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Molecular Epidemiology of Group B Streptococcal Infections: Use of Restriction Endonuclease Analysis of Chromosomal DNA and DNA Restriction Fragment Length Polymorphisms of Ribosomal RNA Genes (Ribotyping) Henry M. Blumberg, David S. Stephens, Carmelo Licitra, Nan Pigott, Richard Facklam, B. Swaminathan, and I. Kaye Wachsmuth
Division ofInfectious Diseases, Department ofMedicine, Emory University School ofMedicine, and Division ofBacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia
Group B streptococci (Streptococcus agalactiae; GBS) cause significant morbidity and mortality in humans, both infants and adults, in the United States and worldwide [1-8]. GBS are the most frequent cause of life-threatening infections in newborns in the United States and cause two distinct syndromes in neonates: early-onset (within the first 5 days of life; mean onset, 20 h) and late-onset disease (from day 7 to 3 months of age) [1]. GBS infections also cause significant pregnancy-related morbidity, which affects 50,000 women each year in the United States [9-11]. Only recently has it been recognized that most bacteremias due to GBS in adults occur in non-pregnancy-related cases [2, 4]. Non-pregnancy-related adult bacteremias are associated with a high mortality rate, 28%-70% in several series [4-7], and usually occur in older individuals with underlying chronic disease or those who are immunosuppressed [4, 6]. Although a large percentage of non-pregnancy-related GBS bacteremic disease in adults is nosocornially acquired, the reservoir for this organism, the mechanism of spread, and the potential for nosocomial transmission in adults are largely unknown [4]. r-
Received 3 January 1992; revised 22 April 1992. Presented in part: 31 st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, October 1991. Reprints or correspondence: Dr. Henry M. Blumberg, Division of Infectious Diseases, Emory University School of Medicine, 69 Butler St., S.£., Atlanta, GA 30303. The Journal of Infectious Diseases 1992;166:574-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0018$01.00
Epidemiologic investigation of GBS infections has been limited by the lack of a discriminatory subtyping system [12]. Serotyping has been the traditional method used to type GBS isolates. GBS can be serotyped with antisera to capsular polysaccharide antigens Ia, Ib, II, and III [13]. Protein antigen c is noted to occur in many serotype la and II strains and among all Ib isolates [14-16]. The conventional serotyping system consists of la, Ia/c, Ib/c, II, II/c, III, and non typeable isolates. The R and X protein antigens classify a few additional human strains but are most useful for serotyping bovine strains, which are usually nontypeable by the above scheme [14]. An additional serotype (IV) has been reported and provisional serotypes (V and candidate NT6) have been proposed [17, 18]. A number ofstudies have reported that GBS isolates recovered from adults, children, and neonates with early-onset disease (i.e., sepsis) are evenly distributed among the three major serotypes: I, II, and III [1, 19-21]. However, 90% of isolates from patients with meningitis and late-onset neonatal disease are serotype III. Overall, serotype III isolates account for more than one-half and in most studies two-thirds of neonatal GBS disease [10, 22]; therefore, the discriminatory power of the serotyping scheme is undesirably low [12]. In addition, in some studies as many as 10%-15% of G BS isolates are non typeable [12, 23], making comparisons with those isolates impossible. Bacteriophage typing has been used as a supplementary typing system but has a number of significant limitations, including variability of results and the fact that ......, 30% of GBS isolates are not typeable by phage [12]. Phage typing is
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Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therfore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. Hhal gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype la/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.
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DNA preparation and digestion and separation offragments. Chromosomal DNA was extracted as described by Pitcher et al. [38] after organisms were grown overnight in Todd-Hewitt broth at 37°C with aeration. DNA of selected isolates was digested with 24 restriction endonucleases (Acel, Alul, Bell, Clal, Dral, EcoRI, Hindlll, Mbol, Pvull, EeoRV, BamHI, KpnI, Smal, Neol, HincH, Haelll, Hpal, BgIH, Aval, Hhal, Neil, Sphl, Sspl, and Xbal) according to the manufacturer's recommendations (New England Biolabs, Beverly, MA). In addition, double digestion with Neil and A val was done. Hhal (which appeared to give the best discrimination of hybridization banding patterns) was used to digest chromosomal DNA from all isolates in the study collection. DNA (2 JLg) was digested by Hhal (I JLL or 10 units) for 2 h at 37°C in a 20-JLL reaction mixture; an additional I JLL (I 0 units) of restriction enzyme was then added, and the reaction mixture was reincubated for 2 h. The digested DNA was electrophoresed in a 1% agarose horizontal gel at 60 V for 18 h in TRIS-acetate buffer (0.04 M TRIS-acetate, 0.002 M EDT A, pH 8.1). After electrophoresis, the gels were stained in ethidium bromide (I JLg/mL) and photographed under ultraviolet light. A l-kb ladder (I JLg) and A phage DNA (2 JLg; GIBCO BRL, Gaithersburg, MD) were used as molecular size standards. Preparation ofSouthern blots. Digested and electrophoresed DNA restriction fragments were denatured and transferred to a nylon membrane (MSI magnagraph; MSI, Westboro, MA) by the method of Southern [39]. On completion of the DNA transfer, nylon membranes were handled as previously described [32, 40] and stored at 4°C until use. Preparation of labeled probe and hybridization. E. coli 16Sand 23S-rRNA (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used as the probe. Hybridization experiments were done with a nonradioactive labeling system (Genius System; Boehringer Mannheim) as previously described [32, 40].
Materials and Methods
Results
Bacterial isolates. We studied 76 GBS isolates (S. agalactiae) associated with human invasive disease (e.g., bacteremia, meningitis) by REAC and ribotyping. Most isolates (n = 62) studied were recovered from patients (adults and infants) at Grady Memorial Hospital (Atlanta) in 1987-1988 and 1990-1991. Fourteen human isolates were referred to the Centers for Disease Control (CDC) from three states (California, Missouri, and Tennessee) in 1986 and 1987. ' Organism identification. GBS isolates recovered from patients at Grady Memorial Hospital were isolated in the Clinical Microbiology Laboratory on the basis of colony morphology, ,a-hemolysis, and gram-positive staining and were confirmed by a latex agglutination procedure (Streptex kit; Burroughs Wellcome, Research Triangle Park, NC). Isolates referred to CDC were confirmed as GBS by Lancefield serologic typing. Serotyping. Serotyping was done by the Lancefield capillary precipitin method for typing GBS [36]. Antisera to polysaccharide antigens la, Ib, II, and III and protein antigen c were used. Antisera were prepared at CDC. Plasmid analysis. Plasmid DNA analysis was done on 18 isolates by the method of Birnboim and Doly [37].
Serotyping ofall 76 isolates studied by REAC and ribotyping demonstrated that 33 were serotype Ia/c, 3 were la, 2 were II/c, 25 were III, 7 were nontypeable (NT), and 6 were nontypeable by antisera to carbohydrate antigens but did give a positive reaction with antisera to protein antigen c (NT/c). Thus most isolates were serotype Ia/c, III, or NT. This distribution reflects the predominance of these serotypes at Grady Memorial Hospital (unpublished data). Plasmid DNA analysis was done on 18 GBS isolates; none contained plasmid DNA. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction endonucleases. HhaI appeared to give the best discrimination of hybridization banding patterns or ribotypes (data not shown) and was used for all study isolates. Digestion with H hal yielded a good distribution of restriction length fragments. REAC patterns of selected GBS isolates and corresponding ribotypes are shown in figure 1. A number of isolates were studied repeatedly and always yielded the same REAC and ribotyping pat-
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not widely available, reagents are not commercially available, and when phage typing was used in one outbreak investigation, it did not substantially add to serotyping data [24]. Plasmid DNA analysis, which has been used to assist epidemiologic investigation of both gram-positive and -negative bacterial pathogens, is generally not useful in typing GBS as most of these isolates do not carry plasmid DNA [14]. In addition, plasmid DNA is extrachromosomal and therefore does not provide definitive evidence that two bacterial strains are related. Multilocus enzyme electrophoresis has been used to estimate chromosomal genetic diversity and relationships among bacterial species (including S. agalactiae) and as a molecular epidemiologic tool [25, 26]. However, this procedure was not useful in strain differentiation of serotype III isolates, as nearly all serotype III isolates collected from patients with invasive disease fell into a single electrophoretic type [26, 27]. Because of the limitations of these other typing methods, we explored the use of two DNA-based technologies to subtype molecularly GBS isolates recovered from humans with invasive disease. The two methods used were restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping). These methods use commercially available reagents, supplies, and equipment and have proved to be useful molecular epidemiologic techniques in the study of a number of other bacterial pathogens, including Salmonella typhi, Haemophilus infiuenzae, Escherichia coli, Campylobacter species, Yersinia enterocolitica, Staphylococcus aureus, and Pseudomonas cepacia [28-35].
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terns, suggesting that these methods are reproducible (data not shown). Overall, 9 ribotypes were noted among the 76 GBS isolates studied (figure IB). Four ribotypes were noted among the laic isolates (ribotypes 1-4, figure 1B); 2 of 3 serotype la isolates were ribotype 1, and 1 was ribotype 2. The 2 serotype Il/c isolates were ribotype 5. Four other distinct ribotypes were noted, among the serotype III isolates (ribotypes 6-9, figure IB). All NT or NTlc isolates studied were recovered from patients at Grady Memorial Hospital. Twelve of 13 NT or NT [c isolates were ribotype 3, a pattern also seen with serotype laic isolates. One NT isolate had ribotype pattern 6 (seen with serotype III isolates). The distribution of the ribotype patterns is shown in table 1. Table 1. Distribution ofGBS ribotypes. Ribotype
Serotype
n n
la. Ia/c, NT. NT/e
5
II/e
III. NT
25 6 16 I 2 15
7 3 I
Discussion 76
Total NOTE.
No. of isolates
NT
=
nontypeable.
Epidemiologically related isolates always had identical ribotype and REAC patterns. This was noted among GBS isolates recovered from 4 mother-infant pairs (figure 2), in which the mothers had postpartum G BS bacteremic disease and their infants had early-onset sepsis, 1 set of twins who had late onset neonatal meningitis and sepsis, and 5 sets of patient isolates from different culture sites (e.g., blood-cerebrospinal fluid, blood-urine). Epidemiologically unrelated isolates of the same ribotype usually had different REAC patterns. This is shown in figure 2 in comparing different mother-infant pairs of the same ribotype. For example, mother-infant pairs 1 and 2 were serotype laic and had ribotype pattern 1. However, on REAC, there was at least one restriction fragment difference between these 2 sets in the high-molecular-size fragments (figure 2A). Similarly, mother-infant pairs 3 and 4 were serotype III and ribotype pattern 6 (figure 2B), but there were differences between the pairs on REAC in the high-molecular-size fragments (figure 2A). Further examples of unrelated isolates of the same ribotype having different REAC patterns are shown in figure 3. These findings suggest that REAC may be a more sensitive technique for strain differentiation. Isolates listed in figures 2 and 3 did not contain plasmid DNA, so differences noted on REAC were due to differences in chromosomal DNA.
Two DNA-based technologies, REAC and ribotyping used in combination, proved to be successful molecular epi-
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Figure 1. A, Restriction endonuclease analysis of chromosomal DNA patterns of selected GBS isolates. B, Corresponding ribotypes (patterns 1-9). Serotypes are shown at bottom. Lane, ribotype, and isolate designations are as follows: 1, 1, 152; 2, 2, 201; 3, 3, GH15; 4, 4, 74; 51> 5, GH-13; 52' 5, 55; 6,6,202; 7, 7, 36; 8, 8, 175; 9, 9, GH-8; M, l-kb ladder (molecular size marker).
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demiologic methods for subtyping GBS. Results were reproducible, and these methods could differentiate among isolates of the same serotype and of different serotypes. These methods should prove useful in assisting epidemiologic investigation of GBS infections such as neonatal nursery outbreaks, nosocomial transmission on adult wards, and the source and reservoir ofGBS (e.g., in late-onset neonatal disease and in non-pregnancy-related adult disease). Previous investigation ofGBS has been limited by the lack ofa discriminatory serotyping [12] or other subtyping system. As shown by our REAC and ribotyping data, there is genetic heterogeneity within GBS serotypes. In addition, all isolates are typeable by REAC and ribotyping, and GBS isolates that are nontypeable by serotyping can be evaluated and compared with other isolates. In our study, epidemiologically related isolates always had identical REAC and ribotype patterns. This included 4 sets of mother-infant paired isolates from mothers with postpartum bacteremia and their infants with early-onset disease (sepsis). The finding of identical mother-infant paired isolates by molecular typing is consistent with the belief that transmission in early-onset disease occurs vertically from mother to child [1]. REAC and ribotyping appear to be the most useful techniques reported to date for subtyping GBS isolates. Multilocus enzyme electrophoresis has been useful in the study of population genetics [25] and has been successfully applied to
molecular epidemiology ofa number of bacterial species [41, 42]. By using electrophoretic patterns of a number of metabolic enzymes, clonal relationships among strains can be determined. However, the use of multilocus enzyme electrophoresis to subtype GBS appears to be limited because of the inability to discriminate among serotype III isolates causing invasive disease. Musser et al. [26] studied 128 GBS isolates, from 5 serotypes, collected from a number of geographic locations. These human isolates (colonizing strains and those causing invasive disease) were categorized into 15 electrophoretic types. However, nearly all (40/44) serotype III isolates causing invasive disease were ofa single electrophoretic type, leading the authors to conclude that a single clone accounts for the overwhelming majority of disease caused by serotype III strains. Our data demonstrated genetic heterogeneity among invasive serotype III isolates (and among other serotypes). Thus, invasive serotype III isolates may not belong to a single clone. As noted, 4 ribotypes were noted among serotype III isolates, and although isolates of the same ribotype often had similar REAC patterns, nearly every epidemiologically unrelated strain had a unique REAC pattern. Two previous studies [14,43] have reported results ofchromosomal digestion patterns ofGBS, and an additional recent study used REAC and ribotyping [44]. These authors also noted that epidemiologically related isolates always had identical REAC patterns [14, 43, 44]. However, they re-
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Figure 2. A, Restriction endonuclease analysis of chromosomal DNA patterns for 4 sets of mother-infant (M-I) paired GBS isolates. Each M-I pair had an identical REAC pattern. Isolates 164, 163, 75, and 69 in lanes M I, II' M2 , and 12 , respectively, were serotype laic; isolates 230, 227, 203, and 202 in lanes M 3 , 13 , M4 , and 14 , respectively, were serotype III. Arrowhead indicates I restriction fragment difference between mother-infant pairs I and 2. B, Corresponding ribotype patterns. Each M-I pair had same ribotype.
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Figure 3. Corresponding restnction endonuclease analysis of chromosomalDNA (A) and ribotype patterns (B) of epidemiologically unrelated GBS isolates. Unrelated isolates of same ribotype had different REAC patterns. Lane numbers in A and B correspond; numbers at bottom (B) are ribotypes. Lane, isolate, and serotype designations are as follows: 1, 1319-87, laic; 2, 2073-86, Ia; 3, GH-4A, laic; 4, GH-6, laic; 5, 3395-86, laic; 6, 2864-86, laic.
Acknowledgments
We thank Portia Williams, Beverly Metchock, John McGowan, and the staff of the Clinical Microbiology Laboratory, Grady Memorial Hospital, for their assistance and Chris Harvey and Monica Farley for their help and suggestions.
References
ported that a number of unrelated strains had the same REAC pattern. They evaluated a limited number of restriction enzymes (two to five) and only Bingen et al. [44] did rRNA hybridization studies. The selection of the restriction enzyme is critical in maximizing the discriminatory power of the REAC and ribotyping systems. Hhal gave the best discrimination among GBS isolates of the 24 restriction enzymes studied; it allowed detection of differences between strains that could not be seen with other endonucleases tested, including those used in other studies. H hal recognizes a 4-base seq'-;lence (5' . . . GCG.C. . . 3') and yielded relatively fewer high-molecular-size fragments than did a number of other restriction enzymes studied. This allowed discrimination of strains by REAC on the basis of differences in the high-molecular-size fragments (e.g., >5.1 kb). The 5-9 fragments detected by rRNA hybridization are simpler to interpret than the larger number of fragments produced by chromosomal endonuclease digestion. Isolates of interest easily can be compared even if run on different gels or on different days. Because rRNA genes are highly conserved across a wide range of bacterial species, E. coli 16Sand 23S-rRNA, which is commercially available, will hybridize with and can be used to probe complementary DNA in S.
I. Baker CJ, Edwards MS. Group B streptococcal infections. In: Remington JS, Klein JO, eds. Infectious diseases of the fetus and newborn infant. 3rd ed. Philadelphia: WB Saunders, 1990:742-811. 2. Harvey RC, Farley MM, Stull T, Smith 10, Schuchat A, Stephens OS. Population-based assessment of Streptococcus agalactiae meningitis and bacteremia in metropolitan adults.In: Program and abstracts of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy (Chicago). Washington DC: American Society for Microbiology. 1991. 3. Klein NC, Schoch PEe Cunha BA. Nosocomial group B streptococcal infections. Infect Control Hosp EpidemioI1989;10:475-9. 4. Opal SM, Cross A, Palmer M. Almazan R. Group B streptococcal sepsis in adults and infants. Arch Intern Med 1988;148:641-5. 5. Verghese A, Mireault K. Arbeit RD. Group B streptococcal bacteremia in men. Rev Infect Dis 1986;8:912-7. 6. Gallagher PG. Watanakunakorn C. Group B streptococcal bacteremia in a community teaching hospital. Am 1 Med 1985;78:795-800. 7. Lerner PI. Gopalakrishna KV. Wolinsky E. McHenry MC, Tan JS. Rosenthal M. Group B streptococcus bacteremia in adults: analysis of 32 cases and review of the literature. Medicine 1977;56:457-73. 8. Schwartz B, Schuchat A. Oxtoby Ml, Cochi SL. Hightower A, Broome CV. Invasive group B streptococcal disease in adults: a populationbased study in metropolitan Atlanta. lAMA 1991;266:1112-4. 9. Baker Cl, Rench MA, Kasper DL. Response to type III polysaccharide in women whose infants have had invasive group B streptococcal infection. N Engl 1 Med 1990;322:1857-60. 10. Institute of Medicine, National Academy of Sciences. New vaccine development: establishing priorities. Vol I. Diseases of importance in
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agalactiae. Stull et al. [34] demonstrated that there are no differences in hybridization banding patterns with speciesspecific or E. coli 16S- and 23S-rRNA. Ribotyping, which has proven useful in determining the taxonomy of a wide range of bacterial species [35], also can detect intraspecies variation, as noted with GBS here and with other bacterial pathogens [28-33]. Although ribotyping could differentiate isolates of the same serotype and 9 ribotypes were noted among the 76 GBS isolates studied, REAC appeared to be an even more sensitive technique for strain differentiation because epidemiologically unrelated isolates of the same ribotype pattern usually had different REAC patterns. Rarely did unrelated isolates have identical REAC patterns. These techniques used in combination allow enhanced discriminatory power. In summary, ribotyping and REAC used in combination were successful and reproducible molecular epidemiologic methods for subtyping GBS and should prove useful in assisting clinical and epidemiologic investigations of GBS infections.
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18.
Molecular Epidemiology of GBS Infections
Molecular Epidemiology of Group B Streptococcal Infections: Use of Restriction Endonuclease Analysis of Chromosomal DNA and DNA Restriction Fragment Length Polymorphisms of Ribosomal RNA Genes (Ribotyping) Henry M. Blumberg, David S. Stephens, Carmelo Licitra, Nan Pigott, Richard Facklam, B. Swaminathan, and I. Kaye Wachsmuth
Division ofInfectious Diseases, Department ofMedicine, Emory University School ofMedicine, and Division ofBacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia
Group B streptococci (Streptococcus agalactiae; GBS) cause significant morbidity and mortality in humans, both infants and adults, in the United States and worldwide [1-8]. GBS are the most frequent cause of life-threatening infections in newborns in the United States and cause two distinct syndromes in neonates: early-onset (within the first 5 days of life; mean onset, 20 h) and late-onset disease (from day 7 to 3 months of age) [1]. GBS infections also cause significant pregnancy-related morbidity, which affects 50,000 women each year in the United States [9-11]. Only recently has it been recognized that most bacteremias due to GBS in adults occur in non-pregnancy-related cases [2, 4]. Non-pregnancy-related adult bacteremias are associated with a high mortality rate, 28%-70% in several series [4-7], and usually occur in older individuals with underlying chronic disease or those who are immunosuppressed [4, 6]. Although a large percentage of non-pregnancy-related GBS bacteremic disease in adults is nosocornially acquired, the reservoir for this organism, the mechanism of spread, and the potential for nosocomial transmission in adults are largely unknown [4]. r-
Received 3 January 1992; revised 22 April 1992. Presented in part: 31 st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, October 1991. Reprints or correspondence: Dr. Henry M. Blumberg, Division of Infectious Diseases, Emory University School of Medicine, 69 Butler St., S.£., Atlanta, GA 30303. The Journal of Infectious Diseases 1992;166:574-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0018$01.00
Epidemiologic investigation of GBS infections has been limited by the lack of a discriminatory subtyping system [12]. Serotyping has been the traditional method used to type GBS isolates. GBS can be serotyped with antisera to capsular polysaccharide antigens Ia, Ib, II, and III [13]. Protein antigen c is noted to occur in many serotype la and II strains and among all Ib isolates [14-16]. The conventional serotyping system consists of la, Ia/c, Ib/c, II, II/c, III, and non typeable isolates. The R and X protein antigens classify a few additional human strains but are most useful for serotyping bovine strains, which are usually nontypeable by the above scheme [14]. An additional serotype (IV) has been reported and provisional serotypes (V and candidate NT6) have been proposed [17, 18]. A number ofstudies have reported that GBS isolates recovered from adults, children, and neonates with early-onset disease (i.e., sepsis) are evenly distributed among the three major serotypes: I, II, and III [1, 19-21]. However, 90% of isolates from patients with meningitis and late-onset neonatal disease are serotype III. Overall, serotype III isolates account for more than one-half and in most studies two-thirds of neonatal GBS disease [10, 22]; therefore, the discriminatory power of the serotyping scheme is undesirably low [12]. In addition, in some studies as many as 10%-15% of G BS isolates are non typeable [12, 23], making comparisons with those isolates impossible. Bacteriophage typing has been used as a supplementary typing system but has a number of significant limitations, including variability of results and the fact that ......, 30% of GBS isolates are not typeable by phage [12]. Phage typing is
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Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therfore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. Hhal gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype la/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.
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DNA preparation and digestion and separation offragments. Chromosomal DNA was extracted as described by Pitcher et al. [38] after organisms were grown overnight in Todd-Hewitt broth at 37°C with aeration. DNA of selected isolates was digested with 24 restriction endonucleases (Acel, Alul, Bell, Clal, Dral, EcoRI, Hindlll, Mbol, Pvull, EeoRV, BamHI, KpnI, Smal, Neol, HincH, Haelll, Hpal, BgIH, Aval, Hhal, Neil, Sphl, Sspl, and Xbal) according to the manufacturer's recommendations (New England Biolabs, Beverly, MA). In addition, double digestion with Neil and A val was done. Hhal (which appeared to give the best discrimination of hybridization banding patterns) was used to digest chromosomal DNA from all isolates in the study collection. DNA (2 JLg) was digested by Hhal (I JLL or 10 units) for 2 h at 37°C in a 20-JLL reaction mixture; an additional I JLL (I 0 units) of restriction enzyme was then added, and the reaction mixture was reincubated for 2 h. The digested DNA was electrophoresed in a 1% agarose horizontal gel at 60 V for 18 h in TRIS-acetate buffer (0.04 M TRIS-acetate, 0.002 M EDT A, pH 8.1). After electrophoresis, the gels were stained in ethidium bromide (I JLg/mL) and photographed under ultraviolet light. A l-kb ladder (I JLg) and A phage DNA (2 JLg; GIBCO BRL, Gaithersburg, MD) were used as molecular size standards. Preparation ofSouthern blots. Digested and electrophoresed DNA restriction fragments were denatured and transferred to a nylon membrane (MSI magnagraph; MSI, Westboro, MA) by the method of Southern [39]. On completion of the DNA transfer, nylon membranes were handled as previously described [32, 40] and stored at 4°C until use. Preparation of labeled probe and hybridization. E. coli 16Sand 23S-rRNA (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used as the probe. Hybridization experiments were done with a nonradioactive labeling system (Genius System; Boehringer Mannheim) as previously described [32, 40].
Materials and Methods
Results
Bacterial isolates. We studied 76 GBS isolates (S. agalactiae) associated with human invasive disease (e.g., bacteremia, meningitis) by REAC and ribotyping. Most isolates (n = 62) studied were recovered from patients (adults and infants) at Grady Memorial Hospital (Atlanta) in 1987-1988 and 1990-1991. Fourteen human isolates were referred to the Centers for Disease Control (CDC) from three states (California, Missouri, and Tennessee) in 1986 and 1987. ' Organism identification. GBS isolates recovered from patients at Grady Memorial Hospital were isolated in the Clinical Microbiology Laboratory on the basis of colony morphology, ,a-hemolysis, and gram-positive staining and were confirmed by a latex agglutination procedure (Streptex kit; Burroughs Wellcome, Research Triangle Park, NC). Isolates referred to CDC were confirmed as GBS by Lancefield serologic typing. Serotyping. Serotyping was done by the Lancefield capillary precipitin method for typing GBS [36]. Antisera to polysaccharide antigens la, Ib, II, and III and protein antigen c were used. Antisera were prepared at CDC. Plasmid analysis. Plasmid DNA analysis was done on 18 isolates by the method of Birnboim and Doly [37].
Serotyping ofall 76 isolates studied by REAC and ribotyping demonstrated that 33 were serotype Ia/c, 3 were la, 2 were II/c, 25 were III, 7 were nontypeable (NT), and 6 were nontypeable by antisera to carbohydrate antigens but did give a positive reaction with antisera to protein antigen c (NT/c). Thus most isolates were serotype Ia/c, III, or NT. This distribution reflects the predominance of these serotypes at Grady Memorial Hospital (unpublished data). Plasmid DNA analysis was done on 18 GBS isolates; none contained plasmid DNA. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction endonucleases. HhaI appeared to give the best discrimination of hybridization banding patterns or ribotypes (data not shown) and was used for all study isolates. Digestion with H hal yielded a good distribution of restriction length fragments. REAC patterns of selected GBS isolates and corresponding ribotypes are shown in figure 1. A number of isolates were studied repeatedly and always yielded the same REAC and ribotyping pat-
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not widely available, reagents are not commercially available, and when phage typing was used in one outbreak investigation, it did not substantially add to serotyping data [24]. Plasmid DNA analysis, which has been used to assist epidemiologic investigation of both gram-positive and -negative bacterial pathogens, is generally not useful in typing GBS as most of these isolates do not carry plasmid DNA [14]. In addition, plasmid DNA is extrachromosomal and therefore does not provide definitive evidence that two bacterial strains are related. Multilocus enzyme electrophoresis has been used to estimate chromosomal genetic diversity and relationships among bacterial species (including S. agalactiae) and as a molecular epidemiologic tool [25, 26]. However, this procedure was not useful in strain differentiation of serotype III isolates, as nearly all serotype III isolates collected from patients with invasive disease fell into a single electrophoretic type [26, 27]. Because of the limitations of these other typing methods, we explored the use of two DNA-based technologies to subtype molecularly GBS isolates recovered from humans with invasive disease. The two methods used were restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping). These methods use commercially available reagents, supplies, and equipment and have proved to be useful molecular epidemiologic techniques in the study of a number of other bacterial pathogens, including Salmonella typhi, Haemophilus infiuenzae, Escherichia coli, Campylobacter species, Yersinia enterocolitica, Staphylococcus aureus, and Pseudomonas cepacia [28-35].
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terns, suggesting that these methods are reproducible (data not shown). Overall, 9 ribotypes were noted among the 76 GBS isolates studied (figure IB). Four ribotypes were noted among the laic isolates (ribotypes 1-4, figure 1B); 2 of 3 serotype la isolates were ribotype 1, and 1 was ribotype 2. The 2 serotype Il/c isolates were ribotype 5. Four other distinct ribotypes were noted, among the serotype III isolates (ribotypes 6-9, figure IB). All NT or NTlc isolates studied were recovered from patients at Grady Memorial Hospital. Twelve of 13 NT or NT [c isolates were ribotype 3, a pattern also seen with serotype laic isolates. One NT isolate had ribotype pattern 6 (seen with serotype III isolates). The distribution of the ribotype patterns is shown in table 1. Table 1. Distribution ofGBS ribotypes. Ribotype
Serotype
n n
la. Ia/c, NT. NT/e
5
II/e
III. NT
25 6 16 I 2 15
7 3 I
Discussion 76
Total NOTE.
No. of isolates
NT
=
nontypeable.
Epidemiologically related isolates always had identical ribotype and REAC patterns. This was noted among GBS isolates recovered from 4 mother-infant pairs (figure 2), in which the mothers had postpartum G BS bacteremic disease and their infants had early-onset sepsis, 1 set of twins who had late onset neonatal meningitis and sepsis, and 5 sets of patient isolates from different culture sites (e.g., blood-cerebrospinal fluid, blood-urine). Epidemiologically unrelated isolates of the same ribotype usually had different REAC patterns. This is shown in figure 2 in comparing different mother-infant pairs of the same ribotype. For example, mother-infant pairs 1 and 2 were serotype laic and had ribotype pattern 1. However, on REAC, there was at least one restriction fragment difference between these 2 sets in the high-molecular-size fragments (figure 2A). Similarly, mother-infant pairs 3 and 4 were serotype III and ribotype pattern 6 (figure 2B), but there were differences between the pairs on REAC in the high-molecular-size fragments (figure 2A). Further examples of unrelated isolates of the same ribotype having different REAC patterns are shown in figure 3. These findings suggest that REAC may be a more sensitive technique for strain differentiation. Isolates listed in figures 2 and 3 did not contain plasmid DNA, so differences noted on REAC were due to differences in chromosomal DNA.
Two DNA-based technologies, REAC and ribotyping used in combination, proved to be successful molecular epi-
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Figure 1. A, Restriction endonuclease analysis of chromosomal DNA patterns of selected GBS isolates. B, Corresponding ribotypes (patterns 1-9). Serotypes are shown at bottom. Lane, ribotype, and isolate designations are as follows: 1, 1, 152; 2, 2, 201; 3, 3, GH15; 4, 4, 74; 51> 5, GH-13; 52' 5, 55; 6,6,202; 7, 7, 36; 8, 8, 175; 9, 9, GH-8; M, l-kb ladder (molecular size marker).
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demiologic methods for subtyping GBS. Results were reproducible, and these methods could differentiate among isolates of the same serotype and of different serotypes. These methods should prove useful in assisting epidemiologic investigation of GBS infections such as neonatal nursery outbreaks, nosocomial transmission on adult wards, and the source and reservoir ofGBS (e.g., in late-onset neonatal disease and in non-pregnancy-related adult disease). Previous investigation ofGBS has been limited by the lack ofa discriminatory serotyping [12] or other subtyping system. As shown by our REAC and ribotyping data, there is genetic heterogeneity within GBS serotypes. In addition, all isolates are typeable by REAC and ribotyping, and GBS isolates that are nontypeable by serotyping can be evaluated and compared with other isolates. In our study, epidemiologically related isolates always had identical REAC and ribotype patterns. This included 4 sets of mother-infant paired isolates from mothers with postpartum bacteremia and their infants with early-onset disease (sepsis). The finding of identical mother-infant paired isolates by molecular typing is consistent with the belief that transmission in early-onset disease occurs vertically from mother to child [1]. REAC and ribotyping appear to be the most useful techniques reported to date for subtyping GBS isolates. Multilocus enzyme electrophoresis has been useful in the study of population genetics [25] and has been successfully applied to
molecular epidemiology ofa number of bacterial species [41, 42]. By using electrophoretic patterns of a number of metabolic enzymes, clonal relationships among strains can be determined. However, the use of multilocus enzyme electrophoresis to subtype GBS appears to be limited because of the inability to discriminate among serotype III isolates causing invasive disease. Musser et al. [26] studied 128 GBS isolates, from 5 serotypes, collected from a number of geographic locations. These human isolates (colonizing strains and those causing invasive disease) were categorized into 15 electrophoretic types. However, nearly all (40/44) serotype III isolates causing invasive disease were ofa single electrophoretic type, leading the authors to conclude that a single clone accounts for the overwhelming majority of disease caused by serotype III strains. Our data demonstrated genetic heterogeneity among invasive serotype III isolates (and among other serotypes). Thus, invasive serotype III isolates may not belong to a single clone. As noted, 4 ribotypes were noted among serotype III isolates, and although isolates of the same ribotype often had similar REAC patterns, nearly every epidemiologically unrelated strain had a unique REAC pattern. Two previous studies [14,43] have reported results ofchromosomal digestion patterns ofGBS, and an additional recent study used REAC and ribotyping [44]. These authors also noted that epidemiologically related isolates always had identical REAC patterns [14, 43, 44]. However, they re-
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Figure 2. A, Restriction endonuclease analysis of chromosomal DNA patterns for 4 sets of mother-infant (M-I) paired GBS isolates. Each M-I pair had an identical REAC pattern. Isolates 164, 163, 75, and 69 in lanes M I, II' M2 , and 12 , respectively, were serotype laic; isolates 230, 227, 203, and 202 in lanes M 3 , 13 , M4 , and 14 , respectively, were serotype III. Arrowhead indicates I restriction fragment difference between mother-infant pairs I and 2. B, Corresponding ribotype patterns. Each M-I pair had same ribotype.
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Figure 3. Corresponding restnction endonuclease analysis of chromosomalDNA (A) and ribotype patterns (B) of epidemiologically unrelated GBS isolates. Unrelated isolates of same ribotype had different REAC patterns. Lane numbers in A and B correspond; numbers at bottom (B) are ribotypes. Lane, isolate, and serotype designations are as follows: 1, 1319-87, laic; 2, 2073-86, Ia; 3, GH-4A, laic; 4, GH-6, laic; 5, 3395-86, laic; 6, 2864-86, laic.
Acknowledgments
We thank Portia Williams, Beverly Metchock, John McGowan, and the staff of the Clinical Microbiology Laboratory, Grady Memorial Hospital, for their assistance and Chris Harvey and Monica Farley for their help and suggestions.
References
ported that a number of unrelated strains had the same REAC pattern. They evaluated a limited number of restriction enzymes (two to five) and only Bingen et al. [44] did rRNA hybridization studies. The selection of the restriction enzyme is critical in maximizing the discriminatory power of the REAC and ribotyping systems. Hhal gave the best discrimination among GBS isolates of the 24 restriction enzymes studied; it allowed detection of differences between strains that could not be seen with other endonucleases tested, including those used in other studies. H hal recognizes a 4-base seq'-;lence (5' . . . GCG.C. . . 3') and yielded relatively fewer high-molecular-size fragments than did a number of other restriction enzymes studied. This allowed discrimination of strains by REAC on the basis of differences in the high-molecular-size fragments (e.g., >5.1 kb). The 5-9 fragments detected by rRNA hybridization are simpler to interpret than the larger number of fragments produced by chromosomal endonuclease digestion. Isolates of interest easily can be compared even if run on different gels or on different days. Because rRNA genes are highly conserved across a wide range of bacterial species, E. coli 16Sand 23S-rRNA, which is commercially available, will hybridize with and can be used to probe complementary DNA in S.
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