Single-Step Capsular Transformation and Acquisition of Penicillin Resistance in Streptococcus pneumoniae

The capsule (cps) locus of Streptococcus pneumoniae is flankedby the pbp2x and pbp1a genes, coding for penicillin-bindingproteins, enzymes involved in cell wall synthesis that are targetsfor ß-lactams. This linkage suggested to us that selectionfor ß-lactam resistance might coselect for capsulartransformants. The recombination event would then involve PBPgenes, as well as the cps operon, and would change both theserotype and the resistance profile of the strain. We transformedß-lactam-susceptible strain TIGR4 by using whole genomicDNA extracted from multidrug-resistant strain GA71, a serotype19F variant of pneumococcal clone Spain^23F-1, and selected ß-lactam-resistanttransformants. Smooth colonies appearing on selective plateswere subcultured, serotyped by the Quellung reaction, and genotypedto confirm the presence of the GA71 pbp2x-cps19-pbp1a locusin the TIGR4 genetic background by restriction fragment lengthpolymorphism analysis of the whole locus and its flanking regions.The results showed that a new serotype, combined with resistanceto ß-lactams, could emerge in a susceptible strainvia a single transformation event. Quantitative analysis showedthat transfer of the cps locus had occurred at an elevated ratein ß-lactam-selected transformants. This suggeststhat in natural settings selection by host immunity and selectionby antibiotics may be interrelated because of "hitchhiking"effects due to linkage of resistance determinants and the capsulelocus.

INTRODUCTION

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Introduction

Seventy-five years ago, in a milestone experiment, Griffithdemonstrated the astonishing ability of Streptococcus pneumoniaeto adapt under the pressure of the host immune system, by acquiringwhat we now know to be DNA encoding biosynthetic enzymes fora polysaccharide capsule, which protects the organism againstphagocytosis during infection (16). In the same year that Griffith'sexperiments were published, Alexander Fleming discovered penicillin—anevent that reshaped the history of both humans and pneumococci(13). Before the antibiotic era, the population of pneumococciisolated from humans was mainly dominated by strains with capsularpolysaccharides of serotypes 1, 2, and 3 (12, 16). Within thelast 25 years, we have witnessed the emergence of penicillin-and multidrug-resistant S. pneumoniae clones, a number of whichhave spread worldwide. Certain serotypes, this time 6B, 9V,14 19A, 19F, and 23F, have also dominated this new population,and many of the worldwide clones have appeared with differentcapsular types (28, 30, 37). The most successful clone in termsof geographical dispersion and prevalence is the multidrug-resistantSpain^23F-1 pandemic clone, nine serotype variants of which havebeen identified so far, including all six of those listed above,in addition to serotypes 3, 9N, and 11 (3, 10, 31; http://www.mlst.net).Such variants are thought to arise through natural transformationinvolving recombinational replacements, within and around thecapsular biosynthesis (cps) locus, of DNA fragments sometimesas large as 25 kb (4-6). Epidemic, multidrug-resistant strainsdominate clinical S. pneumoniae isolates in many regions ofthe world today (28).

In all of the pneumococcal strains analyzed so far, cps genesare clustered and located between the dexB and aliA genes (14,24; http://www.sanger.ac.uk/Projects/S_pneumoniae). Genes fortwo penicillin-binding proteins (PBPs) are located on eitherside of this region, pbp2x upstream of dexB and pbp1a downstreamof aliA, each at a remove of $>=$ 10 kb (23, 40, 41).The PBPs are enzymes involved in cell wall synthesis that aretargets for ß-lactam drugs (20). For example, thecps locus of penicillin-susceptible strain TIGR4 is a 15.2-kbregion encoding 13 open reading frames, flanked by the pbp2xgene 10.9 kb upstream and by the pbp1a gene 11.6 kb downstream,and genes coding for all other PBPs are located at least 450kbp away from the cps locus (40).

There are six PBPs in a pneumococcal cell (PBP1a, -1b, -2a,-2b, -2x, and -3), of which PBP2x and PBP2b have been confirmedto be essential for cell growth (20, 21, 25). The pbp genesin S. pneumoniae have a mosaic structure and can undergo inter-and intraspecies recombination (5, 8, 19). Strains resistantto ß-lactams have some of these enzymes modified toexpress reduced affinity for a ß-lactam (20). In general,the resistance profile of the particular isolates results froman interaction between PBPs of various degrees of modification(9, 22), and probably between these and a modified muropeptidebranching enzyme, encoded by the murM gene (11, 39).

Resistance to ß-lactam drugs used in the treatmentof pneumococcal infections requires, in all of the cases studiedso far, alterations in at least PBP2x or -2b (22). Modified,low-affinity PBP2x is sufficient for the cell to become nonsusceptibleto cephalosporins (e.g., cefotaxime) and is also necessary forpenicillin resistance to emerge. Resistance to penicillin requiresmodified, low-affinity forms of at least PBP1a in addition tomodifications in PBP2x (2, 38). The presence of modified formsof PBP2b, PBP2a, and MurM seems to increase further the levelof resistance to ß-lactams (2, 9, 22, 39).

The existence of a selectable phenotype, resistance to ß-lactams,requiring replacements in pbp2x or in both pbp1a and pbp2x,which flank the cps locus, suggested that it might be possibleto select single-step capsular transformants in vitro underselective pressure of cefotaxime or penicillin. The recombinationevent would then involve one or both PBP-encoding genes, aswell as the capsule locus, and would change both the serotypeand the resistance profile of the strain. In case of selectionfor resistance to penicillin, a single event like this wouldrequire replacement of 42 kb or almost 2% of the TIGR4 straingenome, twice the size of any naturally transformed fragmentof S. pneumoniae homologous DNA previously reported (4). Althoughsome transposons of comparable size have been reported to integrateinto the S. pneumoniae genome following transformation, theirmechanism of integration into the host genome is different (36,42).

(These results were presented in part at the 43rd InterscienceConference on Antimicrobial Agents and Chemotherapy, Chicago,Ill., 2003 [abstr. C1-1113].)

MATERIALS AND METHODS

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Materials and Methods

S. pneumoniae strains. The penicillin-susceptible TIGR4 strain of serotype 4 used inthis study was obtained from the American Type Culture Collection(40). Penicillin-resistant serotype 19F strain GA71 was kindlydonated by Brian Spratt (6). Bacterial strains were routinelygrown at 37°C in 5% CO₂ on tryptic soy agar II supplementedwith 5% defibrinated sheep blood or in Todd-Hewitt broth supplementedwith 0.5% yeast extract (Becton Dickinson, Sparks, Md.), storedin Todd-Hewitt broth supplemented with 0.5% yeast extract supplementedwith 10% glycerol, and frozen at –70°C. MICs of cefotaximeand penicillin G were evaluated with E-test strips (AB Biodisk,Solna, Sweden).

Genetic transformation. Growth media and culture methods for genetic transformationhave been used as previously described (33). The transformationof TIGR4 was induced by competence-stimulating peptide variant2, and the donor DNA concentration was 1 µg/ml of thetransformation culture. The genomic DNA of GA71 was preparedwith the QIAGEN Genomic DNA system (QIAGEN, Valencia, Calif.).Samples containing approximately 1 µg of chromosomal DNAwere evaluated for fragment size distribution by pulsed-fieldgel electrophoresis with the CHEF-DRIII system (Bio-Rad, Hercules,Calif.) (35). Transformants were selected on blood agar basemedium 2 (Becton Dickinson) supplemented with 8% defibrinatedsheep blood and various concentrations of penicillin G, trimethoprim(ICN Biochemicals Inc., Aurora, Ohio), and cefotaxime (Sigma-Aldrich,St. Louis, Mo.) alone or in combinations.

Serotyping of isolates. Polysaccharide capsule types were determined on the basis ofQuellung tests with factor-specific typing sera (Statens SerumInstitute, Copenhagen, Denmark) (27).

RFLP. Restriction fragment length polymorphism (RFLP) analysis ofpbp2x, pbp2b, and pbp1a and of the cps locus cpsA-cpsB fragmentwas performed as previously described (15, 26), as a genotypicscreen for the presence of donor PBPs and capsule in transformants.The DNA templates used for this screening were obtained as previouslydescribed (43). To evaluate the sizes of DNA fragments replacedduring the transformation and to identify recombination crossoverpoints, RFLP analysis of the pbp2x-pbp1a region in the donor,the recipient, and five transformants was performed. DNA templateswere prepared with the QIAGEN Genomic DNA system, and productswere amplified with the TripleMaster PCR system (Eppendorf,Hamburg, Germany) under the cycling conditions recommended bymanufacturer. The following pairs of primers were used: TTM13(5'ATGAAGGCAATATCTGGGGTGACTAC-3') and pbp2xr2 (15) to amplifyfragments from the position 10,075 bp upstream to that 32 bpdownstream of pbp2x in the TIGR4 genome (and corresponding fragmentsfrom other isolates), pbp2xf (15) and TTM07 (41) to amplifythe fragment from base 226 of pbp2x to base 1504 of dexB, 1430and 1402 (24) to amplify the fragment from base 1475 of dexBto base 48 of aliA, TTM08 (41) and pbp1af (15) to amplify thefragment from position 21 of aliA to the position 159 bp downstreamof pbp1a, and pbp1ar (15) and TTM15 (5'ATCGCGGTTGTAAGCATCTGTAAT-3')to amplify the fragment from the position 89 bp upstream tothat 5,865 bp downstream of pbp1a. Products of primer pair TTM13-pbp2xr2(pbp2x locus and upstream) were digested with the DdeI enzyme,and those of primer pair 1430-1402 (dexB-cps-aliA locus) weredigested with the RsaI enzyme. Products of the pbp2xf-TTM07and TTM08-pbp1af primer pairs (pbp2x-dexB and aliA-pbp1a loci,respectively) were digested with AluI, DdeI, HaeIII, HinfI,MseI, and RsaI, and those of primer pair pbp1ar-TTM15 (pbp1alocus and downstream) were digested with DdeI, HaeIII, HinfI,and RsaI (all restriction enzymes supplied by New England Biolabs,Beverly, Mass.). Fingerprints generated for transformants werecompared with those of strains TIGR4 and GA71 with DNAStar software.The regions of possible crossover sites were identified between(i) the last restriction site identified in recipient strainTIGR4 but not in donor strain GA71 or in the transformant and(ii) the first site next to this one that was present in therecipient and in the transformant but not in the donor.

RESULTS AND DISCUSSION

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Results and Discussion

We transformed the ß-lactam-susceptible, serotype4 TIGR4 strain with whole genomic DNA extracted from GA71, apenicillin- and cefotaxime-resistant, serotype 19F variant ofthe pandemic Spain^23F-1 clone. Transformants were selected forthe ability to grow on solid media supplemented with penicillin(0.04 mg/liter) or cefotaxime (0.1 mg/liter) (see Table 1 forthe penicillin and cefotaxime MICs for the strains describedin this study). New serotype 19F polysaccharide capsule variantsof TIGR4 were selected among resistant colonies on the basisof the smooth, viscous morphology of the large colonies typicalof GA71 but not observed for TIGR4. The presence of the cps19Flocus was detected by RFLP analysis of the cpsA-cpsB regionin all resistant transformants with colony morphology typicalof serotype 19F, and the presence of the new 19F capsular polysaccharidewas confirmed by the Quellung reaction (26, 27).

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TABLE 1. Penicillin and cefotaxime MICs for the isolates analyzed in this study

In serotype 19F transformants selected for further analysis(Table 1), resistance phenotypes matched PBP profile changesidentified in RFLP analysis of pbp alleles. For all of them,cefotaxime MICs were equal to those observed for GA71 (1 mg/literin all of these isolates), suggesting that all of the determinantsnecessary for emergence of the full cefotaxime resistance phenotypein strain TIGR4 were transformed. On the other hand, while penicillinMICs increased in all of the transformants, they remained morethan 10 times lower than that for the GA71 strain, suggestingthat not all of the donor's resistance determinants were involvedin the transformation. RFLP analysis of pbp1a, pbp2b, and pbp2xshowed that in all of these transformants recombinational replacementsled to changes in the pbp1a and pbp2x genes but not in pbp2b.The possible role of other ß-lactam resistance determinantshas not been investigated.

The complete genomic sequence of TIGR4 (40) and part of thesequence of the Sanger serotype 23F S. pneumoniae strain, whichis a member of the same pandemic Spain^23F-1 clone as GA71 butdiffers in serotype, were available at the time of this study(http://www.sanger.ac.uk/Projects/S_pneumoniae). This sequenceinformation was used to measure the sizes of replaced fragmentsand to track crossover points by RFLP analysis of large PCRproducts. Figure 1 shows the results of mapping of the 58.1-kbfragment of the TIGR4 genomic DNA, covering the whole pbp2x-cps-pbp1alocus and its flanking regions, the corresponding 51-kb fragmentof GA71, and the corresponding fragments of five serotype 19Fß-lactam-resistant transformants of TIGR4 generatedin independent transformation experiments (isolates are describedin Table 1). The sizes of the replaced fragments were calculatedfor each transformant on the basis of the RFLP patterns, andresults are presented in Fig. 1. For each of the five transformants,mapping results were consistent with the hypothesis that a singletransformation event led to recombinational replacement of thecps locus and at least one of the flanking pbp genes. In allof the transformants analyzed, regions in which recombinationalcrossover points were predicted to have occurred were localizedwithin the regions of expected high homology between donor andrecipient strains. The largest single replacement was identifiedin isolate TIGR19Fe. Fingerprints of the whole pbp2x-cps-pbp1aregion of TIGR19Fe exactly matched those of the donor strain,and possible crossover points were identified upstream of pbp2xand downstream of pbp1a, indicating that in a single event bothPBP genes and the capsule locus might have been replaced andat least a 47.8-kb fragment of the TIGR4 genome has been replacedwith a GA71 fragment of more than 43.0 kb. The size of the replacement,covering more than 2% of the pneumococcal genome, highlightsthe considerable ability of S. pneumoniae to adapt under theselective pressure of antimicrobial use and host immune responses.Pulsed-field gel electrophoresis of the donor DNA used for transformationshowed a high concentration of fragments in the range of 35to 250 kb. Thus, it seems unlikely that cotransformation ofsmall DNA fragments was the main mechanism for the replacementsobserved or that the sizes of replacements were limited by thelength of the donor DNA.

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FIG. 1. Graphic representations of the recombinational replacements within and outside the pbp2x-cps-pbp1a region identified by RFLP analysis in five isolates of TIGR4 transformed independently to penicillin nonsusceptibility and serotype 19F with chromosomal DNA of strain GA71. Horizontal lines at the top represent products of PCR amplified with the oligonucleotides indicated above (forward primer) and below (reverse primer) the line. See Materials and Methods for detailed descriptions of the primers used. The sizes of the amplicons generated for the recipient (TIGR4) and donor (GA71) strains with a particular pair of primers are shown above the lines representing PCR products. Horizontal bars represent regions of genomic DNA in the strains analyzed (indicated on the left). The cross-hatched regions represent cps loci, and the black rectangles depict genes present in the regions flanking the cps locus in all natural isolates of S. pneumoniae. Gray regions indicate indels located outside the dexB-aliA locus. The possible sites of the indels were identified on the basis of the Spain^23F-1 clone (serotype 23F isolate genome sequences were made available by the Sanger Institute [http://www.sanger.ac.uk/Projects/S_pneumoniae]). The top bar presents the genetic organization of the 58.1-kbp fragment of the TIGR4 strain from position 296804 to position 354876 of its published genome sequence (GenBank accession number NC003028) (40). The single star marks a 1,709-bp indel at position 300888 to position 302596, and the double star marks a 553-bp indel at position 304674 to position 305226, both upstream of pbp2x in TIGR4. The second horizontal bar represents the organization of the same region in isolate GA71, with a size of 51.0 kbp, calculated on the basis of the PCR products generated in this study. The triple star marks the 559-bp indel present in the Sanger Spain^23F-1 clone strain at position 3809 bp downstream of pbp2x, also identified in GA71. Arrows above and below bars point at the restriction sites identified as the limits of possible crossover, as described in Materials and Methods. Lack of an enzyme name next to the restriction site genome position number indicates that the crossover site was eliminated by the indel. Inner, solid boxes represent the minimal replacements identified by RFLP analysis in capsule and PBP transformants of TIGR4. Estimated minimal TIGR4 genomic DNA fragments involved in recombinational replacements are depicted above boxes. Outer, dashed boxes represent maximal replacements identified the same way, and the estimated sizes of maximal replacements are depicted below boxes. Fingerprints generated for isolates TIGR19Fa, TIGR19Fc, and TIGR19Fd indicate the presence of two recombinational replacements. The RFLP technique used did not allow us to narrow the sizes of replacements in TIGR19Fc. Region A, extending from a position 611 bp upstream of aliA to one 159 bp downstream of pbp1a, marks the possible site of the additional recombinational crossover(s) in this transformant. Because of the presence of these additional crossovers, the sizes of the replacements were not calculated.

Analysis of the RFLP patterns showed that a single transformationevent could lead to simultaneous exchange of the capsule andPBPs in S. pneumoniae. Thus, loss of ß-lactam susceptibilityand the simultaneous emergence of a new capsule type in pneumococciare feasible. However, RFLP analysis showed also that in a majorityof the transformants examined, more than one recombinationalevent took place.

The results obtained thus far did not quantitatively addressthe hypothesis that capsular transformants would "hitchhike"during selection for resistance to ß-lactams, i.e.,that selection for resistance would increase the frequency ofcapsular transformants. To test this hypothesis rigorously,it was necessary to show that capsular transformants were agreater proportion among strains selected for ß-lactamresistance than among transformants selected for an unlinkedresistance determinant. Table 2 shows that serotype 19F cellsreached frequencies 50 to 400 times higher among strains transformedwith GA71 genomic DNA and selected for ß-lactam resistancethan among strains transformed with the same DNA and selectedfor resistance to trimethoprim, which is mediated by alterationsin dhfr, a gene located 0.9 Mb away from the cps locus in TIGR4(40).

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TABLE 2. Frequency of recombinational replacements in cells of penicillin-susceptible S. pneumoniae strain TIGR4 transformed with chromosomal DNA of penicillin-resistant serotype 19F strain GA71 on the basis of the number of colonies selected for resistance to different concentrations of antimicrobial agents

In principle, at least two mechanisms could result in an abundanceof transformants bearing one replacement (cps) among cells bearinga second replacement (PBP loci). The mechanism we originallyhypothesized was a single transformation bearing both replacements,which are closely linked on the genome. An alternate hypothesisis that some cells are more transformable than others; if thiswere the case, any two markers would be present together moreoften than if their transformation were independent, becausethe presence of either would indicate a highly transformablerecipient cell. Porter and Guild (32) showed that there aretwo "transformable units" per cell in pneumococci, so that thenull expectation, if transformations occur independently andall cells are competent, is that the frequency of dual transformantsbearing unlinked markers would be one-half of the product ofthe frequencies of single transformants. Our results closelymatch that expectation. We found that transformants resistantto both trimethoprim (25 mg/liter) and cefotaxime (0.3 mg/liter)occurred at a frequency of 2.0 x 10^–9, 0.43 times theproduct of the single transformation frequencies. Similarly,dual transformants resistant to penicillin (0.1 mg/liter) andtrimethoprim (25 mg/liter) occurred at a frequency of 1.8 x10^–8, 0.45 times the product of the single transformationfrequencies. Since the hypothesis that the small minority ofcells was highly transformable under the protocol used wouldpredict ratios substantially larger than 0.5, we rejected thishypothesis.

On the basis of these quantitative considerations, we concludedthat selection for ß-lactam resistance results inselection for capsular switches owing to genetic "hitchhiking"effects. For the ß-lactams used in this study, thestrongest effect (indicated by the proportion of serotype 19Fcells among resistant transformants [last column of Table 2])was observed among transformants selected for the high concentrationof cefotaxime, probably because low-affinity forms of PBP2xand PBP1a, but not PBP2b, are necessary for the emergence ofthis particular phenotype (29). A weaker effect was observedfor penicillin, for which resistance also requires low-affinityforms of two PBPs, but not necessarily the two flanking thecps locus. The weakest effect was observed among transformantsselected for the low concentration of cefotaxime, where low-affinityPBP2x is the only replacement necessary for the resistance toemerge. This indicates that the hitchhiking effect was moreapparent in penicillin-susceptible TIGR4 when capsule transformantswere selected for the presence of two flanking resistance markersat once.

Table 2 also shows a decrease in the number of transformantsselected for resistance to increasing concentrations of penicillinor cefotaxime, probably representing the complexity of interactionsbetween transformed PBPs. The complexity is present not onlybecause various PBPs may undergo transformation but also becausethe size of the replacement within each particular gene affectsthe level of resistance conferred. Whereas a single amino acidsubstitution within dhfr is sufficient for a pneumococcal isolateto become resistant to trimethoprim, usually changes withinthree different regions of any pbp gene are necessary to synthesizea modified, low-affinity form of the PBP (1, 9).

Our results showed that a new serotype, combined with ß-lactamresistance, could emerge in a susceptible strain via a singletransformation event. The possibility that a single transformationevent can lead to both a capsular polysaccharide type and PBPexchange in S. pneumoniae has been previously anticipated (4,18) since regions of homology that putatively served as recombinationalcrossover points for replacement of capsule biosynthetic geneshave been identified within the cps locus and in pbp1a of theserotype variants of the pandemic Spain^9V-3 clone (4). However,all of these replacements took place among isolates alreadyresistant to penicillin, and to our knowledge there are no publisheddata documenting simultaneous transformation of penicillin-susceptibleS. pneumoniae to a new serotype and to penicillin nonsusceptibility.Integrons and plasmids carrying multiple determinants encodingboth virulence and antimicrobial resistance have been describedin a number of bacterial species (17, 34). In S. pneumoniae,the major mechanism of horizontal gene transfer is transformation,and the organization of the pbp2x-cps-pbp1a region in S. pneumoniaemay provide an analogous opportunity for cotransfer and associatedlinkage selection of determinants of resistance and pathogenicity.It is also well documented that both the cps operon and thePBP-encoding genes are highly variable and show a mosaic structure,changing frequently and independently from other nearby regions(4, 6, 19).

Our finding suggests that selection by host immunity and selectionby antibiotics may be interrelated because of hitchhiking effectsdue to linkage of resistance determinants and the capsule locus;in natural settings, it suggests that creation of new strains,with different capsular types, may be enhanced by selectionby ß-lactams. It has long been observed that penicillinresistance is found only in a limited number of pneumococcalserotypes, but no compelling mechanism for this restrictionhas been proposed. Our observations suggest one hypothesis:that strains acquiring penicillin resistance will often do soby acquiring a new polysaccharide capsule type from the donorstrain, thereby preserving the association between capsule andpenicillin resistance. Such a hypothesis generates the predictionthat the pbp2x and pbp1a genes of penicillin-resistant strainswill be more closely related to corresponding alleles from otherpenicillin-resistant strains of the same serotype than fromother penicillin-resistant strains of other serotypes. The abilityof the pbp2x-cps-pbp1a region to move as a block also suggeststhat selection by the host immune system (for novel capsuletypes) could indirectly select for resistance (or susceptibility)to penicillin.

Recently published data show that introduction of the heptavalentconjugate vaccine has led not only to a reduction in the proportionof vaccine serotypes among clinical isolates of pneumococcibut also to a reduction in the proportion of resistant strains,since the vaccine serotypes are most prevalent among resistantstrains (7, 44). Concern has arisen that this decline in resistancemay be short-lived, because antimicrobial resistance may arisein non-vaccine-type pneumococci as the latter become more common,or resistant clones may acquire non-vaccine-type capsules. Ourfindings suggest that once resistance to penicillin becomesestablished in non-vaccine types, selection by both vaccine-inducedimmunity and continuing antimicrobial use may work togetherto promote the spread of both resistance and non-vaccine-typecapsule genes in the pneumococcal population.

ACKNOWLEDGMENTS

We thank Brian G. Spratt for the GA71 strain, Susan K. Hollingsheadfor critical discussion of the results, and Howard Gold andLata Venkataraman for access to the pulsed-field apparatus.

This work was supported by National Institutes of Health grant1R01AI48935 and by a New Investigator Award to M.L. from theEllison Medical Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Harvard School of Public Health, Department of Epidemiology, Room 903, Building 1, 665 Huntington Ave., Boston, MA 02115. Phone: (617) 432-3269. Fax: (617) 432-3259. E-mail: ktrzcins{at}hsph.harvard.edu.

REFERENCES

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