Serotype III Streptococcus agalactiae from Bovine milk and human neonatal infections (1).

Streptococcus agalactiae (group B streptococcus [GBS]) causes invasive human infections and bovine mastitis. This study examined the genetic relationship between bovine and human serotype III GBS by using molecular techniques that classify human serotype III GBS into four distinct phylogenetic lineages. Bovine serotype III GBS were largely contained in two lineages, which are distinct from the two major lineages (restriction digest types III-2 and III-3) that infect human neonates. One of the bovine lineages closely resembles the human III-1 lineage, whose members occasionally cause human neonatal infections. The bovine strains in the other lineage characteristically have an initiation factor IF2 gene (infB) H allele and multilocus sequence types that are not found in human GBS strains. Evidence suggests that this "H allele" lineage is related to the human III-3 lineage. These results support the assertion that human and bovine GBS are largely unrelated and provide further insight into the genetic relation between human and bovine GBS.

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Streptococcus agalactiae (group B streptococcus [GBS]) is the major etiologic agent of invasive neonatal infections in humans in industrialized countries, causing sepsis, pneumonia, meningitis, osteomyelitis, and soft tissue infections (1). GBS has also been increasingly recognized as an important pathogen in immunocompromised and elderly persons (2,3). GBS emerged as an important cause of neonatal infections in the 1960s; before this time, it was mainly recognized as a cause of bovine mastitis (4). Most data suggest that GBS strains that infect humans are distinct from strains isolated from bovine sources, since bovine strains frequently cannot be typed with antisera to determine capsular polysaccharide serotype, often express protein antigens not found on human isolates, and tend to have different biochemical properties (5 8). The possibility remains, however, that subgroups of GBS infect both humans and cows. If so, these two closely associated hosts could act as reservoirs for each other and sites for the emergence of novel pathogens.

A number of molecular methods, including multilocus enzyme electrophoresis, pulsed-field gel electrophoresis (PFGE) of restriction enzyme digest products of genomic DNA, randomly amplified polymorphic DNA (RAPD) analysis, and multilocus sequence typing (MLST), have been used to demonstrate that the population of GBS that infects humans is highly clonal and limited to a relatively small number of phylogenctic lineages (9-13). Martinez and colleagues reported in 2000 that a large sample of GBS isolated from cow's in Quebec Province in Canada could be classified into five major RAPD groups, which indicates that this sample of bovine GBS also comprised a limited number of lineages (7).

The Quebec sample is particularly useful for further investigating the relationship between bovine and human GBS because most typeable bovine isolates in the sample were serotype III. Serotype III GBS strains account for a substantial proportion of early-onset neonatal human GBS infections and almost all late-onset neonatal infections (2). Four distinct phylogenetic lineages of human serotype III GBS have been identified by PFGE of restriction digest patterns and designated restriction digest patterns type III-1, III-2, III-3, and III-4 (11). Human GBS strains can also be assigned to each restriction digest pattern type by a distinct set of molecular markers, which include analysis of nucleotide substitutions in the centrally conserved region of the translation infB, the presence or absence of the inserted sequences GBSi1 and IS1548 in three chromosomal loci, and MLST (9,11).

RAPD analysis of human GBS isolates collected with the Quebec bovine isolates suggested that the serotype III bovine strains and human GBS strains were largely unrelated, although a definite conclusion was hindered by the small number of human isolates in the study. The obstacle presented by the small human sample size can now be circumvented by the use of the molecular markers described above that identify human phylogenetic lineages of GBS, but which have not yet been applied to the study of GBS from nonhuman sources. We reexamined the Quebec sampie with these molecular markers to better understand the genetic relationship between bovine serotype III GBS and human serotype III GBS.

Methods

Bacterial Isolates

The serotype III GBS were isolated from bovine milk or from vaginal and rectal swab specimens from asymptomatic pregnant women in Quebec Province, Canada, during 1996 and 1997, as previously described (7). RAPD analysis of the 224 bovine GBS isolates had assigned 210 of the isolates to four RAPD groups (I-IV); the remaining 14 isolates were ungrouped (Figure). A total of 70 of the 82 original serotype III GBS strains were recovered for this study. The remaining GBS in this collection were not serotype III or were nontypeable. Bovine isolates were studied from all RAPD groups except RAPD group I, which contained a single bovine serotype III GBS isolate that could not be recovered. Genomic DNA was extracted with the Qiagen DNeasy Tissue Kit (Qiagen, Valencia, CA) from individual colonies grown overnight in broth.

[FIGURE OMITTED]

Molecular Analyses

The central portion of infB was amplified from bacterial DNA by polymerase chain reaction (PCR) with oligonucleotide primers, as described by Hedegaard et al. (14). Each amplicon was purified and sequenced.

The presence or absence of the inserted sequences GBSi I and IS 1548 in the respective chromosomal loci was determined by using PCR of bacterial DNA with primer pairs flanking each of three sites, as previously described (11). In human serotype III GBS, the first locus is an internal region of hylB, the gene encoding hyaluronidase, which is either interrupted by IS1548 or is intact. The second locus is the region between scpB and lmb, the genes encoding streptococcal C5a-ase and a laminin-binding protein, which either contains GBSi1, IS1548, or no insert. The third locus lies between ftsY and sag0728, two open reading frames (ORFs) with gene products of unknown function, which contains GBSi1 or no insert. The latter locus is either referred to as the AW-10 locus (as we refer to it) or the Y locus, according to Luan et al. (15).

MLST was carried out on 46 selected strains as previously described, and a sequence type was assigned to each strain (9). The sequences of the alleles that make up the novel MLST sequence types described in this manuscript can be found at http://sagalactiae.mlst.net (9). DNA dot blots were performed by using a 96-chamber vacuum manifold and [[sup.32]p]-dCTP labeled probes as previously described (16).

The complete sequences of the infB gene H allele and the novel IS1563-like insertion sequence described in this article are available in the GenBank database under accession nos. AY429663 and AY437915, respectively.

Results

Analysis of infB Alleles in Bovine GBS

All human serotype III GBS have either the A or C allele of the gene encoding translation-initiation factor IF2 (infB allele, Table 1). Twenty-five of the 62 Quebec bovine isolates carried the A allele, and 2 carried the C allele, which raises the possibility that these 27 bovine GBS could be closely related to human GBS (Table 2). No strains bearing the B allele were identified, and only one strain carried the D allele. Serotype III GBS that bear the D allele are unlikely to be isolated from humans because all human GBS strains bearing the D allele have thus far been serotype la. The remaining 34 strains contained a previously unidentified infB allele, which we have designated the H allele. The infB H allele differs from other previously identified infB alleles (designated A-G) by two, three, or five nucleotide substitutions within the central conserved portion of the infB gene and most closely resembles the infB A allele (11,14). All 34 of the strains bearing the H alleles are bovine in origin (Table 2). The infB H allele has not been previously encountered in approximately 700 GBS isolates isolated from human sources studied in our laboratory, including over 150 serotype III strains (unpub. data; [11]). These observations suggest that GBS bearing the infB H allele rarely, if ever, colonize or infect humans.

Relationship between infB Allele and RAPD Group

The possibility that the A or C allele bovine strains could be related to human GBS was investigated by examining the distribution of the bovine A or C allele isolates among the groups previously determined by RAPD analysis (Figure) (7). Of the 25 bovine A allele isolates, 19 were found in RAPD group IV-A, and 2 were found in RAPD group IV-C, which suggests that at least some of the bovine A allele isolates could be genetically related to human GBS since human A allele isolates are also found in these two RAPD clusters (Table 2, Figure). The two bovine C allele isolates are in RAPD group IV-C clustered with two human C allele isolates and thus appear closely related to human C allele isolates based on RAPD analysis. As expected, neither the single bovine D allele isolate nor the 34 bovine II allele isolates cluster with human isolates by RAPD analysis (Table 2).

Identification of Bovine Serotype III GBS Related to Type III-1 GBS

The bovine A and C allele isolates were further examined by using techniques that distinguish the four known human serotype III GBS lineages. These techniques include determining the presence or absence of inserted sequences in three previously identified loci and determining the strains' sequence types by MLST (Table 1). Twenty-three of the 25 A allele bovine isolates appear closely related to human restriction digest pattern type III-1 strains because the isolates lack inserted sequences at any of the three loci examined and had a sequence type (ST) identical to those of previously studied III-1 strains (ST-23) or that was different by only one allele (ST-90, ST-92, or ST-94) (Table 3). Thus, a substantial proportion of the serotype III bovine GBS in this sample appear to come from a lineage that is associated with invasive neonatal disease, albeit rarely (9,17,18). Bovine III-1 strains appear to be genetically heterogeneous since they are found in RAPD groups III, IV-A, and IV-C, but no human III-1 strains were found in this sample; thus, the bovine III-1 strains most closely related to human III-1 strains could not be identified in this sample. Nineteen of the III-1 bovine isolates appear to lack the scpb-lmb locus because PCR with primers flanking the intergenic locus of lmb and scpB did not produce an amplicon.

Seven of the A allele strains appear to be restriction digest pattern type III-2 strains on the basis of an analysis of inserted sequences and MLST (Table 3). Only one of these seven isolates, NI-96-2836, is of bovine origin, however, and is found in RAPD group III, whereas the six human A allele isolates are found in RAPD groups IV-A and IV-C. These data suggest that the bovine NI-96-2836 strain is genetically divergent from human III-2 strains, despite sharing the same infB allele, inserted sequences, and ST with human III-2 strains. These data also suggest that restriction digest pattern type III-2 strains, a major cause of human neonatal infections, are rarely, if ever, isolated from bovine milk.

One of the two remaining A allele strains, SH-96-4807, appears indistinguishable from III-3 strains on the basis of MLST because it has ST-17, which is characteristic of III-3 strains. Unlike III-3 strains, however, SH-96-4807 has no inserted sequences in any of the three sites and contains an infB A allele instead of the C allele typical of III-3 strains. The remaining A allele isolate, SH-96-3696, appears to be most closely related to H allele strains, on the basis of its RAPD group, inserted sequences, and ST. No bovine isolate related to restriction digest pattern type III-4 was found in this sample.

The four C allele isolates have the typical inserted sequences and ST (ST-17) found in restriction digest pattern type III-3 strains, with the exception of human isolate 1004A, which has a truncated form of IS1548 in the scpb-lmb intergenic region (Table 4). These strains, which cluster together in RAPD Group IV-C, were isolated from both human and bovine sources. These data indicate that strains from the restriction digest pattern type III-3 lineage infect both humans and bovine udders but that bovine III-3 strains are rare. The only D allele strain has ST-93, an ST which differs by at least three alleles from all the STs previously described for human GBS, which again indicates that D allele serotype III GBS rarely or never colonize humans.

Analysis of H Allele Isolates

Ten H allele isolates were selected to represent strains from RAPD Group II, Group III, and the ungrouped isolates and analyzed in the same fashion. As shown in Table 5, all of the isolates studied have large inserts in the AW-10 site. The 1,700-bp inserts would be the correct size for the GBSi1 insert found in III-3 strains, and sequencing demonstrated an intact copy of GBSi1 in a 1,700-bp amplicon from one of the strains. A 3,000-bp insert from one strain was amplified and sequenced and found to comprise GBSi1, interrupted by an IS3-like insertion sequence identical to sequences found in both the genome MI strain of S. pyogenes and the genome serotype V strain of S. agalactiae (19,20).

A 3,400-bp insert was sequenced and found to consist of an inserted sequence that has identical direct and inverted repeat sequences to the insertion sequence IS1563, but it has a predicted amino acid sequence that is 75% identical to that of IS1563. This IS1563-like insertion sequence is located upstream of GBSi1 exactly as IS1563 is found upstream of GBSi1 in this locus in restriction digest pattern type II-2 strains (11).

The AW-10, hylB, and scpb-lmb loci were studied in the remaining H allele strains not shown in Table 5. No inserted sequences were found in the AW-10 site in seven isolates, while the remaining strains have either the 1,700-bp, 3,000-bp, or 3,400-bp inserts at AW-10. No inserted sequences were found in the hylB and scpb-lmb sites in any of the H allele strains. Two of the H allele strains, both of which are in RAPD group IV-B, appear to lack the scpb-lmb locus.

MLST shows that 8 of these 10 H allele isolates are ST-61. ST-61 isolates are found in RAPD groups II, IV, and in ungrouped isolates, which indicates genetic divergence among H allele strains that is detected by RAPD but not by MLST. ST-61 differs from ST-67 by one allele, which confirms that SH-96-3696, the A allele strain found in RAPD II that has ST-67, is in the same clonal complex as the H allele serotype III strains. SH-96-3696 also has a 3,400-bp insert at AW- 10, and no insert at the other two sites, which provides further evidence that this isolate is related to H allele strains. The other two STs found in the H allele strains, ST-91 and ST-105, differ from ST-61 by two alleles. Thus, the H allele strains are clonally related.

III-3-specific Sequence Tags in H Allele Serotype III GBS

ST-61, the most common ST of the H allele strains, differs by two alleles from ST-17, the most common ST of III-3 strains. This two-allele difference in ST and the observation that isolates from these two lineages both have GBSi1 in the AW-10 site led us to search for other evidence that the two lineages are related.

We previously described 10 short sequence tags that are found in all III-3 strains but not in III-1 or III-2 strains (16). We therefore performed dot-blot hybridization with eight of these probes on a selection of A, H, and C allele strains from this sample to determine the distribution of the III-3-specific sequences among these various lineages. As expected, all of the III-3-specific probes hybridized with every C allele strain tested, whereas the III-3 probes hybridized rarely or never with the A allele strains. In contrast, seven of the eight III-3 probes hybridized with almost all the H allele strains (Table 6).

Discussion

A key finding of this investigation is that the serotype III GBS strains isolated from bovine milk in this sample are largely genetically distinct from the serotype III GBS strains that commonly infect humans. The two most common lineages of serotype III GBS that colonize women and infect infants in the United States and Japan are restriction digest pattern types III-2 and III-3, whereas III-1 and III-4 strains are rarely isolated from the genitourinary tract of women and are rarely associated with invasive disease (11,16).

Only one bovine strain that resembled RDP type III-2 GBS was identified in the 67 isolates, and RAPD analysis indicates that this isolate is distinct from the 7 human III-2 strains in the sample. Thus, III-2 strains that infect humans are unlikely to infect bovine udders. Only two bovine strains were identified that had the genotypic characteristics of restriction digest pattern type III-3 strains. These two strains appear closely related to two human III-isolates by RAPD analysis, which makes it likely that III-3 strains can infect bovine udders, but they appear to do so infrequently.

We also found that the vast majority of bovine serotype III GBS in this sample belong to one of two major phylogenetic lineages. A separate study of bovine GBS isolates collected in the United Kingdom used MLST to identify two clonal complexes that are similar, if not identical, to the two lineages identified here; this finding suggests that two clones of GBS may predominate among bovine mastitis caused by GBS in North America and England (21).

The data presented here show that the first bovine lineage is closely related to restriction digest pattern type III-1 GBS (Table 7). The lack of human III-I strains in this sample makes it difficult to use the RAPD analysis to determine how closely these III-1-like bovine strains resemble human III-1 strains. That no III-1 human isolates were found in this small sample is not surprising because human III-1 isolates rarely colonize the female genitourinary tract (although they occasionally cause neonatal infections) (9,17,18). Most III-1 bovine isolates in the sample studied here appear to lack the scpb-lmb locus, a finding consistent with that reported by Francken et al., who showed that absence of a putative composite transposon that contains the scpB and lmb genes is a feature of bovine GBS (22). Possibly only a few of the III-1 bovine GBS are capable of infecting both humans and cows, since all human GBS appear to have the scpb-lmb locus.

The other major bovine serotype III GBS lineage in this sample is composed of strains that possess an infB H allele, with the exception of a single A allele strain that, on the basis of RAPD and MLST, appears closely related to the H allele strains. The H allele strains have STs that differ from each other by no more than two alleles, which indicates that these strains are likely to have a recent common ancestor.

We believe that strains in this lineage are unlikely to colonize or infect humans since we have not identified an infB H allele strain in >160 human serotype III GBS isolates, and since the STs of the bacteria in this lineage were not found in a large sample of human GBS obtained from diverse geographic areas (9). However, the major ST (ST-61) found in this lineage differs from the major ST of III-3 strains by only two alleles, which suggests that strains from this group may share a relatively recent common ancestor, H allele strains were found to contain previously identified III-3-specific sequence tags, which supports this hypothesis.

The identification of two III-3 strains of bovine origin and a third bovine strain (SH-96-4807) that is genetically distinct from III-3 strains but with the ST typical of III-3 strains (ST-17) supports the concept that III-3 strains share a recent common ancestor with H allele strains and retain some genetic traits necessary for bovine udder colonization. No bovine III-2 strains were identified, but a single bovine strain (NI-96-2836) appears to be related to strains in the III-2 lineage by all molecular markers (although RAPD analysis found it to be clearly distinct from human III-2 isolates). These III-3 like and III-2-like bovine strains are both in RAPD group III (Table 3) and thus appear by RAPD analysis to be more related to each other than to other major bovine lineages or to human isolates. RAPD analysis also suggests that human III-3 strains are more related to human III-2 strains than to the H allele strains, despite the observation that MLST puts human III-3 strains at a greater phylogenetic distance from III-2 strains than from the H allele strains.

The clustering together of human III-2 and III-3 isolates by RAPD analysis, despite their clear distinction by MLST, leads us to hypothesize that common genetic determinants that account for host tropism (human versus bovine) have been acquired by both the III-2 and III-3 lineages and strongly influence the clustering of isolates, as shown by RAPD analysis. If so, the two bovine isolates that closely resemble III-3 and III-2 strains by MLST, but which resemble each other more closely by RAPD analysis, may represent intermediate genotypes between GBS lineages that have a more clear-cut tropism for either humans or bovines. The exact relationship between these human and bovine lineages, and genes important for host tropism, could be clarified by comparative genomics. Such studies, along with further studies of bovine GBS lineages of other serotypes, could also provide insight into the exact relationship between human and bovine strains and help determine whether these hosts act as reservoirs for each other's pathogenetic lineages and for the emergence of new pathogenic clones.

Table 1. Characteristics of human serotytpe III GBS lineages (a)

                               Inserted sequence sites (b)

                 infB
RDP type (c)   allele (d)     AW-10       hylB      scpb-lmb    ST (e)

III-1              A        No insert   No insert   No insert   23, 25
III-2              A        No insert    IS 1548     IS 1548    19, 21
III-3              C          GBSi1     No insert     GBSi1     17, 29
III-4              A        No insert   No insert     GBSi1       1

(a) GBS, group B streptococcus.

(b) The presence of inserted sequences is determined at the three
chromosomal locations (see Methods).

(c) RDP type refers to phylogenetic lineages originally defined by
analysis of restriction digest patterns of chromosomal DNA.

(d) infB is the highly conserved gene encoding initiation-translation
factor IF2.

(e) ST refers to the isolates' sequence type, based on the alleles of
seven housekeeping genes identified by multilocus sequence typing.

Table 2. Distribution of infB alleles in serotype III GBS from
different RAPID groups (a)

                                               infB allele

                  Species of   Isolates
RAPID group (b)     origin     studied     A   B   C   D    H

II                  Bovine        31       1   0   0   0   30
III                 Bovine        4        3   0   0   1    0
IV-A                Bovine        19      19   0   0   0    0
                    Human         1        1   0   0   0    0
IV-B                Bovine        2        0   0   0   0    2
IV-C                Bovine        4        2   0   2   0    0
                    Human         7        5   0   2   0    0
Ungrouped           Bovine        2        0   0   0   0    2
                    Total         70      31   0   4   1   34

(a) GBS, group B streptococcus.

(b) RAPD group identified by randomly amplified polymorphic DNA
analysis.

Table 3. Inserted sequences and sequence type (ST) of bovine strains
containing the infB A allele (a)

                                       Inserted sequence sites

RAPD group      Isolate      Origin     AW-10          hylB

II            SH-96-3696     Bovine   3,400 (b)        None
III           NI-96-2836     Bovine     None          IS 1548
              SH-96-4807     Bovine     None           None
              RF-96-2997     Bovine     None           None
IV-A             1003A       Human      None          IS 1548
              AL-97-0498     Bovine     None           None
              SH-96-3417
             All 17 others   Bovine     None           None
IV-C          NI-96-3213     Bovine     None           None
              ASS-96-666     Bovine     None           None
                 1007B       Human      None          IS 1548
             1009A, 15888    Human      None          IS 1548
             13228, 1009B    Human      None          IS 1548
                                                  (truncated) (f)

                                Inserted
                             sequence sites

RAPD group      Isolate        scpb-lmb          ST       RDP type

II            SH-96-3696          None           67      Unknown (c)
III           NI-96-2836        IS 1548          19      III-2-like
              SH-96-4807          None           17      III-3-like
              RF-96-2997          None           94         III-1
IV-A             1003A          IS 1548          19         III-2
              AL-97-0498          None           23         III-1
              SH-96-3417
             All 17 others   No product (d)   23,90,92   III-1-like
IV-C          NI-96-3213     No product (d)      23      III-1-like
              ASS-96-666     No product (d)      23      III-1-like
                 1007B         IS 1548/IS        86         III-2
                                1381 (e)
             1009A, 15888       IS 1548          19         III-2
             13228, 1009B       IS 1548          19         III-2

(a) RAPD, randomly amplified polymorphic DNA; RDP, restriction digest
pattern.

(b) A 3,400-bp insert is also found in other strains from randomly
amplified polymorphic DNA (RAPD) group II (see text and Table 5).

(c) This strain appears to be related to the H allele strains
described in Table 5.

(d) No polymerase chain reaction product was produced from these
strains in multiple attempts.

(e) In this strain, the insertion sequence IS 1381 is upstream from
IS 1548 in the scpb-lmb locus.

(f) S 1548 in the hylB locus is missing 657 bp from the 3' end of its
open reading frame in this strain.

Table 4. Analysis of inserted sequences of strains containing
the infB C allele (a)

                               Inserted sequence site

RAPID   Isolate      Origin   AW-10   hylB   scpb-lmb

IV-C    SF 96-5547   Bovine   GBSi1    700   GBSi1
        1004A        Human    GBSi1    700   IS 1548 (b)
        1000B        Human    GBSi1    700   GBSi1
        SF-96-4054   Bovine   GBSi1    700   GBSi1

RAPID   Isolate      ST   RDP type

IV-C    SF 96-5547   17   III-3
        1004A        17   III-3
        1000B        17   III-3
        SF-96-4054   17   III-3

(a) RAPID, randomly amplified polymorphic DNA; ST, sequence type; RDP,
restriction digest pattern.

(b) An insert highly homologous to IS 1548 but lacking by 603-1,301
was found in th is strain. IS 1548 has not previously been found in
this locus in III-3 strains.

Table 5. Inserted sequences and sequence types of infB H allele
strains (a)

                       Size of PCR poroduct from
                      inserted sequence sites (bp)

RAPD          Isolate       AW-10      hylB      scpb-lmb      ST

II          RF-96-2834    3,000 (b)   700 (c)    650 (d)     61
            ASS-97-0701   3,000       700        650         61
            AL-96-1653    1,700 (e)   700        650         61
            SF-96-6312    3,400 (f)   700        650         61 (g)
            SF-96-4396    1,700       700        650         91
            NI-96-2521    3,000       700        650         105
IV-B        ASS-96-659    3,000       700       No product   61 (g)
            SH-96-5461    1,700       700       No product   61
Ungrouped   AL-96-2049    3,000       700        650         61
            NI-96-3329    3,000       700        650         61

(a) RAPD, randomly amplified polymorphic DNA; ST, sequence type.

(b) A 3,000-bp amplicon was sequenced and found to contain an IS3like
insertion sequence interrupting GBSi1.

(c) A 700-bp product from the hylB site indicates that there is no
inserted sequence in the site.

(d) A 650-bp product from the scpb-lmb site indicates that there is no
inserted sequence in the site.

(e) A 1,700-bp product was amplified and found to contain an intact
copy of GBSi1.

(f) A 3,400-bp amplicon was sequenced and found to contain GBSi1 and
an IS 1563-like insertion sequence.

(g) The glcK (glucose kinase) gene in these two strains contains a
mobile genetic element in the portion of glcK used to determine the ST
of GBS strains. These two strains were found to be sequence type 61
(ST-61) after the inserted sequence was removed. The identical
inserted sequence in the glcK gene has been found in other ST-61
bovine strains and is described elsewhere (21).

This study was supported by the following grants: USPHS NIH RO1 AI 40918 (J.F.B., E.E.A.); Thrasher Research Fund (J.F.B.); Cancer Center Support CORE Grant P30 CA 21765 (E.E.A.); and the American Lebanese Syrian Associated Charities (E.E.A.).

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Dr. Bohnsack received research training at the University of Washington, in the National Institute for Allergy and Infectious Diseases Intramural Program at National Institutes of Health, Bethesda, Maryland, and at Scripps Research Institute. He began his studies of group B streptococcus shortly after joining the faculty of the University of Utah in 1988.

Address for correspondence: John F, Bohnsack, Department of Pediatrics, University of Utah Health Sciences Center. 50 North Medical Drive, Salt Lake City. UT 84132, USA; fax: [phone]; email: john.bohnsack@ hsc.utah.edu

John F. Bohnsack, * April A. Whiting, * Gabriela Martinez, ([dagger]) Nicola Jones, ([double dagger]) Elisabeth E. Adderson, ([section]) Shauna Detrick, * Anne J. Blaschke-Bonkowsky, * Naiel Bisharat, ([double dagger]) and Marcelo Gottschalk ([dagger])

* University of Utah Health Sciences Center, Salt Lake City, Utah, USA; ([dagger]) Faculte de Medicine Veterinaire, Universite de Montreal, Saint-Hyacinthe, Quebec, Canada; ([double dagger]) John Radcliffe Hospital, Oxford, United Kingdom; and ([section]) St. Jude Children's Research Hospital, Memphis, Tennessee, USA

(1) Presented in part at the XVth Lancefield International Symposium on Streptococci and Streptococcal Diseases, October 6-11, 2002, Goa, India.

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