colonization with N. lactamica may be responsible for the increase in immunity to meningococcal disease that occurs during childhood, when rates of meningococcal carriage are low. This observation has led to the suggestion that N. lactamica whole cells or components are potential constituents of novel meningococcal vaccines. However, the dynamics of carriage and population diversity of N. lactamica in children are poorly understood, presenting difficulties for the choice of representative isolates for use in vaccine development. This problem was addressed by the multilocus sequence typing of N. lactamica isolates from two longitudinal studies of bacterial carriage in infants. The studies comprised 100 and 216 subjects, with N. lactamica carriage monitored from age 4 weeks until age 96 weeks and from age 2 weeks until age 24 weeks, respectively. The maximum observed carriage rate was 44% at 56 weeks of age, with isolates obtained on multiple visits for the majority (54 of 75, 72%) of carriers. The N. lactamica isolates were genetically diverse, with 69 distinct genotypes recovered from the 75 infants. Carriage was generally long-lived, with an average rate of loss of under 1% per week during the 28 weeks following acquisition. Only 11 of the 75 infants carried more than one genotypically unique isolate during the course of the study. Some participants shared identical isolates with siblings, but none shared identical isolates with their parents. These findings have implications for the design of vaccines based on this organism.
INTRODUCTION
The genus Neisseria comprises gram-negative, oxidase-positive diplococci that are frequently isolated from the mucosal surfaces of humans and animals (39). Two members of the genus cause distinct human diseases: Neisseria meningitidis, which can be responsible for septicemia and meningitis (8), and Neisseria gonorrhoeae, the etiological agent of gonorrhea (44). Neisseria lactamica is closely related to both of these species (24) but is associated with invasive disease only in exceptional circumstances (13, 35, 42). Carriage of N. lactamica throughout the world is high in infants and young children but declines with age. This is the converse of the carriage of the meningococcus, which is low during infancy and rises to high levels in adolescents and young adults (2, 9, 21, 26, 41, 43).
The observation that antimeningococcal bactericidal titers rise steadily in infants, despite low rates of meningococcal carriage, has provided indirect support for the concept that carriage of N. lactamica by children (9) may confer protection against disease caused by N. meningitidis (7). This suggestion has in turn led to the proposal that N. lactamica could be used to induce protection against meningococcal infection, by using killed whole cells, outer membrane vesicles, or outer membrane proteins, as demonstrated in a murine model (40), or by the deliberate inoculation of infants with live N. lactamica cultures (23).
One of the factors that has proved to be a hindrance to the development of vaccines against the meningococcus is its genetic and antigenic diversity (11, 12, 15). Although progress has been made in the development of vaccines to serogroup A and C meningococci, with a polysaccharide-protein conjugate vaccine to serogroup C meningococci found to be effective in young children (3), there is still no comprehensive vaccine for N. meningitidis. Of particular concern are serogroup B organisms, as the capsular polysaccharide is not considered to be suitable for inclusion in a vaccine due to its structural similarity with human embryonic carbohydrates (20) and poor immunogenicity (55).
To date the variation present in natural populations of N. lactamica has not been fully investigated, and the dynamics of carriage in individuals and populations are unknown. This makes an informed choice of isolates for investigation as potential vaccine candidates and the planning of any intervention strategy difficult. High-throughput nucleotide sequence determination provides a means for the rapid and comprehensive investigation of bacterial population diversity and structure (37). These techniques have been exploited in the development of isolate characterization techniques such as multilocus sequence typing (MLST) for a number of bacterial pathogens, including the meningococcus (38). Data from MLST studies have measured the high diversity of meningococcal populations and established the role of horizontal genetic exchange in the generation of this diversity (5, 16, 19, 25, 30). Although there have been no previous population studies of N. lactamica that used MLST or multilocus enzyme electrophoresis, it is plausible to suggest that this population is also diverse and recombining, on the basis of gene sequences obtained from a number of N. lactamica isolates (4, 14, 17, 36, 46, 47, 50, 54) and a previous study of N. lactamica diversity (1). An improved understanding of the population biology of N. lactamica will provide valuable insights for investigating the role that N. lactamica plays in inducing immunity against the meningococcus and in choosing isolates and subcellular components that might be suitable for inclusion in meningococcal vaccines.
The MLST scheme for N. meningitidis was adapted and optimized in order to analyze the genotypic diversity and carriage dynamics of N. lactamica isolates obtained from two longitudinal studies of bacterial carriage in infants. These data established that the population of N. lactamica was diverse and that individual infants carried single genotypes for prolonged periods.
MATERIALS AND METHODS
Bacterial isolates. (i) Study 1. Throat and nasopharyngeal swab samples were collected from a cohort of 100 infants from Oxfordshire, United Kingdom, between December 1996 and February 1999. Throat swab samples were taken at 4-week intervals from age 4 weeks during the first 24 weeks of life and at 8-week intervals for the subsequent 24 weeks. Throat swab samples were also taken from 15 infants at 56 weeks of age and from 78 infants at age 96 weeks. In addition, nasopharyngeal swab samples were taken from the majority of children at 4-week intervals from age 4 weeks until age 24 weeks and then at 8-week intervals until 96 weeks of age. Each swab collected was vortex mixed briefly in brain heart infusion (BHI) broth with 20% (vol/vol) glycerol, and the resultant suspension was stored at –80°C. These stored samples were used as a source of N. lactamica isolates.
(ii) Study 2. N. lactamica isolates were obtained from a longitudinal study of bacterial carriage in infants conducted between August 1999 and May 2001 in the same geographic area as study 1. In this survey of 216 infants, throat swab samples were taken at 2-week intervals from age 2 weeks until age 12 weeks and then at 4-week intervals until age 24 weeks. Throat swab samples were also obtained at week 2 from 166 mothers, 55 fathers, and 50 siblings of the infant participants.
Appropriate consent was obtained for all of the individuals sampled, and the studies conducted with ethical approval from the Oxford Ethics Committee, with study reference numbers C99.044 and 96.152.
Isolation and identification of N. lactamica. Samples from study 1 were plated onto a medium containing 0.04 to 0.08 mg of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) per liter dissolved in N,N-dimethylformamide (Sigma)-41 g of Columbia agar per liter-5 g of yeast extract per liter-10% horse serum-3 mg of vancomycin per liter-25,000 U of polymixin B per liter. X-Gal, a lactose analogue, was included in the growth medium to enable rapid identification of the lactose-fermenting N. lactamica species without the necessity of a further step to detect -galactosidase activity. Samples were also plated on meningitidis nutrient agar (MNA) containing 40 g of blood agar base (Oxoid) per liter, 10% horse blood, 3 mg of vancomycin per liter, and 25,000 U of polymixin B per liter so that direct comparisons could be made between the two media. The inoculated samples were incubated for 48 h at 37°C in a 5% CO2 atmosphere. Potential N. lactamica colonies were subcultured onto blood agar and further identified by oxidase reaction, Gram staining, and microscopic morphology. For colonies that grew only on MNA plates, -galactosidase activity was tested with ortho-nitrophenyl--D-galactopyranoside as the substrate. Samples from study 2 were directly inoculated onto MNA plates and incubated for 48 h at 37°C in a 5% CO2 atmosphere. Potential N. lactamica colonies were subcultured and identified by oxidase reaction, Gram staining, microscopic morphology, and ortho-nitrophenyl--D-galactopyranoside reaction. Axenic cultures of N. lactamica were stored in brain heart infusion broth with 20% (vol/vol) glycerol at –80°C.
DNA preparation and MLST. Frozen isolates were revived on blood agar, and opaque suspensions of N. lactamica cells were made in deionized water (1 ml) and immediately boiled for 15 min to lyse the cells and inactivate nucleases. Amplification of the seven MLST housekeeping gene fragments was carried out directly from the boiled cell suspensions by PCR with the oligonucleotide primers detailed in Table 1. Amplification reaction mixtures contained reaction buffer (10 mM Tris-Cl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.001% [wt/vol] gelatin); 200 μM (each) dATP, dCTP, dGTP, and dTTP; a 1 μM concentration of each primer; 1.25 U of Taq polymerase (Amplitaq; Applied Biosystems) per 50 μl; and 0.5 μl of template DNA (from the boiled cell suspensions) per 50 μl. The PCR conditions consisted of an initial denaturation step at 94°C for 2 min, followed by 35 to 40 cycles of denaturation (94°C for 1 min), annealing (55 to 70°C for 1 min), and extension (72°C for 2 min) and then a final extension step at 72°C for 2 min. The PCR products were precipitated by incubation at room temperature for 30 min with 20% (wt/vol) polyethylene glycol 8000-2.5 M NaCl. After centrifugation for 60 min at 2,750 x g, the precipitates were washed twice in 70% (vol/vol) ethanol, dried, and resuspended in 5 to 10 μl of sterile distilled water.
The nucleotide sequences of the amplified gene fragments were determined at least once on each DNA strand by cycle sequencing with BigDye Ready Reaction Mix (Applied Biosystems), used in accordance with the manufacturer's instructions. Sequencing reactions were performed with the oligonucleotide primers listed in Table 1. Unincorporated dye terminators were removed by precipitation of the termination products with 95% (vol/vol) ethanol, and the labeled extension products were separated and detected with either a Prism 3700 DNA analyzer or a Prism 377 XL DNA analyzer (Applied Biosystems). Sequences were assembled from the resultant chromatograms with the Staden suite of computer programs (49). Allele numbers and sequence types (STs) were assigned by using the Neisseria MLST database (http://pubmlst.org/neisseria/) (28).
Genetic analyses. The number of alleles, percentage of polymorphic sites, and ratio of nonsynonymous to synonymous nucleotide substitutions (dN/dS) were calculated by using the program START (29). Presumptive mosaic gene structures were visualized by using MEGA version 2.1 (34). The STs were assigned to clonal complexes by using a combination of analysis techniques. Central genotypes were identified by using the BURST algorithm (18) implemented in the START package (29), and split decomposition was identified by using SplitsTree version 3.1 (27). Members of a clonal complex were defined as isolates with STs that shared identical alleles at four or more loci with the central genotype.
Sensitivity of detection on each visit. The sensitivity of swabbing and culture to carriage was estimated among individuals who exhibited at least two isolates sharing the same ST with one or more intermediate sampling episodes. The proportion of intermediate swabbings which were positive between the first and last positive isolations was the sensitivity rate. The 95% confidence intervals were estimated, allowing for the effect of clustering within individual children, by using the STATA Svy command suite (StataCorp).
Sensitivity of studies to acquisition. Acquisition of N. lactamica might not be detected if either (i) carriage was very short, with acquisition and loss occurring between successive sampling visits, or (ii) carriage was present at a swabbing but was not detected due to imperfect sensitivity of swabbing and culture for prevalent carriage as outlined above. Using the sampling attendance and culture results for each individual, the proportion of incident infections likely to be detected in these studies was calculated by using the sensitivity of detection on each visit as described above and the rate of carriage loss estimated as described below. Assumptions were that the rate of acquisition was constant during the generally short intervals between successive swab samples and that any variation in an individual's probability of incident infection was unrelated to the completeness of his or her attendance for sampling. The overall estimates were a pooled average of the sensitivity for each participating child weighted by the duration of follow-up time in the study. Only the first 48 weeks of study 1 were considered, since sampling became infrequent during the second year.
Calculations of carriage dynamics. Acquisition rates among those without current or previous N. lactamica carriage were estimated by using survival analysis and Poisson regression. Study groups were compared graphically by using Kaplan-Meier survival curves and statistically by estimation of study effect in a Poisson regression controlling for age at acquisition. The rate of loss of carriage was estimated in the same way. Following detection, carriage was considered to extend until the last positive swab for that genetic type. For individuals with a positive swab on their last visit, the time of loss of carriage was not available. These individuals were included in the survival analysis up to this time, with censoring at the date of their last swabbing. All estimates were made both from the first detected carriage and in a corrected form to allow for the estimated delay in initial detection based on an assumption that the acquisition rate was constant in the interval prior to detection. The extent to which strains carried by parents and siblings of the study individuals overlapped with the strains carried by the study individuals was assessed.
RESULTS
Isolation of N. lactamica. In study 1, a total of 1,353 nasopharyngeal and 938 throat swab samples were analyzed. Where both throat and nasopharyngeal swab samples were taken, there were no nasopharyngeal swab samples positive for N. lactamica when the throat swab samples were negative, and only 4 (0.3%) of the nasopharyngeal swab samples were culture positive for N. lactamica, compared to 145 (15.5%) of the throat swab samples.
No meningococci were isolated from the children included in either of these two studies. A total of 250 N. lactamica isolates were obtained from the 316 infants, and 21 isolates were obtained from the 271 family members sampled. The sensitivity of each swabbing between the first and last isolations was 84% (95% confidence interval, 74 to 94%). Growth of N. lactamica appeared to be inhibited when it was grown on X-Gal medium, compared with colonies grown in parallel on MNA. In each case growth was more robust on the MNA plates, and on occasion growth was absent on X-Gal plates when a few colonies were observed on MNA. An X-Gal concentration of 0.06 mg/liter in the isolation medium gave optimal identification of N. lactamica, with colonies recognizable by a light blue coloration.
Optimization of Neisseria MLST for N. lactamica. For the aroE and pdhC gene fragments, the previously published N. meningitidis oligonucleotide primers (30) were employed for amplification and sequencing. The amplification and sequencing of the abcZ, fumC, and pgm gene fragments were performed with the published N. meningitidis sequencing primers, and additional primers were designed for the amplification and sequencing of the adk and gdh gene fragments (Table 1). The annealing temperature for PCR amplifications was 55°C, except for the pgm locus, for which an annealing temperature of 70°C gave optimal results in terms of sensitivity and specificity.
Genetic diversity of isolates. The total number of alleles present at each locus varied between 12 for abcZ and pdhC and 27 for gdh, and the percentage of polymorphic sites ranged from 3.3 for pdhC to 21.3 for pgm. The ratio of nonsynonymous to synonymous substitutions (dN/dS) in each case was less than one, ranging between 0.0229 (pdhC) and 0.4643 (aroE) (Table 2). Inspection of the polymorphic sites in the nucleotide sequences of the alleles at each locus identified possible mosaic structures (data not shown).
A total of 72 unique allelic profiles were identified among the 96 N. lactamica carriers (75 infants and 21 family members), 69 of which were obtained from infants. Of these, 54 unique allelic profiles were found in single individuals. Each of these allelic profiles was assigned a unique ST, and related STs were assigned to four clonal complexes (Table 3), with the ST-624 complex being the largest, comprising 18 STs, with 22 independent isolations. The remaining clonal complexes contained fewer STs, with the ST-613 complex consisting of five STs and the ST-595 and ST-640 complexes having three each. There were 43 STs which could not be assigned to complexes on the basis of these data alone. Given the diversity of this organism and the sample size analyzed, these clonal complex definitions must be regarded as preliminary.
Genotypic differences in carried isolates. Isolates with a single genotype were obtained from the majority (64 of 75, 85%) of carriers (Fig. 1). In study 1, most of the genotypic differences within isolates obtained from single carriers were due to multiple changes. A single child, infant 1-058, carried isolates with three unrelated genotypes; the second genotype identified shared no alleles with the first, and the third genotype identified shared one allele (abcZ40) with the first genotype but none with the second. Two different genotypes were identified from isolates obtained from throat swab samples from each of eight infants in study 1. Infant 1-051 carried isolates of ST-622 and ST-625, which are members of the ST-624 complex. ST-625 is a double-locus variant of ST-622, having 11 nucleotide substitutions in the fumC gene fragment and a single nucleotide substitution in the pdhC gene fragment. Infant 1-081 carried isolates characterized as ST-633 and ST-634. These genotypes differed by a single nucleotide substitution in the gdh gene fragment. The genotypes isolated from the other six infants (1-005, 1-014, 1-032, 1-044, 1-066, and 1-094) were unrelated, sharing between no and two alleles. One child, infant 1-097, carried two related isolates at age 40 weeks, with ST-623 obtained from the throat and ST-1191 obtained from the nasopharynx, which differed by two nucleotides in gdh. In study 2, infant 2-044 carried isolates with identical genotypes except for the isolate obtained at age 16 weeks, which had a single synonymous nucleotide substitution in the pdhC gene fragment. Infant 2-110 appeared to carry isolates with only one genotype, but due to the presence of a second abcZ gene with a deletion, this could not be confirmed.
Carriage prevalence and rate of carriage loss. Of the 316 infants (study 1 and study 2), 75 (23%) carried N. lactamica during the study period, with the majority (54, 72%) carrying the organism on multiple, usually successive, visits (Fig. 1). Carriage rates at a given point in time ranged from 1.8% (children age 2 weeks in study 2) to 43.8% (children age 56 weeks in study 1), and the age-specific carriage rates were consistent between the two studies (Table 4). Of the 54 individuals from whom the bacterium was isolated more than once, the largest number of identical isolates (nine), as defined by MLST, were obtained from infant 2-065 (study 2), who carried N. lactamica for the duration of the study. Infant 1-017 (study 1) was culture positive for identical isolates of N. lactamica for the longest recorded duration, from 8 to 96 weeks of age. Of the 21 individuals from whom N. lactamica was isolated only once, 12 were culture positive on the last visit of the study (age 96 weeks for study 1 and 24 weeks for study 2).
The rates of loss of carriage during the first 28 weeks following acquisition are summarized in Fig. 2; later loss was not estimated because the data became increasingly sparse. Carriage was persistent, with overall loss rates of 0.77% (95% confidence interval, 0.32 to 1.85%) per week during the 28 weeks following acquisition. Loss of carriage was estimated to be threefold higher in the second 14-week period than in the first, but given the relatively small numbers, there was statistical uncertainty in this estimate, with a 95% confidence interval extending from 0.5- to 18-fold.
Acquisition rates. The acquisition rates were similar for each study for the age range covered by both studies, and statistical testing did not indicate any evidence for a difference between studies (log rank P = 0.46). Modeling of acquisition rates by Poisson regression adjusted for age group again indicated no evidence for a study effect. The detected acquisition rate of 41 acquisitions per 100 infants per year over the first 48 weeks (95% confidence interval, 32 to 52) was little changed by adjustment for imperfect sensitivity of the study design to incident acquisition (adjusted result, 42 [95% confidence interval, 33 to 54]). The acquisition rate was relatively constant up to age 32 weeks, with detected acquisition rates per 100 infants per year of 34 (95% confidence interval, 19 to 61) up to age 16 weeks and 36 (21 to 63) between 16 and 32 weeks, which then increased to 102 (68 to 153) between 33 and 48 weeks of age. The estimated underlying acquisition rate was 3.33 (1.62 to 6.83) times higher in the period from 33 to 48 weeks compared to pre-16 weeks. The rate of acquisition of a second strain, measured from the time of acquisition of the first, was 28% per year (95% confidence interval, 11 to 76%). This was similar to the rate of acquisition for a first strain, but the estimate was imprecise and consistent with identical or substantially different acquisition rates for first and second strains.
Carriage by family members. Isolates of N. lactamica were obtained, at week two of study 2, from throat swab samples from one (1 of 55, 1.8%) of the fathers, six (6 of 166, 3.6%) of the mothers, and 14 (14 of 50, 28%) of the siblings of 12 infant participants. None of the strains isolated from the parents were shared with the infants; 7 of the 12 infants shared a strain with a sibling (P < 0.02 by Fisher's exact test).
DISCUSSION
The dynamics of carriage and population diversity of N. lactamica will influence any immunity to meningococcal disease that is generated by the asymptomatic carriage of this commensal during infancy (6, 21, 22). As N. lactamica is acapsulate (22), this immunity would have to be active against subcapsular components of the meningococcus, and given the antigenic and genetic diversity of the meningococcal populations (10), any active protection induced by N. lactamica would also have to be cross-protective against many meningococcal strains. Such broad protection could, for example, be a consequence of exposure to many different N. lactamica strains, resulting in an immune response against many variants of a diverse Neisseria cell component, such as one of the porins (14). Alternatively, exposure to a single N. lactamica strain could result in immunity to cell components that are highly conserved among members of the genus Neisseria. These alternative scenarios have different implications for vaccine development; the former implies that a multicomponent vaccine, based on many different N. lactamica strains, would be required to mimic natural immunity, while the latter suggests that a vaccine based on a single strain could be effective. The molecular genetic typing of the N. lactamica isolates obtained from the longitudinal sampling of infants reported here suggests that N. lactamica populations are highly diverse and that the prevalence of carriage among infants is high. As most children were colonized by a single N. lactamica strain for a prolonged period of time, these data provide support for the concept of antimeningococcal immunity arising from exposure to a single N. lactamica isolate. This raises the question of whether the long duration of carriage is important in conferring this immunity.
The age at which carriage was highest was consistent with previous reports of N. lactamica carriage (2, 9, 21, 41, 45), with rates increasing through the first 2 years of life and no detectable carriage of N. meningitidis. The efficiency of N. lactamica isolation (84%) on repeat sampling was comparable to that obtained in a previous study (41), and the isolation rates in the two studies described here were indistinguishable, notwithstanding the fact that the swab samples from study 1 had been frozen prior to culture for N. lactamica, whereas the isolates from study 2 were obtained after direct plating. Although the samples were collected in the context of studies on the carriage of other respiratory pathogens and as a consequence did not fully cover the ages at which maximum carriage of N. lactamica have been reported (2, 41, 45), the observed isolation rates support the contention that swab samples taken from the throat are more efficient for the isolation of this organism than those taken from the nasopharynx of young children.
The N. meningitidis MLST scheme (25, 38), was easily adapted to N. lactamica, providing the first isolate characterization scheme for this organism. Given the close genetic relationship between these two bacteria (24) and the reported sharing of gene sequences between them (17), the N. lactamica data were deposited in the same database as the meningococcal MLST data (http://pubmlst.org/neisseria) (28, 53). This avoided the possibility of the same allele sequence being assigned to different MLST allele numbers in different schemes. Further, N. lactamica isolates, especially those from carriage, are occasionally misidentified by microbiological techniques (52), and inclusion of both species in the same database has the advantage that such misclassifications are immediately apparent. MLST data may assist in future sequence-based species definitions of closely related organisms such as these (48).
The MLST analysis revealed patterns of genetic diversity largely similar to those observed in meningococci (10, 38) with respect to numbers of polymorphic sites, dN/dS ratios, numbers of alleles at each MLST locus, and numbers of STs (25, 30, 38), although there were some differences in locus-by-locus comparisons. The high levels of diversity seen were consistent with previous studies using phenotypic analysis (21), macrorestriction analysis (45), and nucleotide sequence-based methods (1). Visual inspection of the N. lactamica gene sequences identified possible mosaic structures at many of the loci, and these observations, together with the sharing of identical alleles in otherwise unrelated sequence types (e.g., the fumC100 allele, which was shared between ST-614 and ST-620 [Table 3]), were consistent with horizontal genetic exchange playing a major role in generating population structure in N. lactamica, as seen in meningococcal populations (25). It is also likely that N. lactamica populations contain clonal complexes, although, as a consequence of the levels of diversity present in these isolate collections, few of these complexes could be defined with confidence from these data alone.
The availability of reliable molecular typing enabled accurate estimates for acquisition rates and the duration of carriage. The data show that the first 2 years of life are characterized by rates of acquisition that exceed rates of loss of carriage, resulting in rising carriage prevalence within individuals. Once an individual had acquired a given genotype, this could be recovered for prolonged periods, and there was no evidence for a rapid acquisition and loss of multiple genotypes. A previous study (21), which did not have the benefit of molecular isolate characterization, reported median carriage durations of 3.8 months in infants 3 to 13 months of age and of 3 months in 6- to 8-year-olds as determined by using a life table method; the persistence of carriage described here is inconsistent with a carriage duration as short as this. Possible explanations for this include inherent differences in the populations studied or differences in the sensitivities of the sampling techniques employed. Persistence of single strains within individuals was reported in the previous study (21), with two-thirds of infants whose cultures were positive more than once in the first year of life appearing to carry the same organism on each occasion when tested with meningococcal typing antisera. The increase in the acquisition rate observed between the ages of 32 weeks and 1 year in the present study might be a consequence of the decline in maternally derived antibodies and/or the increased opportunity for exposure to other children carrying N. lactamica at this age.
Study 2 provided some information on within-family transmission, suggesting that infants are more likely to acquire N. lactamica from siblings and other children than from their parents. The high prevalence of N. lactamica carriage in young children reported in this study and others may be a consequence of their high milk consumption (22) and the ability of N. lactamica to ferment lactose. Carriage of N. lactamica is low in young adult males (33) and in the elderly, both male and female (32), who are less likely to have prolonged contact with children. In adults age 21 to 59 years, the majority of N. lactamica was isolated from women (32), possibly reflecting their longer or closer contact with children, but as N. lactamica has evolved in a niche where lactose is prevalent, it is possible that carriage in adults may be lower as a consequence of differences in adult and infant diets.
Despite the interest in the exploitation of this bacterium in vaccines, the mechanisms responsible for the age-related variation in carriage of N. lactamica and N. meningitidis are not well understood. Further, the mechanisms for the induction of immunity to meningococcal disease by N. lactamica carriage have not been established. It has been documented that antibodies that are cross-reactive against meningococci are present in infants after carriage of N. lactamica (6, 21, 22), and it has been suggested that bacterial interference is a protective factor, with the more predominant N. lactamica inhibiting colonization by N. meningitidis (21). If this is correct, then the widespread intentional colonization by N. lactamica strains could enhance natural protection (23) and inhibit colonization by N. meningitidis. It has been suggested that live attenuated meningococci could be used in a vaccine (51), but as the virulence factors associated with N. meningitidis are poorly understood, a less hazardous approach would be to employ N. lactamica, which has been shown to have cross-reactive surface antigens (22, 31), in vaccines. Whatever the mechanisms or extent of immunity induced by N. lactamica against meningococcal disease, this study demonstrates that such immunity is likely to be induced by the prolonged carriage of a single genotype rather than from the short-term carriage of multiple genotypes of this highly diverse commensal bacterium.
ACKNOWLEDGMENTS
We are grateful to the staff of the Oxford Vaccine Group, and Linda Diggle in particular, for supporting the collection of samples. This publication made use of the Neisseria multilocus sequence typing website (http://pubmlst.org/neisseria/) developed by Keith Jolley and Man-Suen Chan and sited at the University of Oxford. We thank all of the families and children who participated in this study.
This study was funded by the Meningitis Research Foundation. M.C.J.M. is a Wellcome Trust Senior Research Fellow, N.M.C. is a Wellcome Trust Clinical Training Fellow, and D.W.C. was a Wellcome Leave Fellow at the time of the study. The nasopharyngeal and throat swab samples were obtained from studies carried out by the Oxford Vaccine Group, supported by the Wellcome Trust (reference number 056886/2/994/Z). The development of the Neisseria multilocus sequence typing website has been funded by the Wellcome Trust and the European Union.
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Nuffield Department of Clinical Microbiology
Oxford Vaccine Group, Department of Paediatrics, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom
ABSTRACT
Neisseria lactamica, a harmless human commensal found predominantly in the upper respiratory tracts of infants, is closely related to Neisseria meningitidis, a pathogen of global significance. Colonization with N. lactamica may be responsible for the increase in immunity to meningococcal disease that occurs during childhood, when rates of meningococcal carriage are low. This observation has led to the suggestion that N. lactamica whole cells or components are potential constituents of novel meningococcal vaccines. However, the dynamics of carriage and population diversity of N. lactamica in children are poorly understood, presenting difficulties for the choice of representative isolates for use in vaccine development. This problem was addressed by the multilocus sequence typing of N. lactamica isolates from two longitudinal studies of bacterial carriage in infants. The studies comprised 100 and 216 subjects, with N. lactamica carriage monitored from age 4 weeks until age 96 weeks and from age 2 weeks until age 24 weeks, respectively. The maximum observed carriage rate was 44% at 56 weeks of age, with isolates obtained on multiple visits for the majority (54 of 75, 72%) of carriers. The N. lactamica isolates were genetically diverse, with 69 distinct genotypes recovered from the 75 infants. Carriage was generally long-lived, with an average rate of loss of under 1% per week during the 28 weeks following acquisition. Only 11 of the 75 infants carried more than one genotypically unique isolate during the course of the study. Some participants shared identical isolates with siblings, but none shared identical isolates with their parents. These findings have implications for the design of vaccines based on this organism.
INTRODUCTION
The genus Neisseria comprises gram-negative, oxidase-positive diplococci that are frequently isolated from the mucosal surfaces of humans and animals (39). Two members of the genus cause distinct human diseases: Neisseria meningitidis, which can be responsible for septicemia and meningitis (8), and Neisseria gonorrhoeae, the etiological agent of gonorrhea (44). Neisseria lactamica is closely related to both of these species (24) but is associated with invasive disease only in exceptional circumstances (13, 35, 42). Carriage of N. lactamica throughout the world is high in infants and young children but declines with age. This is the converse of the carriage of the meningococcus, which is low during infancy and rises to high levels in adolescents and young adults (2, 9, 21, 26, 41, 43).
The observation that antimeningococcal bactericidal titers rise steadily in infants, despite low rates of meningococcal carriage, has provided indirect support for the concept that carriage of N. lactamica by children (9) may confer protection against disease caused by N. meningitidis (7). This suggestion has in turn led to the proposal that N. lactamica could be used to induce protection against meningococcal infection, by using killed whole cells, outer membrane vesicles, or outer membrane proteins, as demonstrated in a murine model (40), or by the deliberate inoculation of infants with live N. lactamica cultures (23).
One of the factors that has proved to be a hindrance to the development of vaccines against the meningococcus is its genetic and antigenic diversity (11, 12, 15). Although progress has been made in the development of vaccines to serogroup A and C meningococci, with a polysaccharide-protein conjugate vaccine to serogroup C meningococci found to be effective in young children (3), there is still no comprehensive vaccine for N. meningitidis. Of particular concern are serogroup B organisms, as the capsular polysaccharide is not considered to be suitable for inclusion in a vaccine due to its structural similarity with human embryonic carbohydrates (20) and poor immunogenicity (55).
To date the variation present in natural populations of N. lactamica has not been fully investigated, and the dynamics of carriage in individuals and populations are unknown. This makes an informed choice of isolates for investigation as potential vaccine candidates and the planning of any intervention strategy difficult. High-throughput nucleotide sequence determination provides a means for the rapid and comprehensive investigation of bacterial population diversity and structure (37). These techniques have been exploited in the development of isolate characterization techniques such as multilocus sequence typing (MLST) for a number of bacterial pathogens, including the meningococcus (38). Data from MLST studies have measured the high diversity of meningococcal populations and established the role of horizontal genetic exchange in the generation of this diversity (5, 16, 19, 25, 30). Although there have been no previous population studies of N. lactamica that used MLST or multilocus enzyme electrophoresis, it is plausible to suggest that this population is also diverse and recombining, on the basis of gene sequences obtained from a number of N. lactamica isolates (4, 14, 17, 36, 46, 47, 50, 54) and a previous study of N. lactamica diversity (1). An improved understanding of the population biology of N. lactamica will provide valuable insights for investigating the role that N. lactamica plays in inducing immunity against the meningococcus and in choosing isolates and subcellular components that might be suitable for inclusion in meningococcal vaccines.
The MLST scheme for N. meningitidis was adapted and optimized in order to analyze the genotypic diversity and carriage dynamics of N. lactamica isolates obtained from two longitudinal studies of bacterial carriage in infants. These data established that the population of N. lactamica was diverse and that individual infants carried single genotypes for prolonged periods.
MATERIALS AND METHODS
Bacterial isolates. (i) Study 1. Throat and nasopharyngeal swab samples were collected from a cohort of 100 infants from Oxfordshire, United Kingdom, between December 1996 and February 1999. Throat swab samples were taken at 4-week intervals from age 4 weeks during the first 24 weeks of life and at 8-week intervals for the subsequent 24 weeks. Throat swab samples were also taken from 15 infants at 56 weeks of age and from 78 infants at age 96 weeks. In addition, nasopharyngeal swab samples were taken from the majority of children at 4-week intervals from age 4 weeks until age 24 weeks and then at 8-week intervals until 96 weeks of age. Each swab collected was vortex mixed briefly in brain heart infusion (BHI) broth with 20% (vol/vol) glycerol, and the resultant suspension was stored at –80°C. These stored samples were used as a source of N. lactamica isolates.
(ii) Study 2. N. lactamica isolates were obtained from a longitudinal study of bacterial carriage in infants conducted between August 1999 and May 2001 in the same geographic area as study 1. In this survey of 216 infants, throat swab samples were taken at 2-week intervals from age 2 weeks until age 12 weeks and then at 4-week intervals until age 24 weeks. Throat swab samples were also obtained at week 2 from 166 mothers, 55 fathers, and 50 siblings of the infant participants.
Appropriate consent was obtained for all of the individuals sampled, and the studies conducted with ethical approval from the Oxford Ethics Committee, with study reference numbers C99.044 and 96.152.
Isolation and identification of N. lactamica. Samples from study 1 were plated onto a medium containing 0.04 to 0.08 mg of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) per liter dissolved in N,N-dimethylformamide (Sigma)-41 g of Columbia agar per liter-5 g of yeast extract per liter-10% horse serum-3 mg of vancomycin per liter-25,000 U of polymixin B per liter. X-Gal, a lactose analogue, was included in the growth medium to enable rapid identification of the lactose-fermenting N. lactamica species without the necessity of a further step to detect -galactosidase activity. Samples were also plated on meningitidis nutrient agar (MNA) containing 40 g of blood agar base (Oxoid) per liter, 10% horse blood, 3 mg of vancomycin per liter, and 25,000 U of polymixin B per liter so that direct comparisons could be made between the two media. The inoculated samples were incubated for 48 h at 37°C in a 5% CO2 atmosphere. Potential N. lactamica colonies were subcultured onto blood agar and further identified by oxidase reaction, Gram staining, and microscopic morphology. For colonies that grew only on MNA plates, -galactosidase activity was tested with ortho-nitrophenyl--D-galactopyranoside as the substrate. Samples from study 2 were directly inoculated onto MNA plates and incubated for 48 h at 37°C in a 5% CO2 atmosphere. Potential N. lactamica colonies were subcultured and identified by oxidase reaction, Gram staining, microscopic morphology, and ortho-nitrophenyl--D-galactopyranoside reaction. Axenic cultures of N. lactamica were stored in brain heart infusion broth with 20% (vol/vol) glycerol at –80°C.
DNA preparation and MLST. Frozen isolates were revived on blood agar, and opaque suspensions of N. lactamica cells were made in deionized water (1 ml) and immediately boiled for 15 min to lyse the cells and inactivate nucleases. Amplification of the seven MLST housekeeping gene fragments was carried out directly from the boiled cell suspensions by PCR with the oligonucleotide primers detailed in Table 1. Amplification reaction mixtures contained reaction buffer (10 mM Tris-Cl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.001% [wt/vol] gelatin); 200 μM (each) dATP, dCTP, dGTP, and dTTP; a 1 μM concentration of each primer; 1.25 U of Taq polymerase (Amplitaq; Applied Biosystems) per 50 μl; and 0.5 μl of template DNA (from the boiled cell suspensions) per 50 μl. The PCR conditions consisted of an initial denaturation step at 94°C for 2 min, followed by 35 to 40 cycles of denaturation (94°C for 1 min), annealing (55 to 70°C for 1 min), and extension (72°C for 2 min) and then a final extension step at 72°C for 2 min. The PCR products were precipitated by incubation at room temperature for 30 min with 20% (wt/vol) polyethylene glycol 8000-2.5 M NaCl. After centrifugation for 60 min at 2,750 x g, the precipitates were washed twice in 70% (vol/vol) ethanol, dried, and resuspended in 5 to 10 μl of sterile distilled water.
The nucleotide sequences of the amplified gene fragments were determined at least once on each DNA strand by cycle sequencing with BigDye Ready Reaction Mix (Applied Biosystems), used in accordance with the manufacturer's instructions. Sequencing reactions were performed with the oligonucleotide primers listed in Table 1. Unincorporated dye terminators were removed by precipitation of the termination products with 95% (vol/vol) ethanol, and the labeled extension products were separated and detected with either a Prism 3700 DNA analyzer or a Prism 377 XL DNA analyzer (Applied Biosystems). Sequences were assembled from the resultant chromatograms with the Staden suite of computer programs (49). Allele numbers and sequence types (STs) were assigned by using the Neisseria MLST database (http://pubmlst.org/neisseria/) (28).
Genetic analyses. The number of alleles, percentage of polymorphic sites, and ratio of nonsynonymous to synonymous nucleotide substitutions (dN/dS) were calculated by using the program START (29). Presumptive mosaic gene structures were visualized by using MEGA version 2.1 (34). The STs were assigned to clonal complexes by using a combination of analysis techniques. Central genotypes were identified by using the BURST algorithm (18) implemented in the START package (29), and split decomposition was identified by using SplitsTree version 3.1 (27). Members of a clonal complex were defined as isolates with STs that shared identical alleles at four or more loci with the central genotype.
Sensitivity of detection on each visit. The sensitivity of swabbing and culture to carriage was estimated among individuals who exhibited at least two isolates sharing the same ST with one or more intermediate sampling episodes. The proportion of intermediate swabbings which were positive between the first and last positive isolations was the sensitivity rate. The 95% confidence intervals were estimated, allowing for the effect of clustering within individual children, by using the STATA Svy command suite (StataCorp).
Sensitivity of studies to acquisition. Acquisition of N. lactamica might not be detected if either (i) carriage was very short, with acquisition and loss occurring between successive sampling visits, or (ii) carriage was present at a swabbing but was not detected due to imperfect sensitivity of swabbing and culture for prevalent carriage as outlined above. Using the sampling attendance and culture results for each individual, the proportion of incident infections likely to be detected in these studies was calculated by using the sensitivity of detection on each visit as described above and the rate of carriage loss estimated as described below. Assumptions were that the rate of acquisition was constant during the generally short intervals between successive swab samples and that any variation in an individual's probability of incident infection was unrelated to the completeness of his or her attendance for sampling. The overall estimates were a pooled average of the sensitivity for each participating child weighted by the duration of follow-up time in the study. Only the first 48 weeks of study 1 were considered, since sampling became infrequent during the second year.
Calculations of carriage dynamics. Acquisition rates among those without current or previous N. lactamica carriage were estimated by using survival analysis and Poisson regression. Study groups were compared graphically by using Kaplan-Meier survival curves and statistically by estimation of study effect in a Poisson regression controlling for age at acquisition. The rate of loss of carriage was estimated in the same way. Following detection, carriage was considered to extend until the last positive swab for that genetic type. For individuals with a positive swab on their last visit, the time of loss of carriage was not available. These individuals were included in the survival analysis up to this time, with censoring at the date of their last swabbing. All estimates were made both from the first detected carriage and in a corrected form to allow for the estimated delay in initial detection based on an assumption that the acquisition rate was constant in the interval prior to detection. The extent to which strains carried by parents and siblings of the study individuals overlapped with the strains carried by the study individuals was assessed.
RESULTS
Isolation of N. lactamica. In study 1, a total of 1,353 nasopharyngeal and 938 throat swab samples were analyzed. Where both throat and nasopharyngeal swab samples were taken, there were no nasopharyngeal swab samples positive for N. lactamica when the throat swab samples were negative, and only 4 (0.3%) of the nasopharyngeal swab samples were culture positive for N. lactamica, compared to 145 (15.5%) of the throat swab samples.
No meningococci were isolated from the children included in either of these two studies. A total of 250 N. lactamica isolates were obtained from the 316 infants, and 21 isolates were obtained from the 271 family members sampled. The sensitivity of each swabbing between the first and last isolations was 84% (95% confidence interval, 74 to 94%). Growth of N. lactamica appeared to be inhibited when it was grown on X-Gal medium, compared with colonies grown in parallel on MNA. In each case growth was more robust on the MNA plates, and on occasion growth was absent on X-Gal plates when a few colonies were observed on MNA. An X-Gal concentration of 0.06 mg/liter in the isolation medium gave optimal identification of N. lactamica, with colonies recognizable by a light blue coloration.
Optimization of Neisseria MLST for N. lactamica. For the aroE and pdhC gene fragments, the previously published N. meningitidis oligonucleotide primers (30) were employed for amplification and sequencing. The amplification and sequencing of the abcZ, fumC, and pgm gene fragments were performed with the published N. meningitidis sequencing primers, and additional primers were designed for the amplification and sequencing of the adk and gdh gene fragments (Table 1). The annealing temperature for PCR amplifications was 55°C, except for the pgm locus, for which an annealing temperature of 70°C gave optimal results in terms of sensitivity and specificity.
Genetic diversity of isolates. The total number of alleles present at each locus varied between 12 for abcZ and pdhC and 27 for gdh, and the percentage of polymorphic sites ranged from 3.3 for pdhC to 21.3 for pgm. The ratio of nonsynonymous to synonymous substitutions (dN/dS) in each case was less than one, ranging between 0.0229 (pdhC) and 0.4643 (aroE) (Table 2). Inspection of the polymorphic sites in the nucleotide sequences of the alleles at each locus identified possible mosaic structures (data not shown).
A total of 72 unique allelic profiles were identified among the 96 N. lactamica carriers (75 infants and 21 family members), 69 of which were obtained from infants. Of these, 54 unique allelic profiles were found in single individuals. Each of these allelic profiles was assigned a unique ST, and related STs were assigned to four clonal complexes (Table 3), with the ST-624 complex being the largest, comprising 18 STs, with 22 independent isolations. The remaining clonal complexes contained fewer STs, with the ST-613 complex consisting of five STs and the ST-595 and ST-640 complexes having three each. There were 43 STs which could not be assigned to complexes on the basis of these data alone. Given the diversity of this organism and the sample size analyzed, these clonal complex definitions must be regarded as preliminary.
Genotypic differences in carried isolates. Isolates with a single genotype were obtained from the majority (64 of 75, 85%) of carriers (Fig. 1). In study 1, most of the genotypic differences within isolates obtained from single carriers were due to multiple changes. A single child, infant 1-058, carried isolates with three unrelated genotypes; the second genotype identified shared no alleles with the first, and the third genotype identified shared one allele (abcZ40) with the first genotype but none with the second. Two different genotypes were identified from isolates obtained from throat swab samples from each of eight infants in study 1. Infant 1-051 carried isolates of ST-622 and ST-625, which are members of the ST-624 complex. ST-625 is a double-locus variant of ST-622, having 11 nucleotide substitutions in the fumC gene fragment and a single nucleotide substitution in the pdhC gene fragment. Infant 1-081 carried isolates characterized as ST-633 and ST-634. These genotypes differed by a single nucleotide substitution in the gdh gene fragment. The genotypes isolated from the other six infants (1-005, 1-014, 1-032, 1-044, 1-066, and 1-094) were unrelated, sharing between no and two alleles. One child, infant 1-097, carried two related isolates at age 40 weeks, with ST-623 obtained from the throat and ST-1191 obtained from the nasopharynx, which differed by two nucleotides in gdh. In study 2, infant 2-044 carried isolates with identical genotypes except for the isolate obtained at age 16 weeks, which had a single synonymous nucleotide substitution in the pdhC gene fragment. Infant 2-110 appeared to carry isolates with only one genotype, but due to the presence of a second abcZ gene with a deletion, this could not be confirmed.
Carriage prevalence and rate of carriage loss. Of the 316 infants (study 1 and study 2), 75 (23%) carried N. lactamica during the study period, with the majority (54, 72%) carrying the organism on multiple, usually successive, visits (Fig. 1). Carriage rates at a given point in time ranged from 1.8% (children age 2 weeks in study 2) to 43.8% (children age 56 weeks in study 1), and the age-specific carriage rates were consistent between the two studies (Table 4). Of the 54 individuals from whom the bacterium was isolated more than once, the largest number of identical isolates (nine), as defined by MLST, were obtained from infant 2-065 (study 2), who carried N. lactamica for the duration of the study. Infant 1-017 (study 1) was culture positive for identical isolates of N. lactamica for the longest recorded duration, from 8 to 96 weeks of age. Of the 21 individuals from whom N. lactamica was isolated only once, 12 were culture positive on the last visit of the study (age 96 weeks for study 1 and 24 weeks for study 2).
The rates of loss of carriage during the first 28 weeks following acquisition are summarized in Fig. 2; later loss was not estimated because the data became increasingly sparse. Carriage was persistent, with overall loss rates of 0.77% (95% confidence interval, 0.32 to 1.85%) per week during the 28 weeks following acquisition. Loss of carriage was estimated to be threefold higher in the second 14-week period than in the first, but given the relatively small numbers, there was statistical uncertainty in this estimate, with a 95% confidence interval extending from 0.5- to 18-fold.
Acquisition rates. The acquisition rates were similar for each study for the age range covered by both studies, and statistical testing did not indicate any evidence for a difference between studies (log rank P = 0.46). Modeling of acquisition rates by Poisson regression adjusted for age group again indicated no evidence for a study effect. The detected acquisition rate of 41 acquisitions per 100 infants per year over the first 48 weeks (95% confidence interval, 32 to 52) was little changed by adjustment for imperfect sensitivity of the study design to incident acquisition (adjusted result, 42 [95% confidence interval, 33 to 54]). The acquisition rate was relatively constant up to age 32 weeks, with detected acquisition rates per 100 infants per year of 34 (95% confidence interval, 19 to 61) up to age 16 weeks and 36 (21 to 63) between 16 and 32 weeks, which then increased to 102 (68 to 153) between 33 and 48 weeks of age. The estimated underlying acquisition rate was 3.33 (1.62 to 6.83) times higher in the period from 33 to 48 weeks compared to pre-16 weeks. The rate of acquisition of a second strain, measured from the time of acquisition of the first, was 28% per year (95% confidence interval, 11 to 76%). This was similar to the rate of acquisition for a first strain, but the estimate was imprecise and consistent with identical or substantially different acquisition rates for first and second strains.
Carriage by family members. Isolates of N. lactamica were obtained, at week two of study 2, from throat swab samples from one (1 of 55, 1.8%) of the fathers, six (6 of 166, 3.6%) of the mothers, and 14 (14 of 50, 28%) of the siblings of 12 infant participants. None of the strains isolated from the parents were shared with the infants; 7 of the 12 infants shared a strain with a sibling (P < 0.02 by Fisher's exact test).
DISCUSSION
The dynamics of carriage and population diversity of N. lactamica will influence any immunity to meningococcal disease that is generated by the asymptomatic carriage of this commensal during infancy (6, 21, 22). As N. lactamica is acapsulate (22), this immunity would have to be active against subcapsular components of the meningococcus, and given the antigenic and genetic diversity of the meningococcal populations (10), any active protection induced by N. lactamica would also have to be cross-protective against many meningococcal strains. Such broad protection could, for example, be a consequence of exposure to many different N. lactamica strains, resulting in an immune response against many variants of a diverse Neisseria cell component, such as one of the porins (14). Alternatively, exposure to a single N. lactamica strain could result in immunity to cell components that are highly conserved among members of the genus Neisseria. These alternative scenarios have different implications for vaccine development; the former implies that a multicomponent vaccine, based on many different N. lactamica strains, would be required to mimic natural immunity, while the latter suggests that a vaccine based on a single strain could be effective. The molecular genetic typing of the N. lactamica isolates obtained from the longitudinal sampling of infants reported here suggests that N. lactamica populations are highly diverse and that the prevalence of carriage among infants is high. As most children were colonized by a single N. lactamica strain for a prolonged period of time, these data provide support for the concept of antimeningococcal immunity arising from exposure to a single N. lactamica isolate. This raises the question of whether the long duration of carriage is important in conferring this immunity.
The age at which carriage was highest was consistent with previous reports of N. lactamica carriage (2, 9, 21, 41, 45), with rates increasing through the first 2 years of life and no detectable carriage of N. meningitidis. The efficiency of N. lactamica isolation (84%) on repeat sampling was comparable to that obtained in a previous study (41), and the isolation rates in the two studies described here were indistinguishable, notwithstanding the fact that the swab samples from study 1 had been frozen prior to culture for N. lactamica, whereas the isolates from study 2 were obtained after direct plating. Although the samples were collected in the context of studies on the carriage of other respiratory pathogens and as a consequence did not fully cover the ages at which maximum carriage of N. lactamica have been reported (2, 41, 45), the observed isolation rates support the contention that swab samples taken from the throat are more efficient for the isolation of this organism than those taken from the nasopharynx of young children.
The N. meningitidis MLST scheme (25, 38), was easily adapted to N. lactamica, providing the first isolate characterization scheme for this organism. Given the close genetic relationship between these two bacteria (24) and the reported sharing of gene sequences between them (17), the N. lactamica data were deposited in the same database as the meningococcal MLST data (http://pubmlst.org/neisseria) (28, 53). This avoided the possibility of the same allele sequence being assigned to different MLST allele numbers in different schemes. Further, N. lactamica isolates, especially those from carriage, are occasionally misidentified by microbiological techniques (52), and inclusion of both species in the same database has the advantage that such misclassifications are immediately apparent. MLST data may assist in future sequence-based species definitions of closely related organisms such as these (48).
The MLST analysis revealed patterns of genetic diversity largely similar to those observed in meningococci (10, 38) with respect to numbers of polymorphic sites, dN/dS ratios, numbers of alleles at each MLST locus, and numbers of STs (25, 30, 38), although there were some differences in locus-by-locus comparisons. The high levels of diversity seen were consistent with previous studies using phenotypic analysis (21), macrorestriction analysis (45), and nucleotide sequence-based methods (1). Visual inspection of the N. lactamica gene sequences identified possible mosaic structures at many of the loci, and these observations, together with the sharing of identical alleles in otherwise unrelated sequence types (e.g., the fumC100 allele, which was shared between ST-614 and ST-620 [Table 3]), were consistent with horizontal genetic exchange playing a major role in generating population structure in N. lactamica, as seen in meningococcal populations (25). It is also likely that N. lactamica populations contain clonal complexes, although, as a consequence of the levels of diversity present in these isolate collections, few of these complexes could be defined with confidence from these data alone.
The availability of reliable molecular typing enabled accurate estimates for acquisition rates and the duration of carriage. The data show that the first 2 years of life are characterized by rates of acquisition that exceed rates of loss of carriage, resulting in rising carriage prevalence within individuals. Once an individual had acquired a given genotype, this could be recovered for prolonged periods, and there was no evidence for a rapid acquisition and loss of multiple genotypes. A previous study (21), which did not have the benefit of molecular isolate characterization, reported median carriage durations of 3.8 months in infants 3 to 13 months of age and of 3 months in 6- to 8-year-olds as determined by using a life table method; the persistence of carriage described here is inconsistent with a carriage duration as short as this. Possible explanations for this include inherent differences in the populations studied or differences in the sensitivities of the sampling techniques employed. Persistence of single strains within individuals was reported in the previous study (21), with two-thirds of infants whose cultures were positive more than once in the first year of life appearing to carry the same organism on each occasion when tested with meningococcal typing antisera. The increase in the acquisition rate observed between the ages of 32 weeks and 1 year in the present study might be a consequence of the decline in maternally derived antibodies and/or the increased opportunity for exposure to other children carrying N. lactamica at this age.
Study 2 provided some information on within-family transmission, suggesting that infants are more likely to acquire N. lactamica from siblings and other children than from their parents. The high prevalence of N. lactamica carriage in young children reported in this study and others may be a consequence of their high milk consumption (22) and the ability of N. lactamica to ferment lactose. Carriage of N. lactamica is low in young adult males (33) and in the elderly, both male and female (32), who are less likely to have prolonged contact with children. In adults age 21 to 59 years, the majority of N. lactamica was isolated from women (32), possibly reflecting their longer or closer contact with children, but as N. lactamica has evolved in a niche where lactose is prevalent, it is possible that carriage in adults may be lower as a consequence of differences in adult and infant diets.
Despite the interest in the exploitation of this bacterium in vaccines, the mechanisms responsible for the age-related variation in carriage of N. lactamica and N. meningitidis are not well understood. Further, the mechanisms for the induction of immunity to meningococcal disease by N. lactamica carriage have not been established. It has been documented that antibodies that are cross-reactive against meningococci are present in infants after carriage of N. lactamica (6, 21, 22), and it has been suggested that bacterial interference is a protective factor, with the more predominant N. lactamica inhibiting colonization by N. meningitidis (21). If this is correct, then the widespread intentional colonization by N. lactamica strains could enhance natural protection (23) and inhibit colonization by N. meningitidis. It has been suggested that live attenuated meningococci could be used in a vaccine (51), but as the virulence factors associated with N. meningitidis are poorly understood, a less hazardous approach would be to employ N. lactamica, which has been shown to have cross-reactive surface antigens (22, 31), in vaccines. Whatever the mechanisms or extent of immunity induced by N. lactamica against meningococcal disease, this study demonstrates that such immunity is likely to be induced by the prolonged carriage of a single genotype rather than from the short-term carriage of multiple genotypes of this highly diverse commensal bacterium.
ACKNOWLEDGMENTS
We are grateful to the staff of the Oxford Vaccine Group, and Linda Diggle in particular, for supporting the collection of samples. This publication made use of the Neisseria multilocus sequence typing website (http://pubmlst.org/neisseria/) developed by Keith Jolley and Man-Suen Chan and sited at the University of Oxford. We thank all of the families and children who participated in this study.
This study was funded by the Meningitis Research Foundation. M.C.J.M. is a Wellcome Trust Senior Research Fellow, N.M.C. is a Wellcome Trust Clinical Training Fellow, and D.W.C. was a Wellcome Leave Fellow at the time of the study. The nasopharyngeal and throat swab samples were obtained from studies carried out by the Oxford Vaccine Group, supported by the Wellcome Trust (reference number 056886/2/994/Z). The development of the Neisseria multilocus sequence typing website has been funded by the Wellcome Trust and the European Union.
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