Division of Genomic Medicine, F Floor, Sheffield University Medical School, Beech Hill Road, Sheffield S10 2RX, UK1
School of Public Health, University of Nottingham, UK2
Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, UK3
Author for correspondence: Robert C. Read. Tel: +44 114 272 4072. Fax: +44 114 273 9926. e-mail: r.c.read{at}shef.ac.uk
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ABSTRACT |
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Keywords: pathogenesis, colonization, Neisseria lactamica, Neisseria animalis
Abbreviations: MBC, mean bactericidal concentration
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INTRODUCTION |
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Humans can be colonized by commensal Neisseria including N. lactamica, which rarely causes disease. Colonization by N. lactamica is frequent in infants but declines to relatively low rates in teenagers and adults (Cartwright et al., 1987 ; Gold et al., 1978
). It is possible that N. lactamica displaces N. meningitidis from the nasopharynx in childhood and produces natural immunity against invasive meningococcal disease (Coen et al., 2000
) as a consequence of immunogenic epitopes shared between N. lactamica and N. meningitidis.
Organ culture permits the study of the interaction of N. meningitidis with a tissue which has physiologically relevant cellular and matriceal components. A number of groups have used organ culture to study biology of the interaction of N. meningitidis with human airway mucosa. Meningococci attach selectively to non-ciliated columnar cells, and during this process microvilli of non-ciliated cells elongate and surround the organisms. Meningococci appear to undergo parasite-directed endocytosis and are observed in subepithelial tissues adjacent to lymphoid tissue after prolonged incubation (Stephens et al., 1983 ), though this is observed in a minority of explants (Read et al., 1995
). Pili and capsular polysaccharide both influence association of N. meningitidis with nasopharyngeal mucosa (Rayner et al., 1995
; Stephens et al., 1993
), but concurrent switching of multiple phase-variable bacterial surface components such as these, and outer-membrane proteins including Opa, appears to occur during successful invasion of this tissue (de Vries et al., 1996
).
In this work, a model of survival of Neisseria spp. within human nasopharyngeal mucosa was developed, and the success of various Neisseria spp. within this tissue was compared. The variation of survival of N. meningitidis with the mucosae of a large number of donor human tissues was then measured.
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METHODS |
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Organ culture.
A modified technique of organ culture using explants of human nasal turbinate mucosa was used (Jackson et al., 1996 ; Read & Goodwin, 2001
). Inferior turbinates derived from patients with non-allergic nasal obstruction were resected in all cases by the same surgeon. All donors gave informed consent and the research was approved by the South Sheffield Research Ethics Committee (96/260). Tissue was transported to the laboratory in Minimal Essential Medium (MEM) containing penicillin (50000 units l-1), streptomycin (50 mg l-1) and gentamicin (50 mg l-1) and dissected to produce 34 mm squares of mucosa, after removal of the anterior pole of the turbinate, which can be heavily populated by squamous epithelium. Tissue was incubated in antibiotic-containing MEM for a total of 4 h before being immersed in 20 ml antibiotic-free MEM for 1 h. Homogenates of explants treated in this way exhibited no antibiotic activity in bioassay (data not shown), suggesting that the methods used did not result in accumulation of antibiotics in tissue. A 3 cm Petri dish was placed within the perimeter of a 10 cm Petri dish, and Visking tubing (30/32; Scientific Instruments) placed across the inner dish to irrigate the explant with antibiotic-free MEM (7 ml) placed in the outer dish. Explants were then placed cut-surface downwards onto the filter paper across the centre of the inner dish and 1% molten agar (at 40 °C) was placed around the explant to hold its orientation. The agar set rapidly, and then tissue was bathed in 200 µl MEM until it was ready for inoculation with bacteria. In experiments to validate the invasion assay, explants were treated with and without cytochalasin D (1 µg ml-1), which was added to all media bathing the tissue from arrival in the laboratory until completion of experiments.
Measurement of survival and penetration by N. meningitidis.
To measure survival of Neisseria spp. within the organ culture system, the MEM bathing the tissue was aspirated and 100 µl PBS containing a suspension of Neisseria spp. was placed onto the surface of the air-exposed explant. The whole was then transferred to an incubator at 37 °C in humidified 5% CO2 and incubated over a period of 24 h. At intervals over the period of incubation, explants were carefully removed from the agar and transferred to PBS (6 ml) in a bijou container. Explants were vortexed and washed through three changes of PBS, then homogenized in a 1-shot cell disrupter (Warwick Systems, Warwick, UK). The total number of bacteria within each homogenate was then estimated by viable counting. A technique described previously (Read et al., 1999 ) was used to estimate bacterial invasion of the tissue. Explants were immersed in 0·25% (w/v) sodium taurocholate (bile salts) (Sigma) for 30 s. Tissue was then immediately transferred to three changes of PBS in a universal container prior to homogenization in the 1-shot cell disrupter. This concentration of sodium taurocholate kills a suspension of 107 N. meningitidis within 30 s (Read et al., 1999
). The mean bactericidal concentration (MBC) of sodium taurocholate for each strain under test was measured by a standard dilutional technique. Prior to homogenization, all tissue samples were weighed and the viable counts of the homogenates of both sodium-taurocholate-treated and untreated explants were expressed per mg tissue. Where required, an invasive fraction was calculated by dividing the viable count of sodium-taurocholate-treated explants by the viable count of untreated explants. Output bacteria were checked by Gram staining. Uninfected control explants were included so that any contamination could be identified.
Statistical analysis.
For multiple comparisons between strains, statistical analysis was conducted by two-way ANOVA. Residuals were checked for normality using the AndersonDarling test. To compare inter-subject variability, data from 40 patients were normalized by logarithmic transformation, prior to analysis of variance. Intra-class correlation between replicates was based on variance components and estimated by two-way ANOVA.
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RESULTS |
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Treatment of tissue with the f-actin polymerization inhibitor cytochalasin D reduced the yield of strain K454 from homogenized explants (Fig. 1b). Following sodium taurocholate treatment, no viable bacteria were recovered from explants exposed to cytochalasin, supporting the utility of sodium taurocholate in isolating bacteria internalized into the mucosa.
The MBC of sodium taurocholate for all strains used in the study was 0·03%. Thus, the concentration of sodium taurocholate used to kill extra-mucosal bacteria (0·25%) was more than eightfold higher than the MBC. To determine whether treatment with sodium taurocholate was killing all extra-mucosal neisseriae without killing those that had penetrated mucosa, explants infected with strain K454 for 24 h were washed, treated with sodium taurocholate and then washed a further three times prior to vigorous rolling over the surface of blood agar. No colonies of Neisseria grew over 24 h incubation (n=3). However, when the same explants were homogenized, viable counts of strain K454 were recovered from all three, at similar concentrations to those shown in Fig. 1(b) (data not shown). To demonstrate that sodium taurocholate did not penetrate the tissue, uninfected explants were treated with sodium taurocholate 0·25% for 30 s, washed, homogenized, and the homogenate was placed in wells cut into blood agar. No bactericidal activity was observed.
Comparison of N. meningitidis, N. lactamica and N. animalis
To determine whether this model discriminates among representative pathogenic and non-pathogenic Neisseria spp., explants were infected with N. meningitidis (strain K454), and NCTC strains of N. lactamica and N. animalis. The strain of N. lactamica used in this study expressed PilC but not Opa, whilst the strain of N. animalis expressed neither (see Table 1). Data from nine repeated experiments each using tissue from a different human host, in which Neisseria recovery after 4 h and 18 h incubation was compared, are shown in Table 2
. Explants weighed in the range 2836 mg, with no significant difference observed between experimental limbs. There was no significant difference between the inoculum of organisms, and no significant difference between viable counts of homogenates of untreated explants after 4 h incubation. In contrast, the counts of N. meningitidis recovered from untreated explants by 18 h of incubation was significantly greater than those of N. lactamica and N. animalis. Very little change in the recovered counts of N. lactamica was seen over 18 h of infection; N. animalis was recovered at much lower counts from untreated explants after 18 h. In these experiments, there was no recovery of bacteria from explants incubated for 4 h and subsequently treated with sodium taurocholate; however, there was recovery of N. meningitidis from sodium-taurocholate-treated explants at 18 h, in contrast to N. lactamica and N. animalis, which were not recovered from sodium taurocholate-treated explants. Minor variations in inoculum into the model were observed but when the analysis was adjusted to account for this, all significant associations remained.
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Variance of recovery of bacteria from explants is illustrated in Fig. 2. Analysis of triplicate data derived from individual donor tissues showed that the methods were highly reliable, with an intraclass correlation at 4 h of 0·988, and at 18 h of 0·968 (of c.f.u. per mg of untreated explants). When recovered viable counts were adjusted for minor variation of the inoculum size, the coefficient of variation of recovered viable counts from untreated explants was 1335% after 4 h incubation, and 77% after 18 h incubation.
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DISCUSSION |
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Overall survival of Neisseria within the mucosa was measured by homogenizing washed explants infected with organisms over variable lengths of time. The use of sodium taurocholate to kill extramucosal bacteria is reasonable; compared with other bile salts, sodium taurocholate has a low pKa and requires specialized active transport for efficient penetration of epithelial cells (Shiau, 1987 ). Invasion of nasopharyngeal explants by N. meningitidis is an active process of parasite-directed endocytosis (Stephens et al., 1983
; Read et al., 1995
). During this process, bacteria are enveloped by microvillous extensions from the cell surface, a process requiring host cell cytoskeletal rearrangement, which in turn requires polymerization of actin filaments. Failure to recover any bacteria from cytochalasin D-exposed explants after treatment with sodium taurocholate (in contrast to explants not exposed to cytochalasin D) suggests that sodium taurocholate kills all bacteria which have not invaded the mucosa as a result of endocytosis, or some other process requiring an active host response. We demonstrated no bactericidal activity of homogenates of sodium-taurocholate-treated explants, indicating that sodium taurocholate did not penetrate this tissue. We also demonstrated absence of meningococci on the surface of sodium-taurocholate-treated infected explants and were only able to retrieve meningococci from such explants once they had been homogenized. This suggests that sodium taurocholate treatment kills extra-mucosal bacteria, but does not kill bacteria within a protected site deep within the mucosa. Sodium taurocholate kills bacteria rapidly, in contrast with gentamicin (which is often used for similar assays of invasion), which requires prolonged periods of incubation for bactericidal activity.
We found that high inocula (107 c.f.u.) of N. meningitidis were required for consistent meningococcal survival in this model. Inocula of 105 c.f.u. or below failed to thrive, whilst an inoculum of 106 c.f.u. did not result in survival of sufficiently high numbers within the tissue. The natural inoculum during droplet transmission between humans cannot be accurately measured, but is probably orders of magnitude below that used in this model. On the other hand, the surface area of mucosa available for epithelial contact by droplet-associated Neisseria during natural transmission is very much higher than that available in this experimental method.
The failure of N. animalis to thrive in this experimental model is consistent with its habitat it has never been isolated from humans but does colonize a range of small mammals including guinea pigs (Morse & Genco, 1998 ). N. lactamica survived in relatively low numbers but, in contrast to N. meningitidis, could not be recovered from sodium-taurocholate-treated explants. The behaviour of single strains should not be overinterpreted but these data suggest that survival of Neisseria spp. within nasal mucosa may correlate with pathogenicity. However, the survival of diverse isolates of N. meningitidis was not uniform in these experiments. Meningococci belonging to the subgroups of serogroup A could not be recovered from an intramucosal site, whereas the representative isolates of the ET-37 complex, lineage III and ET-5 complex did invade and grow intramucosally. These observations are intriguing, given the diverse epidemiologies of different meningococcal lineages. Strains belonging to the subgroups of serogroup A cause large-scale epidemics and pandemics in China and Africa, but have caused virtually no disease in Western Europe and North America since the Second World War (Schwartz et al., 1989
). By contrast, ET-37 complex meningococci cause up to 40% of meningococcal disease in European and American countries and are commonly associated with institutional outbreaks of serogroup C disease. Members of the ET-5 complex were responsible for the spread of hyperendemic meningococcal disease during the 1970s and 1980s in Western Europe, but were largely replaced as a major cause of meningococcal disease in many countries by organisms belonging to lineage III. The reasons for these diverse epidemiologies are not completely understood, but it is thought that ET-5 complex and lineage III organisms, for example, may have prolonged carriage compared to meningococci belonging to the subgroups of serogroup A. The t
of carriage of serogroup A meningococci is 1 month, whereas the t
of carriage of meningococci belonging to other serogroups is about 3 months (Blakebrough et al., 1982
).
Our data suggest that differences in the potential of genetically diverse meningococci to penetrate human nasopharyngeal mucosa can be measured in this model system, potentially yielding information valuable to the understanding of meningococcal epidemiology and pathogenesis. This diversity of invasion and intramucosal growth is likely due to a combination of several meningococcal cellular components, including outer-membrane proteins, capsule, IgA protease and pili (Nassif et al., 1999 ). Although Opa and PilC have been clearly shown to influence successful attachment to epithelial cell monolayers and organ culture mucosa (Virji et al., 1992
, 1993
; Rayner et al., 1995
) the data presented here suggest that other meningococcal determinants are also required for survival within the mucosa. More experimentation using organ culture systems is required to address these issues, perhaps employing isogenic meningococcal constructs. The availability of whole genome sequences for comparison of serogroup A subgroup (IV) (Parkhill et al., 2000
) with a member of the ET-5 complex (Tettelin et al., 2000
) will open up new possibilities for developing further these observations in this in vitro model.
One remarkable finding was the very wide variation in the recovery of meningococci from nasopharyngeal explants derived from different humans, which was initially apparent in experiments investigating differences between Neisseria species and clonal groups (Tables 2 and 3
). This was then formally tested using tissue from 40 human donors (Fig. 2
). Experiments were conducted in triplicate and there was very little variation between explants derived from an individual host. However, the coefficient of variation across the group of 40 individuals was 1335% after 4 h and 77% after 24 h incubation. This is dramatic inter-subject variability in comparison, the coefficient of variation of the human haematocrit is 9%, and that of the height of 17-year-old males is only 3·4% (Lentner, 1984
). As the experimental methodology was uniform across the 40 individuals studied, the variation that was observed was likely due to genetic or environmental influences upon the host tissue. All of the individuals who donated tissue in this survey were suffering from non-allergic nasal obstruction, mainly due to septal deviation. None were receiving intranasal topical steroids, though clearly we cannot exclude the influence of disease comorbidities secondary to the upper respiratory tract obstruction. Other possible environmental influences that could explain this variation include recent respiratory tract infection, though there was no correlation with season. It is unlikely that anti-meningococcal antibody present in the mucosae was responsible for the variation observed, as the tissue employed is not lymphoid, and we have not detected antibody to common viruses (e.g. influenza) in tissue homogenates (Read et al., 1999
).
Epidemiologically, there is support for the notion of a genetic influence on successful colonization and invasion. Within communities, nasopharyngeal colonization by N. meningitidis is only rarely complicated by invasive disease, even amongst those who do not possess serogroup- or serotype-specific antibodies (Goldschneider et al., 1969 ). Likewise, individuals differ in the characteristics of nasopharyngeal carriage of meningococci. Rake (1934)
followed the nasopharyngeal carriage of meningococci of groups of individuals over a period of 2 years. This included one group in a confined meningococcal laboratory amongst whom 50% were never colonized despite the fact that they are likely to have been engaged regularly in oral pipetting of meningococcal cultures. Amongst those who became colonized, there were three patterns: some were transient, some were intermittent and others were chronic carriers of the meningococcus. This pattern was also observed in young children in a nursery school environment (Rake, 1934
).
We conclude that the distinctive colonization and disease potential of Neisseria spp. may be partially a consequence of their ability to invade and survive within human nasopharyngeal mucosa, but that this is influenced greatly by host or environmental factors.
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ACKNOWLEDGEMENTS |
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We would like to thank the Theatre Sister and Staff of the ENT Theatre, Royal Hallamshire Hospital, and also Pauline Whitaker and Ian Geary for help with the preparation of this manuscript. We are grateful to Janet Suker of the National Institute for Biological Standards and Control, and to Xavier Nassif, Philippe Morand and James Moir for provision of antibodies.
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Received 20 September 2001;
accepted 2 January 2002.