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Institute for Hygiene and Microbiology, University of Würzburg, Würzburg, Germany
Peter Medawar Building for Pathogen Research and Departments of Zoology and Statistics, University of Oxford, Oxford, United Kingdom
Background.
Neisseria meningitidis is a diverse commensal bacterium that occasionally causes severe invasive disease. The relationship between meningococcal genotype and capsular polysaccharide, the principal virulence factor and vaccine component, was investigated in carried meningococci isolated from 8000 children and young adults in Bavaria, Germany.
Methods.
Of the 830 meningococci isolated (carriage rate, 10.4%) by microbiological techniques, 822 were characterized by serogrouping, multilocus sequence typing, and genetic analysis of the capsule region. Statistical and population genetic analyses were applied to these data.
Results.
The rapid increase in carriage rates with age of carrier, the low prevalence of hyperinvasive meningococci, and the relative prevalence of the 4 disease-associated serogroups were consistent with earlier observations. There was no genetic structuring of the meningococcal population by age of carrier or sampling location; however, there was significant geographic structuring of the meningococci isolated in civil, but not military, institutions. The rate of capsule gene expression did not vary with age of carrier or meningococcal genotype, except for serogroup C, for which increased expression was associated with ST-11 (formerly ET-37) complex meningococci.
Conclusions.
Serogroup C capsule expression during carriage may contribute to the invasive character of ST-11 complex meningococci and to the high efficacy of meningococcal serogroup C conjugate polysaccharide vaccine.
Neisseria meningitidis, although responsible for bacterial meningitis and septicemia worldwide, causes disease infrequently relative to the prevalence of its asymptomatic carriage [1, 2]. Phenotypic and genotypic investigations have shown that carried meningococci are more diverse [3, 4] than disease-associated meningococci [5, 6]. For example, carried meningococci may be either encapsulatedsuch that they express 1 of 13 capsular polysaccharides, each of which corresponds to a particular serogroup [7]or acapsulate, because of genetic down-regulation of capsule expression [8, 9], inactivation of genes in the capsule gene cluster (cps), or the absence of cps in capsule null (cnl) meningococci [10, 11]. In contrast, the majority of disease-associated isolates express polysaccharide of 1 of 5 serogroups: A, B, C, W-135, and Y [12]. The polysaccharide acid capsules corresponding to serogroups B, C, W-135, and Y predominate in Europe, Australia, and the Americas, whereas the polysialic acid capsules corresponding to serogroup A are predominant in Africa and Asia [12, 13]. The cps regions of serogroups B, C, W-135, and Y differ at the siaD locus, where 4 distinct allele classes occur (siaDB, siaDC, siaDW, and siaDY), each of which encodes serogroup-specific polysialyltransferases [14, 15].
Analysis of meningococcal genotypes by multilocus enzyme electrophoresis [5] and multilocus sequence typing (MLST) [6] has demonstrated that relatively few genotypes, termed "hyperinvasive lineages," are responsible for most disease. Disease-causing meningococci belong to particular groups of related genotypes, termed "clonal complexes" [6, 13]for example, the ST-11 (formerly ET-37) complex [6, 16, 17]that are overrepresented in disease isolates relative to their carriage prevalences [3]. The nature of meningococcal transmission suggests that disease outbreaks follow the spread of carried hyperinvasive meningococci [1, 18]. In many countries, disease mainly occurs in infants and young children, whose carriage rates are low, although increased disease incidence also occurs in young adults, whose carriage rates are much higher [19, 20].
The most widely used meningococcal vaccines are composed of purified polysaccharide [21] and have the ability to contain disease outbreaks; however, they are ineffective in infants and do not confer memory responses in adults [22]. Polysaccharide-protein conjugates address these problems for serogroup C and probably for serogroups A, W-135, and Y, but poor immunogenicity and similarity to host antigens have hampered the development of serogroup B vaccines [23, 24]. A number of protein-based outer membrane vesicle vaccines have been developed [2527], but they are poorly cross-protective [28]. Because disease is not necessary for meningococcal transmission [29, 30], understanding the carrier state is important for resolving the epidemiology of meningococcal disease, which will enable the population effects of immunizationsuch as herd immunity and vaccine escapeto be exploited in immunization campaigns [1, 31].
The present study investigated meningococcal carriage in individuals 326 years old in Bavaria, Germany, at a time when meningococcal disease rates reported in Germany were <1/100,000 persons and when (in 2001) 20% of disease isolates expressed serogroup C capsule. The few disease isolates available from Bavaria at the time of this study were consistent with the national data. Disease clusters caused by serogroup C meningococci of the ET-15 variant of the ST-11 complex had occurred in Bavaria before the study was initiated [32]. Combining genotypic and phenotypic typing data for the carriage isolates elucidated relationships among capsular operons, capsule expression, and clonal complexes and determined the relative prevalence of genotypes among regions, institutions, and age groups.
PARTICIPANTS, MATERIALS, AND METHODS
Isolation of carried meningococci.
Sampling was conducted from November 1999 to March 2000. The study protocol was approved by the ethics committee of the Medical Faculty of the University of Würzburg (study 137/99). At each sampling location, several educational institutions covering different age groups were chosen by the local health authorities, in consultation with school directors. In many cases, an informational event was organized for parents and guardians of students at the school, and the parents and guardians were provided with written information, including a consent form. At military camps, the commander chose the company to be sampled; military recruits were sampled within 2 weeks of arrival. Participation was voluntary, which was documented via a signed consent form; consent from parents and guardians was obtained for participants <18 years old. Most of the individuals who were approached agreed to participate. Retropharyngeal swab sampling was performed at each institution, and a single meningococcus was isolated from each culture-positive sample by direct plating onto Martin-Lewis agar plates (gift from Becton Dickinson). Gram-negative, oxidase-positive colonies were tested for -galactosidase and -glutamyltransferase activity, and the identity of the bacterium was confirmed by use of the API NH system (gift from BioMerieux).
MLST.
Isolates were characterized by MLST, as described elsewhere [4, 6]; an ABI Prism 3700 automated sequencer was used to separate the labeled extension products. The data were assembled by use of the Staden suite of computer programs [33]. Alleles, sequence types, and clonal complexes were assigned on the basis of the Neisseria MLST database (available at: http://pubmlst.org/neisseria) [34].
Serogroup analysis.
Previously described methods [35] were employed to measure the expression of serogroup A, B, C, W-135, and Y capsules (by ELISA with monoclonal antibodies); the presence of the mynB gene specific to serogroup A meningococci; and the presence of the siaD allele classes specific to serogroup B, C, W-135, and Y meningococci (the latter 2 by dot-blot hybridization with specific probes) [36]. The positive control isolates were as follows: serogroup A isolate Z2491 (ST-4) (gift from M. Achtman, Max-Planck Institut für Infektionsbiologie, Berlin, Germany), serogroup B isolate MC58 (ST-74), serogroup C isolate 2120 (ST-11), serogroup W-135 isolate 171 (ST-11), and serogroup Y isolate 172 (ST-166). The negative controls were gonococcal strain FA1090 (gift from M. Achtman) and Neisseria lactamica strain 4691 (German Collection of Microorganisms and Cell Cultures).
Statistical analyses.
The association between siaD allele class and age was evaluated for serogroups B, C, W-135, and Y. Age and 2-term fractional polynomial forms of age were used to test for monotonic or polytonic continuous associations [37]. Age was grouped as follows: 39, 1014, 1519, and 20 years. The 2 test was used to test for the association between age group and siaD allele class. The association between age and capsule expression was evaluated by use of a logistic regression model, with 2-term fractional polynomial forms of age as explanatory variables. Analyses were conducted for all 4 siaD allele classes and for each siaD allele class on its own.
The association between siaD allele class and capsule expression was estimated by use of logistic regression, to provide odds ratios with 95% confidence intervals, and by Fisher's exact test, to provide P values. A logistic regression model was used to assess the degree to which carrier age group might have confounded the results. The association between clonal complex and capsule expression was estimated by likelihood ratio tests across all 4 serogroups. Within each siaD allele class, this association was assessed by Fisher's exact test with the Bonferroni correction for multiple comparisons.
Analysis of molecular variance (AMOVA).
Significant genetic differentiation among groups of isolates was assessed by AMOVA [38], as implemented in Arlequin software (version 2.000) [39]. This program computed an F statistic [40, 41] by applying a permutation test to assess statistical significance. AMOVAs were performed on the data as grouped by institution, institution excluding military camps, military camps versus all other institutions, age of carrier, carrier age group, and geographic location of the institutions. Two further analyses were performed on geographic subsets of the data, one excluding military camps and one including military camps only. For the analysis by carrier age group, the age groups described above were used. The locations of the institutions sampled were used for all geographic analyses in this study. Although the locations of origin (mainly Bavaria) were known for most of the military recruits, this was not amenable to analysis, because they were distributed throughout Bavaria.
The following genetic characteristics were analyzed: nucleotide sequence of an individual locus, concatenated nucleotide sequences of all 7 loci, allele designations, concatenated allele designations of all 7 loci (known as the "allelic profile"), and sequence type. For the small genetic differences detected, analysis of the nucleotide sequences of individual loci lacked statistical power, but power was recovered when the concatenated nucleotide sequences, concatenated allele designations, and sequence types were used. Where appropriate, the Bonferroni correction was applied, to account for multiple comparisons.
Mantel test.
Correlation between genetic distance and geographic distance was assessed by use of the Mantel test [42]. Square n-by-n matrices were generated, where n was the number of isolates and, with the exception of the diagonal elements, each element of the matrix corresponded to an isolate pair. The correlation coefficient was calculated for the distance matrices, and its significance was assessed by permutation [35]. Mantel tests were performed on the complete MLST data set (n = 822), the data set excluding military camps (n = 441), and the data set including military camps only (n = 381). The geographic distance for a pair of isolates was taken to be the geographic distance between the towns at which the isolates were collected. For a pair of isolates, the genetic distance was defined variously, as follows: the proportion of nucleotide sites that differed at an individual locus, the proportion of nucleotide sites that differed across all 7 loci, a number that indicated whether the allele designation for a particular locus was the same (0) or different (1), the proportion of alleles that differed across all 7 loci, and a number that indicated whether the sequence types were the same (0) or different (1). All definitions of genetic distance were used to investigate the effect on the analysis; where appropriate, the Bonferroni correction was applied, to account for multiple comparisons.
RESULTS
Meningococcal carriage rates.
From the 8000 children and young adults sampled, 830 (10.4%) meningococci were isolated; carriage rates were lowest in young children and highest in participants 25 years old (figure 1 and table 1). The 6821 participants who provided samples at schools (age range, 321 years) yielded 446 isolates (München, 75 isolates; Ingolstadt, 61 isolates; Erlangen, 57 isolates; Coburg, 38 isolates; Passau, 47 isolates; Würzburg, 47 isolates; Rottal-Inn, 47 isolates; Augsburg, 23 isolates; Sonthofen, 20 isolates; Dinkelsbühl, 17 isolates; and Weiden, 9 isolates). The remaining 384 meningococci were isolated from 1179 military recruits 1826 years old at 6 camps (Roth, 109 isolates; Volkach, 96 isolates; Bayreuth, 95 isolates; Kempten, 50 isolates; Sonthofen, 21 isolates; and Ebern, 10 isolates).
Meningococcal capsule genes and serogroups.
The majority of the 822 MLST-typed meningococci (677/822 [82%]) were also characterized at cps. Most isolates (541/822 [66%]) possessed a siaD gene, with 136 (17%) containing cnl in place of cps [10]. The most prevalent serogroup was B (261/822 [32%]), and the most common siaD allele class was siaDB (333/822 [41%], corresponding to 4% of participants sampled). Serogroup C was of much lower prevalence (20/822 [2%]), as was the siaDC allele class (56/822 [7%] of all meningococci, corresponding to 0.7% of participants sampled) (table 1). Among the 4 siaD allele classes, siaDB serogroup B meningococci comprised the majority of isolates in all age groups (range, 55%68%). There was some variation among age groups in excess of that expected by chance (P = .02, 2 test). The main difference was a higher proportion of siaDC isolates found in the participants 1519 years old and 20 years old (overall, 12%; for each group, 9% and 16%, respectively) than in the participants 39 years old and 1014 years old (overall, 4%; for each group, 1% and 9%, respectively), with associated reductions in the proportion of siaDB isolates found in the participants 1519 years old and siaDY isolates in the participants 20 years old. The proportion of meningococci expressing their capsule genes was calculated from the prevalence of the serogroup-specific siaD allele classes and serogrouping data (table 2). Expression of capsule genes was much less common among siaDC and siaDY isolates than among siaDB and siaDW isolates (table 2). Adjustment for age group had no substantial effect on this pattern (data not shown). There was no evidence for an effect of age of carrier on capsule expression, either overall or within any serogroup.
Genetic diversity.
Among the 822 MLST-typed isolates, there were 323 unique sequence types, 221 of which occurred only once. The number of alleles per locus varied from 30 (adk) to 57 (pdhC). Most isolates (543/822 [66%]) were assigned to 1 of 20 previously identified clonal complexes, with 153 sequence types among the 279 unassigned isolates. With the exception of the serogroup Aassociated clonal complexes (ST-1, ST-4, and ST-5), most of the previously described hyperinvasive lineages were present: ST-41/44 complex (138 isolates [17%]); ST-23 complex (71 isolates [9%]); ST-32 complex (41 isolates [5%]); ST-11 complex (8 isolates [1%]); and ST-8 complex (2 isolates) (table 3). The full data set, including isolate details, is available as an MLST dbNet database [34] at http://pubmlst.org/neisseria. A small number (21) of Bavarian disease-associated isolates contemporary to the present study were available; of these, 3 were ST-8 complex, serogroup C; 4 were ST-11 complex, serogroup C; 3 were ST-32 complex, serogroup B; and 5 were ST-42/44 complex, serogroup B. The remaining 6 isolates, all serogroup B, belonged to various unrelated sequence types.
Relationship between clonal complex and capsule genes.
The distribution of cps variants was not random among clonal complexes; certain clonal complexes were associated with particular cps genotypes. Furthermore, siaDC ST-11 complex isolates were more likely to express their capsule than were siaDC isolates that belonged to the other clonal complexes (table 3). All ST-53 complex (n = 59) and ST-198 complex (n = 44) isolates were cnl meningococci, as reported elsewhere [10]. The 401 siaD genecontaining isolates that were assigned to a known clonal complex were distributed among 16 clonal complexes (table 3), and there was no strong evidence showing an association between clonal complex and capsule expression (P = .30, by a likelihood ratio test from a logistic regression model that excluded 15 isolates because their clonal complexes were represented by 3 isolates). In a logistic regression model (n = 386) that included the siaD allele class, there was weak statistical support for an overall association between clonal complex and capsule expression (P = .09). No formal interaction test of reasonable power was feasible for this data set, given the strong correlation between clonal complex and serogroup and the limited number of isolates for some clonal complexes. Within individual serogroups, there was an association between clonal complex and capsule expression in siaDC isolates that remained significant after application of the Bonferroni correction for 4 comparisons (P = .03; n = 34) but not in any other siaD allele class, with P > .1 for siaDB (n = 252), siaDW (n = 31), and siaDY (n = 84). This association was due to the high level of expression among ST-11 complex and ST-8 complex isolates and the low level of expression among other siaDC isolates (table 3).
Genetic structuring.
AMOVA provided no evidence for structuring of the concatenated nucleotide sequence data for any of the groupings tested except geographic location, where low but statistically significant levels of structuring were observed (table 4). Further AMOVAs showed that there was significant geographic structuring for the nucleotide sequences of 3 alleles, for all but 1 allele designation (aroE), for the allelic profile, and for the sequence type (table 5). When the data were categorized by institution type (military or school), the evidence for geographic structuring remained for the schools but not for the military camps. The correlation coefficients derived from the Mantel test () provided similar evidence of geographic structuring for the concatenated nucleotide sequence, the allele designations, the allelic profile, and the sequence type for the complete data set and for the schools. These analyses demonstrate that the degree of genetic differentiation correlated with geographic distance excepting only military camps, for which there was no support for geographic structuring (table 5).
DISCUSSION
Meningococcal vaccines protect individuals from disease by eliciting serum bacterial antibodies [43] but can also induce mucosal immunity, affecting carriage and transmission [44]. In terms of public health, the resultant population effects can be either advantageous, if transmission of the disease-associated meningococcus is interrupted, or disadvantageous, if they promote the emergence of vaccine escape variants [31]. Quantitative description of meningococcal carriage and transmission in terms of capsule expression and genotype is, therefore, necessary to understand these effects.
Here, a carried meningococcal population is analyzed in terms of host characteristics, genotype, and capsule expression. The age-specific carriage prevalence and frequency of serogroups observed were consistent with the findings of previous studies, which have consistently shown low rates of carriage in young children that rise dramatically with age [11, 19, 4548], presumably as a result of behavioral changes. Our results also confirmed that some cps genotypes are associated with particular clonal complexes [10, 13]. Except for a marginal increase in the carriage of serogroup C meningococci with age, there was no evidence for associations between meningococcal serogroups and geographic regions, institutions, or age groups. Meningococci with the siaDC or siaDY allele classes were less likely to express capsule than were those with the siaDB or siaDW allele classes. There were significant differences among the meningococci with the siaDC allele class, with evidence for a possibly very strong association between the ST-11 complex and siaDC expression. This novel observation is potentially important, because, notwithstanding their low carriage prevalence, members of the ST-11 complex have caused elevated levels of disease on numerous occasions, recently spreading across several continents [49]. Because capsule expression is a major virulence determinant [12], this property may contribute to the invasive character of ST-11 siaDC meningococci. If these high levels of capsule expression are also necessary for fitness in terms of transmission, these meningococci may be particularly vulnerable to the mucosal immunity generated by conjugate polysaccharide vaccines [48]. This may have contributed to the high efficacy of the meningococcal serogroup C conjugate polysaccharide vaccines recently introduced in the United Kingdom [50] and elsewhere.
The only genetic structuring observed was by geographic location, with the observed FST indicating that 0.7% of the total genetic diversity could be attributed to differences between locations. This statistic has not been widely used in bacterial populations, but the level was lower than the differentiation (1.5%9.4%) observed between environmental and farm-animal isolates of the zoonotic pathogen Campylobacter jejuni [51]. Although the AMOVA showed that there was a strong signal of association between particular genotypes and given geographic locations (P < .001), the magnitude was not great. This low but statistically significant result for meningococci was consistent with the idea that meningococcal populations contain distinct genotypesthe clonal complexeswhose members have global distribution but are unevenly distributed among different geographic regions at any given time. The data also suggested that this genetic differentiation is very recent, perhaps arising as a consequence of the fact that clonal complexes spread faster than novel clonal complexes emerge. For an obligate inhabitant of humans such as the meningococcus, the spread of genotypes is likely to be related to host demographic behavior. In this context, the present data suggest that the rate of spread is sufficiently slow to result in detectable geographic structuring among major population centers in a region the size of Bavaria, providing some indication of the relative intensity of transmission between and within population centers.
Further analysis of geographic structuring by AMOVA and the Mantel test confirmed that significant but low levels of geographic structuring were present and that structuring was the result of isolation by distance. Indeed, this structuring was often undetectable when individual loci were analyzed, caused by a lack of statistical power. MLST is a typing procedure, optimized for epidemiological applications rather than population-genetics analysis [52], and the present results suggest that, for meningococcal populations, contiguous nucleotide sequences in excess of the 500 bp used in MLST are required to detect geographic structuring at the level of individual loci. Geographic structuring was, however, detectable at the level of concatenated nucleotide sequences, allele designations, allelic profile, and sequence types. Each of these levels of analysis emphasized differences among the regions, allowing them to be detected against the background of high genetic variation that is present in meningococcal populations [4]. These analyses further established that the geographic structuring was present among the schools but absent among the different military centers, which was consistent with the wide geographic catchment areas of the latter institutions, with the majority of recruits originating in diverse locations in Bavaria.
The present study provides support for the idea that variations in disease incidence are due to the gradual spread of hyperinvasive meningococci, with intensity of transmission declining with geographic distance. Geographic structuring by distance is a likely explanation for the differences in the predominant clonal complexes that cause invasive disease in different European countries at any given time [13]. Furthermore, notwithstanding the differences in carriage rates among different cohorts, the meningococcal transmission system is the community as a whole, rather than a subpopulation of a particular age or peer group.
Although a large number of individuals were included in the present study, the high diversity of the meningococcal population, the low levels of carriage of the ST-11 complex, and the lack of a large set of contemporary disease isolates limited some of the conclusions that could be drawn. In particular, although the finding that ST-11 meningococci with the siaDC allele class may be unusual in terms of capsule expression has implications for disease control, because the number of isolates on which this observation was based is small, the finding has to be confirmed in a study that includes more isolates. Nonetheless, the present study has indicated the types of analyses that can be performed on these data; it has also generated hypotheses and data that will be essential to the design of the future studies needed to further explore the associations described here.
Acknowledgments
The Bavarian Government and the German Armed Forces are gratefully acknowledged for their assistance. We also thank Man-Suen Chan, for help with data analysis; Dirk Alber, Marc Oberktter, Silke Getzlaff, and Carmen Kantelberg, for assistance with sampling; Gabi Heinze, for providing expert technical assistance; Rainer Maag, for providing help with serogrouping; and Man-Suen Chan, for developing, with K.A.J., the Neisseria multilocus sequence typing database (available at: http://pubmlst.org/neisseria).
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Fluoroquinolone antibiotics provide activity against a broad range of pathogens, including gram-negative, gram-positive, atypical, and multidrug-resistant organisms [1]. As an antibiotic class, fluoroquinolones are widely used in adults, because of their broad spectrum of activity, low toxicity, and oral availability; use in children has been limited because of concerns about cartilage toxicity [24].
Within a relatively short period after the introduction of the fluoroquinolone ciprofloxacin in 1987, resistance was noted in both gram-positive and gram-negative bacteria. Among gram-positive species, resistance was first recognized in Staphylococcus aureus and became widely prevalent, especially in methicillin-resistant strains [5]. A subsequent disturbing development was the detection of ciprofloxacin resistance in Streptococcus pneumoniae, with rates as high as 12%15% in some countries [6, 7]. Most recently, clinically significant resistance has been reported in a few cases of infection caused by Streptococcus pyogenes [8, 9].
In S. pneumoniae, ciprofloxacin resistance develops in a stepwise fashion. Mutations generally appear first in the topoisomerase IV gene, parC, leading to reduced susceptibility or "low-level" resistance as the result of an 8-fold increase in the MIC of ciprofloxacin (MIC, 4 to 8 g/mL). "High-level" resistance occurs as a result of additional mutations in the DNA gyrase gene, gyrA, with associated MICs of >8 g/mL [1013]. Mutations identical or similar to those described in S. pneumoniae were present in recently described isolates of S. pyogenes that possessed either low-level or high-level resistance to fluoroquinolones [8, 9, 14].
During a recent survey of antibiotic resistance in S. pyogenes, we were surprised to find a relatively large population of isolates that exhibited reduced susceptibility to ciprofloxacin. Our studies of those isolates revealed that most were a single serotype, M type 6, and possessed a serine-to-alanine substitution within the quinolone resistancedetermining region (QRDR) of topoisomerase IV, at amino acid position 79 (serine79alanine). Additional isolates of this serotype from diverse locations and time periods possessed the same mutation, including the M type 6 reference strain, isolated in 1918 and characterized by Dr. Rebecca Lancefield [15]. Thus, we have demonstrated that S. pyogenes of M type 6 possesses intrinsic reduced susceptibility to fluoroquinolone antibiotics.
METHODS
Group A streptococcal isolates.
We studied 311 isolates recovered from clinical samples submitted to the microbiology laboratories of St. Louis Children's Hospital (SLCH) and Barnes-Jewish Hospital (BJH) in 2002 (288 from children and 23 from adults) and 73 isolates from samples submitted to the BJH laboratory in 2003 (72 from adults). Ninety-one percent of the isolates were from throat swabs. Isolates were identified on the basis of growth of -hemolytic colonies on sheep blood agar after incubation for 1824 h at 35°C in 5% CO2. Each isolate was confirmed as S. pyogenes on the basis of a positive L-pyrrolidonyl--naphthylamide (PYR) test (Lifesign PYR) and specific agglutination with latex particles coated with antigroup A streptococcal antibodies (Lifesign Streptolex).
Antibiotic susceptibility testing.
Kirby-Bauer disk-diffusion susceptibility testing using NCCLS procedures and breakpoints was performed for the following antibiotics: clindamycin, erythromycin, penicillin, tetracycline, and vancomycin (BD BBL Sensi-Disc; Becton Dickinson). A ciprofloxacin disk (BD BBL) was also placed, and the zone of inhibition was measured. S. aureus ATCC 25923 was used as a control. The double disk-diffusion test using erythromycin and clindamycin disks was performed, as has been described elsewhere [16], on all isolates found to be resistant to erythromycin.
MIC determination.
All isolates with a ciprofloxacin zone of inhibition <18 mm in diameter and a subset of isolates with a ciprofloxacin zone of inhibition 18 mm in diameter were tested for the MIC of ciprofloxacin by use of Etest strips (AB Biodisk North America), in accordance with the procedure recommended by the manufacturer. For all isolates with a ciprofloxacin MIC 1.5 g/mL and for a subset of isolates with a ciprofloxacin MIC 0.75 g/mL, the MICs of gatifloxacin, levofloxacin, moxifloxacin, and ofloxacin were also determined, by use of Etest strips. S. aureus ATCC 29213 was used as a control. The cutoffs defined for S. pyogenes by the NCCLS as indicating nonsusceptibility are >2 g/mL for levofloxacin and ofloxacin and >1 g/mL for gatifloxacin. The NCCLS does not have recommended cutoffs for S. pyogenes for ciprofloxacin and moxifloxacin [17].
parC and gyrA sequencing.
Nucleotide sequencing of the relevant portions of the parC and gyrA genes was performed on DNA from 26 isolates of S. pyogenes with ciprofloxacin MICs 1.5 g/mL and from 9 isolates with ciprofloxacin MICs 0.75 g/mL. For each procedure, each isolate was grown overnight on sheep blood agar at 37°C. Colonies were harvested in 100 L of Sigma Water PCR Reagent (Sigma-Aldrich) and heated for 1520 min at 95°C. Portions of the parC and gyrA genes were amplified from each strain by polymerase chain reaction (PCR) using the primers previously described by Yan et al. [9]. The numbering of the sequence was adjusted to correlate with the coordinates of S. pneumoniae [14]. PCR products were purified using the QIAGEN QIAquick PCR purification kit (Qiagen) and then were sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Sequences were analyzed using an ABI PRISM 377 DNA Sequencer (Applied Biosystems). Wild-type sequences were obtained from GenBank (accession no. AF220945 for gyrA and AF220946 for parC), and comparisons were made using BLAST.
Typing.
All isolates with reduced susceptibility to ciprofloxacin and a subset of isolates that were susceptible to ciprofloxacin (15 from SLCH and 11 from BJH 2003) were sent to the World Health Organization (WHO) Collaborating Center for Reference and Research on Streptococci at the University of Minnesota in Minneapolis, for serological classification. The serogroups of all isolates were confirmed, and opacity factor detection, T-protein serotyping, and M-protein typing was performed according to standard methods [18]. Each of the isolates was also typed by DNA sequencing of the M-protein gene (emm) [19]. Thus, the serotype is referred to as the "M/emm type." If the serotype was determined strictly by sequencing, the serotype is referred to as the "emm type."
Archived isolates.
Because of the association found between ciprofloxacin resistance and M/emm type 6, we obtained additional isolates of M/emm type 6 S. pyogenes from diverse locations worldwide. These included 14 isolates from the WHO Collaborating Center for Reference and Research on Streptococci at the University of Minnesota in Minneapolis, as well as 9 isolates from a longitudinal study of S. pyogenes infections in school-aged children in Pittsburgh, of which 4 were erythromycin susceptible and collected during 19981999 and 5 were erythromycin resistant and collected during 2001 [16]. Also evaluated were 14 isolates of other M/emm types from the Pittsburgh and University of Minnesota collections. MIC determination was performed on all isolates, as described above. Sequencing of the relevant portions of parC and gyrA were performed on a sample of these isolates, as described above.
RESULTS
Disk-diffusion susceptibility testing.
Kirby-Bauer disk-diffusion susceptibility testing of the isolates from St. Louis revealed uniform susceptibility to penicillin and vancomycin and low rates of resistance to erythromycin, clindamycin, and tetracycline (table 1). Testing of ciprofloxacin revealed a bimodal distribution in the diameters of the zones of inhibition surrounding ciprofloxacin disks (figure 1), with isolates with smaller zones of inhibition accounting for 10.9% (range, 9.4%16.4%) of isolates tested (table 1). These isolates had zones of inhibition <18 mm in diameter (mean, 14.9 mm), in contrast to the majority of strains, whose zones of inhibition were 18 mm in diameter (mean, 21.9 mm).
MIC determination.
The isolates with reduced ciprofloxacin zones of inhibition were all found to have elevated MICs of ciprofloxacin (1.56 g/mL) and levofloxacin (24 g/mL), compared with those of fully susceptible strains; their MICs were in the nonsusceptible range for ofloxacin (412 g/mL), and, thus, they were considered to have reduced susceptibility to these antibiotics (table 2). The MICs of gatifloxacin and moxifloxacin were within the susceptible range (on the basis of the NCCLS interpretive standards for gatifloxacin MICs in Streptococcus species) but were elevated compared with those of fully susceptible strains.
Gene sequencing.
To determine the genetic basis for reduced susceptibility to ciprofloxacin, we determined the nucleotide sequence of the QRDRs of the parC and gyrA genes in 26 isolates with ciprofloxacin MICs 1.5 g/mL and in 9 isolates with ciprofloxacin MICs 0.75 g/mL. Compared with the parC nucleotide sequence of an M type 1 reference strain of S. pyogenes (ATCC 700294 [GenBank accession no. AF220946]), 24 of the 26 isolates with reduced susceptibility to ciprofloxacin had a base change from thymine to guanine at parC position 366, resulting in a serine79alanine substitution. A causal relationship between this substitution and resistance to fluoroquinolones has been demonstrated by experiments in which a fluoroquinolone-susceptible strain of S. pneumoniae became resistant upon transformation with DNA containing the substitution [14].
Of the 2 isolates with reduced susceptibility to ciprofloxacin that lacked the serine79alanine substitution, 1 had a serine79phenylalanine substitution without additional base changes, and the other had 2 amino acid changes in the parC QRDR: aspartic acid83asparagine and aspartic acid91asparagine. Only 1 amino acid substitution, aspartic acid91asparagine, was detected in the QRDR of parC in any isolate with a ciprofloxacin MIC 0.75 g/mL. This substitution by itself has not been shown to confer fluoroquinolone resistance. No coding changes were found in the gyrA gene of any isolate.
Typing.
All 24 of the isolates with reduced susceptibility to ciprofloxacin that were sequenced and shown to have the serine79alanine substitution were found to be M/emm type 6. The isolate with the serine79phenylalanine substitution was emm type 44/61, and the isolate with the aspartic acid83asparagine and aspartic acid91asparagine substitutions was emm type 75. The 25 fluoroquinolone-susceptible isolates that were typed included 4 of M/emm type 1; 3 each of M/emm types 3, 12, 18, and 75; 2 each of M/emm types 4, 22, and 73; and 1 each of M/emm types 2, 28, and 41.
Analysis of archived M type 6 isolates.
To further explore the link between the serine79alanine substitution and M/emm type 6, we studied 23 isolates of M/emm type 6 from diverse locations worldwide. Included were 4 isolates of M/emm type 6 that were recovered from patients >20 years ago, before the licensure of ciprofloxacin in 1987. One of these isolates was the reference strain of M type 6, isolated in 1918 and characterized by Dr. Rebecca Lancefield [15]. In addition to testing an isolate of this strain from the WHO Collaborating Center for Reference and Research on Streptococci at the University of Minnesota, we also obtained and tested an isolate of the same strain from the American Type Culture Collection (ATCC 12348). Each of the archived M/emm type 6 isolates was found to be nonsusceptible to ciprofloxacin, and all of the isolates that were sequenced (n = 18) possessed the serine79alanine substitution (table 3). Of the 14 nonM type 6 isolates examined, none had elevated ciprofloxacin MICs, and no amino acid substitutions were present in the QRDR of the parC or gyrA gene of any sequenced isolate.
Other relationships between the M/emm type and the sequence of parC were also apparent. Numerous noncoding base substitutions were present within and surrounding the parC QRDR in ciprofloxacin-susceptible as well as ciprofloxacin-nonsusceptible isolates, and these substitutions correlated with M/emm type. For example, each of 42 isolates of M/emm type 6 that were sequenced had an identical set of 7 noncoding base substitutions in addition to the change associated with the serine79alanine substitution. M/emm type 75 was an exception, with 3 different patterns of base substitutions in the 4 isolates that were sequenced (table 4).
DISCUSSION
In this article, we report the novel finding that M/emm type 6 S. pyogenes possesses inherent reduced susceptibility to ciprofloxacin as well as to other fluoroquinolone antibiotics. The basis for this assertion is that all isolates of M/emm type 6 S. pyogenes that we studied had elevated ciprofloxacin MICs (10-fold higher than those of fully susceptible isolates), and all had the identical amino acid substitution in the parC gene at a position that has been associated with fluoroquinolone resistance in S. pneumoniae [1014]. Perhaps most striking was that these same findings were present in the M type 6 reference strain, characterized by Dr. Rebecca Lancefield after its isolation in Texas in 1918 [15], decades before the development of the first fluoroquinolone antibiotic, nalidixic acid, in 1965. Interestingly, this finding suggests that reduced susceptibility to ciprofloxacin in M/emm type 6 is unrelated to selective pressure from antibiotic usage.
M/emm type 6 S. pyogenes is recognized as a cause of streptococcal pharyngitis and is one of the streptococcal M types associated with rheumatic fever [20]. It is also a relatively common M type, as shown by a recent population survey of S. pyogenes from all body sites, in which it accounted for 3.0% of all isolates [21]. The finding that M/emm type 6 possesses intrinsic reduced susceptibility to ciprofloxacin as well as to the other fluoroquinolone antibiotics raises the concern that widespread use of this class of antibiotics may select for this serotype. Extensive data that would enable us to determine whether this is occurring are not available, but, interestingly, Kaplan et al. reported a statistically significant increase in the proportion of M type 6 isolates causing pharyngitis when comparing data from 19881990 with data from 19911993 [22]. However, streptococcal M type prevalence is in constant flux, both temporally and geographically, making any observed changes difficult to interpret [2226].
Even though fluoroquinolone antibiotics are not frequently used at present to treat infections caused by S. pyogenes, which remains susceptible to penicillin, there are several other reasons to be concerned about reduced susceptibility to fluoroquinolones among S. pyogenes. One reason is that the presence of intrinsic reduced susceptibility to ciprofloxacin in M/emm type 6 S. pyogenes as a result of an amino acid polymorphism in the parC gene sets the stage for the emergence of the high-level resistance that would be present if a single point mutation were to occur in the gyrA gene. High-level resistance would be clinically significant, because it would preclude the use of ciprofloxacin and a number of newer widely used fluoroquinolone antibiotics for the treatment of S. pyogenes infections. This would eliminate a therapeutic option that is occasionally important at the present time, particularly in penicillin-allergic patients, and that may be of greater importance in the future, because resistance to other classes of antibiotics (such as macrolides) is increasing [16, 2629]. Additionally, fluoroquinolones may be used in the empiric treatment of patients with serious illnesses, and, if S. pyogenes is among the organisms causing infection, resistance might cause therapeutic failure.
Another reason for concern is that M/emm type 6 S. pyogenes could become the backbone for multidrug-resistant strains of S. pyogenes. Multidrug resistance is currently an important clinical and public health issue, since a number of important bacterial pathogens, including S. aureus, S. pneumoniae, Enterococcus faecium, and Mycobacterium tuberculosis, have developed resistance to multiple classes of antibiotics with unrelated mechanisms of action. An association between ciprofloxacin resistance and macrolide resistance in S. pyogenes has been suggested by previous authors [30]. Our results in the present study demonstrate that the strain of emm type 6 S. pyogenes responsible for a recent outbreak of macrolide-resistant S. pyogenes infections in Pittsburgh [16] also exhibits reduced susceptibility to ciprofloxacin and carries the serine79alanine substitution, as well as the noncoding base changes that were present in all of the M/emm type 6 isolates we have tested. Macrolide resistance in the Pittsburgh strain was based on the presence of the mef gene, which confers resistance only to the macrolide class of antibiotics. However, a significant number of macrolide-resistant S. pyogenes from other regions of the world possess the erm gene, which confers resistance to the lincosamide and streptogramin classes of antibiotics as well as to the macrolides [31, 32].
A final concern is that a parC gene that encodes a fluoroquinolone-resistant topoisomerase in S. pyogenes could be a source for the transfer of genetic material to other clinically important species, such as S. pneumoniae [33, 34], for which fluoroquinolone antibiotics are an important part of the therapeutic armamentarium, especially in adults. Indeed, evidence already exists supporting the exogenous acquisition, by strains of S. pneumoniae, of parC genes encoding fluoroquinolone resistance [14, 35]. Children serve as the primary community reservoir for both S. pyogenes (including M/emm type 6) and S. pneumoniae. Resistance to fluoroquinolones increases in conjunction with increasing use of these drugs within a population [36]. Widespread use of fluoroquinolones in children could promote strong selective pressure favoring resistant strains of either species, and this effect could potentially be amplified through interspecies genetic transfer.
The presence of at least 1 serotype of S. pyogenes with a polymorphism in the parC gene that confers reduced susceptibility to ciprofloxacin gives this species (and possibly others, such as S. pneumoniae) an evolutionary "head start" in the development of high-level fluoroquinolone resistance [37] that could become widespread under the selective pressure of fluoroquinolone usage. Combined with the presence of other antimicrobial-resistance determinants, fluoroquinolone resistance in S. pyogenes further limits the therapeutic armamentarium for treatment of this very common pathogen.
Acknowledgments
We thank Monique Gaudreault-Keener, for assistance with sequencing; James Connelly, Patricia Sellenreik, and the staff of the Microbiology Laboratory at St. Louis Children's Hospital, for assistance with collecting streptococcal strains and for susceptibility testing; W. Michael Dunne and the Barnes-Jewish Hospital Microbiology Laboratory, for collecting streptococcal strains; Joseph St. Geme III, Jeffrey McKinney, and Gillian and Jonathan McDunn, for review of the manuscript and valuable discussion; and Barbara Hartman, for preparation of the manuscript.
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