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EPIDEMIOLOGY OF ESCHERICHIA COLI K1 IN HEALTHY AND DISEASED NEWBORNS
Saturday
I7
EPIDEMIOLOGY OF ESCHERICHIA COLI K1
g 4,5,10-12
IN HEALTHY AND DISEASED NEWBORNS
gens of E.
LARRIE D. SARFF
GEORGE H. MCCRACKEN, JR.
Department of Pediatrics, University of Texas Health Science
Center Dallas Southwestern Medical School, Dallas, Texas, U.S.A.
at
MARY P. GLODE MARK S. SCHIFFER JOHN B. ROBBINS*
Developmental Immunology Branch, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, Maryland IDA ØRSKOV
FRITS
ØRSKOV
W.H.O. Collaborative Centre for Reference and Research on Escherichia, Statens Seruminstitut, Copenhagen, Denmark at least different 100 Escherichia coli capsular antigens have been recognised, strains possessing the K1 antigen are responsible for 77% of neonatal E. coli meningitis cases. K1 strains were found in 20-40% of rectal swab cultures from healthy infants, children, and adult women. Vertical transmission from mother
Although
Sum ary
infant
the most common means of acquiring K1 organisms in term infants. Premature babies in a nursery with little maternal contact acquired K1 strains later than did term infants, and this acquisition may have been related to carriage by nursery staff. Capsular content and fermentation reactions of cerebrospinal-fluid K1 organisms were comparable to those found in rectal strains from healthy individuals. E. coli K1 with identical O and H antigens were found in maternal and infantile cultures of babies with E. coli meningitis. It seems very likely that host immune mechanisms play a significant role in the pathogenesis of neonatal E. coli K1 meningitis.
to
was
Introduction SOME Escherichia coli strains possess capsular polysaccharide K antigens that are morphologically and physiochemically similar to the capsular polysaccharides of Streptococcus pneumonice, Neisseria meningitidis, Hcemophilus influenzce, and Klebsiella pneumoniae.1-12 These capsular antigens are related to E. coli invasiveness in man and in some other ani* Present address: Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Washington, D.C.
7916
May I975
Although there
are at
least 100 K anti-
coli, the Kl capsule was detected in -approxi-
mately 80% of E. coli obtained from cerebrospinal fluid (c.s.F.)- of neonates with meningitis (table 1).11,12 This predominance of Kl capsule was not observed for E. coli strains isolated from blood cultures of septic newborn and adult patients without meningitis, from the urinary tract of pregnant females or children, or from stool cultures of healthy adults. Comparable to studies of capsular polysaccharides of pneumococci, meningococci, and H. influenza, the concentration and persistence of the Kl capsular polysaccharide in serum and C.S.F. of infants with meningitis were found to be directly correlated with the morbidity and mortality of this disease.12,17 These findings pose several questions regarding the pathogenesis of neonatal E. coli Kl meningitis. What is the prevalence of E. coli Kl strains in infants, children, and adults? What is the origin of the Kl strain in neonatal meningitis? Do other structural components, such as the somatic (0) and flagellar (H) antigens, of E. coli Kl strains from diseased neonates differ from those of Kl strains obtained from healthy newborns and adults? Are Kl strains isolated from C.S.F. and blood more encapsulated than those found in healthy individuals? Do the biochemical characteristics of Kl strains differ in health and disease? To answer these questions we have studied the prevalence, capsular content, fermentation reactions, and serotypes of E. coli strains isolated from blood, c.s.F., urine, and rectal cultures of diseased and healthy individuals of varying ages.
Materials and Methods Bacterial Strains E. coli strains were collected from the following sources: (1) urine cultures of patients at Parkland Memorial HosTABLE I-ASSOCIATION OF CAPSULAR Kl ANTIGEN WITH E. COLI STRAINS CAUSING DISEASE
1100
pregnant women at delivery (maternal culture), from nonpregnant females 16-31 years of age attending a familyplanning clinic, and from nursing personnel at Parkland Memorial Hospital. Rectal, aural, and nasopharyngeal cultures were obtained with sterile cotton swabs moistened with sterile phosphate-buffered saline. Rectal swab cultures obtained outside Dallas were mailed in Stuartt transport media.
This antiserum, containing B (strain B-ll). approximately 0-5 mg. of anti-K1 capsular polysaccharide antibody per ml., was mixed (1/10 v/v) with trypticase soy broth (T.s.B.) plus agarose at a final concentration of 1-5%. The cultures were streaked onto antiserum-agar and incubated overnight at 37°C. After initial examination for haloes of precipitation, the plates were incubated an additional 24 hours at 4°C and reinspected, using a high-intensity spotlight against a dark background. This second inspection occasionally revealed a previously undetected halo-producing colony. Analysis of 664 rectal swab cultures yielding E. coli Kl strains revealed pure or almost pure growth in 35%, mixed heavy growth (greater than 10 to a majority of E. coli Kl-colonies) in 31 %, and a mixed light growth (less than 10 halo-producing colonies) in 34% of the cultures. This is illustrated in the figure.
Antigenic Analysis
KI
E. coli were identified by routine bacteriological methods and serotyped by agglutination and immunoelectrophoresis at the W.H.O. Collaborative Centre for Reference and Research on Escherichia.4
coli strains, taken from overnight cultures on T.s.B. agar, were inoculated into 250 ml. volumes of sterile-filtered casein hydrolysate 20 g. per litre (Nutritional Biochemicals), D-glucose 2-5 g. per litre, dipotassium hydrogen phosphate 2-5 g. per litre, and sodium chloride 5 g. per litre. The flasks were incubated with agitation for 9 hours at 37 °C. The pH (range 6-5-7-5) and D-glucose were maintained by addition of 5 ml. 1’OM " tris " buffer at 4, 6, and 8 hours, and of 5 ml. 10% D-glucose at 6 hours. The optical density at 545 nm. was determined at 9 hours, and cultures with readings between 2-5 and 4-5 were then stored at -20°C for assay. K1 was measured in the whole cultures by radial immunodiffusion using equine antiserum raised against the crossreacting capsular antigen of meningococcus group B, with purified group-B polysaccharide as a reference standard.7.18 Use of meningococcal group-B antiserum facilitated Kl analysis because it avoids multiple precipitation reactions with non-capsular antigens observed with E. coli
pital
and Children’s Medical Center of Dallas; (2) rectal swabs from healthy newborns in Boston, Cincinnati, Cleveland, Dallas, Durham, Los Angeles, Madison, Pittsburgh, Salt Lake City, San Francisco, Montreal, Winni-peg, Mexico City, and Medellin, Colombia; (3) c.s.F. cultures of neonates with meningitis who were part of the Cooperative Neonatal Meningitis Study or cared for at other medical centres; and (4) rectal swabs obtained from
Antiserum-aga Technique for Detecting E. coli Kl E. coli Kl polysaccharide is immunochemically indistinguishable from meningococcal group-B polysaccharide.7 Equine meningococcal group B antiserum was prepared by intravenous injection of formaldehyde-fixed meningococcus
group
Capsular Content Single colonies of E.
antiserum.10 Cultures were also assayed for N-acetyl neuraminic acid, the monomeric unit of the Kl capsular polysaccharide.7 The casein hydrolysate medium had no detectable neuraminic acid. 100 Al. of culture fluid was incubated with 400 1. of 0-2M acetate buffer, pH 4-6, containing 0-025 units of neuraminidase (Clostridia, Worthington Biochemicals) and 10 Ag. of human serum albumin for 24 hours.19 200 jul. was assayed using purified N-acetyl neuraminic acid as a standard (Sigma, type IV).20 The results of these two assays are expressed as /g. per ml. per O.D.
unit.
Results
Prevalence
of E. coli Kl Strains in Rectal Cultures ofHealthy Infants Two methods of detecting E. coli Kl strains were studied. In the first rectal swabs were plated onto eosin/methylene-blue (E.M.B.) agar and different appearing E. coli colonies were picked and inoculated onto antiserum-agar. The second method involved direct inoculation of rectal swabs onto antiserum-agar. To date, only E. coli Kl yielded haloes. A distinctive thin precipitin band was observed surrounding an occasional Staph. aureus colony presumably due to secretion of protein A. Halo-producing organisms were confirmed as E. coli Kl by agglutination and immunoelectrophoresis.4 These two methods were compared in 22 rectal swab cultures obtained from 10 healthy neonates colonised with E. coli Kl. When plated directly on antiserum-agar, 11 swabs contained a mixed light growth, 6 a mixed heavy growth, and ’
E. coli Kl colonies identified by halo precipitation reaction in meningococcal group B antiserum-agar.
Photographs demonstrate mixed light, mixed heavy, and growth of E. coli Kl in rectal swab cultures from neonates.
pure
1101 TABLE II-PREVALENCB-RATES OF E. COLI Kl IN RECTAL SWABS OF NEWBORNS FROM VARIOUS NURSERY POPULATIONS
I
I
5 swab cultures a pure or almost pure growth of E. coli Kl. The identical swab from each infant was streaked concurrently on E.M.B. agar. When 10 E. coli colonies were randomly picked from E.M.B. and inoculated on antiserum-agar, the chance of selecting a Kl colony was 45 % for swabs containing a mixed light growth, 93% for those with a mixed heavy for swabs having pure or almost growth, and of coli Kl as determined by the antiE. growth pure concluded that the antimethod. We serum-agar is more sensitive in detecting serum-agar technique Kl strains. Table 11 shows the prevalence of E. coli Kl in rectal swabs from fourteen nursery populations. Colonisa-
100%
TABLB III-VARIATION IN PREVALENCE-RATES OF E. COLI Kl RECTAL COLONISATION OF PREMATURE INFANTS*
tion-rates varied from 7%
to 38% with a mean of The rates varied with time. For example, Kl strains were detected in only 1 of 100 E. coli strains from neonates in Mexico City in December, 1973, and May, 1974, but 7 of 20 (35%) strains obtained in October, 1974, possessed Kl antigen. Table ill shows the variation in the weekly prevalence-rates of E. coli Kl in the premature nursery at Parkland Memorial Hospital. Every week from Dec. 10, 1973, to July 15, 1974, rectal swabs were taken from all infants in the premature nursery. There were no changes in the routine of handling these babies. During the first 19 weeks, 10% to 32% (mean, 20%) of infants had detectable E. coli Kl. During the next 8 weeks, Kl prevalence rates were 0 to 11% (mean, 66 %). For the last 4 weeks of the study, Kl strains were found in 11 to 16% (mean, 13-5%) of rectal swabs. These data demonstrate the variability of E. coli Kl prevalence-rates in an individual nursery.
19 °/ .
by Neonates Cultures were performed on full-term babies admitted immediately after birth to an observation unit of Parkland Memorial Hospital where all personnel observed strict isolation techniques. Nasal, aural, and rectal swab cultures taken within the first 9 hours of life yielded E. coli Kl strains from 4 of 101 neonates. These strains were isolated from ear cultures only.
Acquisition of
E. coli KI
Babies were transferred from the observation unit to the general nursery by 12 hours, and were in contact with their mothers before 24 hours of age. 29 % of rectal swabs taken on the second day of life yielded Kl strains. 20 of 50 hospital personnel had Kl organisms on rectal culture; 13 of 20 had identical serotypes of E. coli Kl as the infants under their care.
*
Determined by antiserum-agar technique. t From Dec. 10, 1973, to July 15, 1974.
Rectal swabs were cultured from 97 mothers at delivery and from their offspring daily while in hospital. 44% of mothers and 36% of infants had E. coli Kl strains. Two-thirds of neonates born to E. coli Kl positive mothers had Kl strains while only 11 % of infants born to Kl negative mothers demonstrated Kl colonisation. Viewed differently, of 35 neonates with E. coli Kl, 83% of their mothers also had Kl organisms. Conversely, only 23% of 62 Kl negative babies had Kl positive mothers. 88 % of E. coli Kl strains cultivated from 51 mother-infant pairs had identical 0 and H serotypes. Among full-term babies who eventually acquired E. coli K1, 77% had positive rectal cultures by the second day and 91 % by the third day of life. Studies comparing the time of acquisition by infants born to mothers with or without E. coli Kl suggested that Kl organisms are acquired later in neonates born to noncolonised mothers. E.. coli Kl rectal colonisation of premature babies occurred later than observed for term infants. Of 71 premature infants who acquired Kl organisms, 45 % were colonised in the first week, 76% in the second week, and 90% by the fourth week of life. Full-term and premature babies with E. coli Kl organisms were indistinguishable clinically from Kl negative infants. The duration of hospital stay was not affected by the colonisation status.
1102 TABLE IV-E. COLI
KI IN RECTAL SWABS OF INFANTS, CHILDREN, AND ADULT WOMEN*
whose infants had E. coli K1 meningitis; 11 mothers had the same serotype of E. coli as that causing the meningitis (table vi). In an additional mother/infant pair (N72, table vi), E, coli 083: Kl : H12 was cultured from multiple sites of the infant and from the maternal urine, but a different serotype was isolated from the maternal rectal swab. In 8 of 12 infants from whom rectal swab cultures were obtained, the 0 and H antigens were identical for the rectal and c.s.F. E. coli Kl isolates.
Capsular Polysaccharide Content E. coli from c.s.F., blood, and stool
KI
of healthy and and blood and stool of adults were assayed for their Kl capsular content (table vn). N-acetyl neuraminic acid determination yielded higher values for capsular content than immunodiffusion. Non-Kl E. coli contain some thiobarbituric-acid reactive material, probably due to the 7-deoxyketose in the cell-wall lipopolysaccharide. Both methods, however, yielded the conclusion that the Kl capsular content was not different among strains isolated from the c.s.F. and blood of sick neonates nor from stools of healthy infants, children, and adults. sick
Determined by antiserum-agar technique.
Prevalence
ofE. coli Kl in Rectal Swabs off Infants, Children, and Adult Women Kl strains were frequently found in rectal. cultures of healthy infants, children, and adult women (table iv). 22-42% of infants and children were colonised with Kl organisms and the highest carriage-rates were in pregnant and non-pregnant women aged 16-31. There were no sex differences in Kl carriage-rates for infants and children.
E. coli Serotypes from Diseased and Healthy Infants Table v shows the 0 and H antigens of E. coli Kl strains isolated from infants with meningitis or septi-
csemia, from rectal cultures of healthy neonates and their mothers, and from urine of pregnant women with urinary-tract infection. 018, 07, and spontaneous agglutination due to deficient polysaccharide sidechains of the 0 antigens were observed most commonly among C.s.F. strains. 59% of all E. coli Kl isolates were non-motile. H7 and H6 antigens were found most frequently among C.S.F. isolates and in rectal swabs of prematures, respectively. Serotypes of E. coli Kl from Neonates with Meningitis and from their Mothers Rectal swab cultures
were
obtained from 17 mothers
TABLE V-0 AND H ANTIGENS FOUND IN E. COLI
*
neonates
Fermentation Reactions
Selected biochemical reactions of the E. coli strains studied. All organisms gave characteristic indol, Simmon’s citrate, urease, lysine/iron/agar, and triplesugar/iron (T.S.I.) agar reactions. Some Kl organisms were colourless or light pink on E.M.B. agar and only slowly fermented lactose in T.S.I. agar. These strains were tested for /3-galactosidase activity using the substrate o-nitrophenyl-&bgr;-D-galactopyranoside. Strains on and T.S.I. fermentation E.M.B. showing delayed agar were
and positive galactosidase activity are designated " slow " or lactose fermenters and this has been found due to deficiency of permease, a transport protein allowing lactose in low concentrations to enter the bacterial cell. Using these criteria, 52 of 207 (25 %) E. coli Kl strains from rectal cultures of
Kl STRAINS
" delayed "
CULTURED FROM DISEASED AND HEALTHY INDIVIDUALS
Number of strains studied shown in parentheses.
t Spontaneous agglutination.
1103 TABLE VI-SEROTYPES OF E. COLI Kl STRAINS FROM NEONATES WITH MENINGITIS
N.D. =No E. coli Kl detected *
TABLE
VII-Kl
Sp.
ag.
=
on antiserum-agar. Spontaneous agglutination.
positive infants. Thus, differences in the rate degree of exposure do not adequately explain the age relationship or the predominance of E. coli Kl meningitis in the neonatal period. Similar Kl prevalence rates in newborns have been reported by ?5rskov and Sørensen.21 These workers found that approximately 25 % of healthy babies had detectable faecal Kl strains and that recovery of these organisms was less common in breast-fed babies. This finding, together with the observation that colostrum frequently contains Kl antibodies, suggests a protective role for breast feeding.22 Because most infants in our study were not breast fed, we are unable to explain the variable prevalence-rates for E. coli Kl strains in
Kl and
CAPSULAR POLYSACCHARIDE CONTENT OF E. COLI
detected. mean±s.D.
N.D. = not *
individuals and 13 of 46 (28%) E. coli Kl strains from c.s.F. or blood of sick babies were classified as slow lactose fermenters. Only 7 of 70 (10%) of E. coli non-Kl strains from blood or c.s.F. of neonates fermented lactose slowly.
healthy
Discussion Our studies have revealed certain similarities and
N.A. =Not available.
a
major difference between neonatal E. coli Kl meningitis and purulent meningitis beyond the newborn period caused by meningococci, H. influenzae type b, and pneumococci. First, most of the pathogens are encapsulated with a polysaccharide. Second, a protective effect of anticapsular antibody can be demonstrated in animal models. Third, mortality and morbidity of meningitis are related to the concentration and persistence of capsular polysaccharides in c.s.F. and/or blood. However, in contrast to the low carrier-rates of encapsulated pneumococci, meningococci, and H. !M/!MCti2e type b, E. coli Kl organisms are detected in rectal swabs of 20-40% of individuals of all ages. Further, Kl strains are the predominant organism in
rectal swab cultures of neonates. Slow lactose fermentation was found more commonly among Kl strains than non-Kl E. coli. It is unknown whether this Kl-associated genetic factor is related to gastrointestinal carriage of this organism. However, slow lactose fermentation was also found in comparable strains isolated from c.s.F. Proteins on the surface outer membranes of encapsulated meningococci are related to the disease potential of these bacterial24 Only a few of at least eleven different outer-membrane proteins were found on strains isolated from c.s.F. whereas the other proteins were found on nasopharyngeal cultures of healthy individuals. The finding of outer-membrane proteins on E. coli 25 emphasises the need for further studies of invasive E. coli Kl strains. Our studies indicate that somatic antigens 018 and 07 are more frequent among c.s.F. strains than those found on rectal cultures of healthy individuals. Analyses of the Kl content of c.s.F. E. coli indicate that the degree of encapsulation cannot distinguish virulent from non-virulent organisms. Although these studies were confined to Kl determinations, similar findings for K antigen concentrations of blood and stool E. coli isolates from adults have been reported.10,26,27 ’
1104
We have shown that vertical transmission from to infant is the principal means of E. coli Kl infection of healthy neonates. There was a greater than 80% likelihood that the same serotype of E. coli Kl found in the neonate would be identified in the maternal rectal culture also. On the other hand, at least 10% of colonised full-term infants were born to Kl-negative mothers, and these babies acquired the Kl organisms later than did neonates born to Klpositive mothers. E. coli Kl strains in rectal swabs of nursery staff had identical 0 and H serotypes as Kl organisms obtained from babies under their care. Infant-to-infant cross-infection via the hands of nursery staff is likely to be common enough to maintain one or more E. coli serotypes within a nursery for 11 long time. For example, two serotypes (O1: Kl : H-and 018ac : Kl :H-) accounted for 52 % of all E. coli Kl strains obtained from healthy full-term babies for 6 months. During the same time period, two different E. coli serotypes (016 : K1: H6 and 07 : K1: H - ) colonised 57% of premature infants in a separate nursery of Parkland Memorial Hospital. The role of nursery staff in cross-infection is more convincingly demonstrated in the premature unit. Of 71 premature babies with E. coli Kl strains, 39 (55 %) acquired the organism after the first week of life. 50% of nursery staff attending to these infants had Kl strains with identical 0 and H antigens. Although maternal rectal cultures were not done routinely in this phase of the study, the mothers had virtually no contact with their babies from delivery to discharge from the nursery. Thus, acquisition of Kl strains primarily of two specific serotypes after the first week of life in infants without detectable Kl organisms in the first 7 days argues strongly for a non-maternal source of infection. Most babies who develop E. coli Kl disease during the first months of life obtain the pathogen from their mother. This route of infection was documented in approximately 70% of infants with meningitis. This percentage may actually have been greater because we did not select more than one halo-producing colony In addition, rectal on antiserum-agar for serotyping. cultures from sick neonates yielded organisms identical to those causing disease in approximately 65% of cases, suggesting that these organisms were most likely acquired by aspiration of infected material at delivery or by the faecal-oral route during the early days of life. Bloodstream invasion with subsequent involvement of the central nervous system probably occurred from the gastrointestinal tract. The high prevalence-rate of E. coli Kl of similar capsular content and fermentation reactions in diseased patients and among healthy infants, children, and adults suggests that host immune factors are critical in the pathogenesis of neonatal E. coli Kl meningitis. We thank the Cooperative Neonatal Meningitis Study Group participants 12 for providing rectal swab cultures from healthy mother
coli strains isolated from blood and c.s.F. of We also acknowledge the assistance of N. Threlkeld and N. Davis in these studies. L. D. S. and G. H. M. are supported by grants from the United Cerebral Palsy Foundation and the John A. Hartford Foundation Inc. Requests for reprints should be addressed to G. H. M., Department of Pediatrics, 5323 Harry Hines Blvd, Dallas, neonates and E. sick neonates.
Texas 75235. U.S.A.
PLASMA-LIPIDS AND GLUCOSE/INSULIN RELATIONSHIP IN NON-INSULINREQUIRING DIABETICS WITH AND WITHOUT RETINOPATHY A. H. KISSEBAH* E. M. KOHNER B. LEWIS
Y. K. SIDDIQ C. LOWY T. R. FRASER
Endocrine Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0HS
Serum-lipid concentrations and their relationship to blood-glucose and serum-insulin were examined in non-insulin-requiring diabetics, 62 with and 45 without retinopathy. The age, sex, body-weight, and duration of known diabetes was comparable in the two groups. All were treated by diet only or diet and oral hypoglycæmic agents. Patients with retinopathy had higher fasting serumtriglyceride and serum-cholesterol levels than those without. Compared with a non-diabetic population, significantly more diabetics with retinopathy had raised serum-lipids. The lipid concentrations did not correlate with body-weight, serum-thyroid-stimulating-hormone levels, renal involvement, or fasting blood-sugar. While the blood-sugar concentrations Summary
similar in the two groups the absolute insulin increment and the relative insulin response to a 50 g
were
*
Present address: London W2.
St.
Mary’s Hospital Medical School;
DR SARFF AND OTHERS: REFERENCES 1. Smith, T., Bryant, G. J. exp. Med. 1927, 46, 133. 2. Kauffman, F. The Bacteriology of Enterobacteriaceæ. Copenhagen 1966. 3. Ørskov, I., Ørskov, F., Jann, B., Jann, K. Nature, 1963, 200, 144 4. Ørskov, F., Ørskov, I., Jann, B., Jann, K. Acta path. microbiol scand. B, 1971, 79, 142. 5. Ørskov, F., Ørskov, I., Jann, B., Jann, K. ibid. 1971, 80, 905. 6. Heidelberger, M., Jann, B., Jann, K., Ørskov, F., Ørskov, I., Westphal, O. J. Bact. 1968, 95, 2415. 7. Kasper, D. L., Winkelhake, J. L., Zollinger, W. D., Brandt, B., Artenstein, M. S. J. Immun. 1973, 110, 262. 8. Schneerson, R., Bradshaw, M., Whisnant, J. K., Parke, J. C., Robbins, J. B. ibid. 1972, 108, 1551. 9. Robbins, J. B., Myerowitz, R. L., Whisnant, J. K., Argaman, M., Schneerson, R., Handzel, Z. T., Gotschlich, E. C. Infect. Immun. 1973, 6, 651. 10. Kaijser, B. J. infect. Dis. 1973, 121, 670. 11. Robbins, J. B., McCracken, G. H., Jr., Gotschlich, E. C., Ørskov, F., Ørskov, I., Hanson, L. New Engl. J. Med. 1974, 290, 1216. 12. McCracken, G. H., Sarff, L. D., Glode, M. P., Mize, S. G., Schiffer, M. S., Robbins, J. B., Gotschlich, E. C., Ørskov, F., Ørskov, I. and the Cooperative Neonatal Meningitis Study Group. Lancet, 1974, ii, 246. 13. Bukantz, S. E., de Gara, P. F., Bullowa, J. G. M. Archs intern. Med. 1942, 69, 191. 14. Alexander, H. E. J. Pediat. 1944, 25, 517. 15. Hoffman, T. A., Edwards, E. A. J. infect. Dis. 1972, 126, 636. 16. Dorff, G. J., Coonrod, J. D., Ryfel, M. W. Lancet, 1971, i, 578. 17. Ingram, D. L., Anderson, P., Smith, D. H.J. Pediat. 1972, 81, 1156. 18. Mancini, G., Carbonara, A. O., Heremans, J. F. Immunochemistry, 1965, 2, 235. 19. Liu, T. Y., Gotschlich, E. C., Dunne, F. T., Jonsenn, E. K. J. biol. Chem. 1971, 246, 4703. 20. Warren, L. ibid. 1959, 234, 1971. 21. Ørskov, F., Sørensen, K. B. Br. med. J. (in the press). 22. Hanson, L., Kaijser, B. Unpublished. 23. Frasch, C. E., Chapman, S. S. J. infect. Dis. 1973, 127, 149. 24. Gold, R., Winklehake, J. L., Mars, R. S., Artenstein, M. S. ibid 1971, 124, 593. 25. Schnitman, C. A. J. Bact. 1970, 104, 882. 26. Glynn, A. A., Brumfitt, W., Howard, C. J. Lancet, 1971, i, 514. 27. McCabe, W. R., Carling, P. C., Bruins, S., Greely, A. J. infect. Dis
1975, 131, 6.
I7
EPIDEMIOLOGY OF ESCHERICHIA COLI K1
g 4,5,10-12
IN HEALTHY AND DISEASED NEWBORNS
gens of E.
LARRIE D. SARFF
GEORGE H. MCCRACKEN, JR.
Department of Pediatrics, University of Texas Health Science
Center Dallas Southwestern Medical School, Dallas, Texas, U.S.A.
at
MARY P. GLODE MARK S. SCHIFFER JOHN B. ROBBINS*
Developmental Immunology Branch, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, Maryland IDA ØRSKOV
FRITS
ØRSKOV
W.H.O. Collaborative Centre for Reference and Research on Escherichia, Statens Seruminstitut, Copenhagen, Denmark at least different 100 Escherichia coli capsular antigens have been recognised, strains possessing the K1 antigen are responsible for 77% of neonatal E. coli meningitis cases. K1 strains were found in 20-40% of rectal swab cultures from healthy infants, children, and adult women. Vertical transmission from mother
Although
Sum ary
infant
the most common means of acquiring K1 organisms in term infants. Premature babies in a nursery with little maternal contact acquired K1 strains later than did term infants, and this acquisition may have been related to carriage by nursery staff. Capsular content and fermentation reactions of cerebrospinal-fluid K1 organisms were comparable to those found in rectal strains from healthy individuals. E. coli K1 with identical O and H antigens were found in maternal and infantile cultures of babies with E. coli meningitis. It seems very likely that host immune mechanisms play a significant role in the pathogenesis of neonatal E. coli K1 meningitis.
to
was
Introduction SOME Escherichia coli strains possess capsular polysaccharide K antigens that are morphologically and physiochemically similar to the capsular polysaccharides of Streptococcus pneumonice, Neisseria meningitidis, Hcemophilus influenzce, and Klebsiella pneumoniae.1-12 These capsular antigens are related to E. coli invasiveness in man and in some other ani* Present address: Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Washington, D.C.
7916
May I975
Although there
are at
least 100 K anti-
coli, the Kl capsule was detected in -approxi-
mately 80% of E. coli obtained from cerebrospinal fluid (c.s.F.)- of neonates with meningitis (table 1).11,12 This predominance of Kl capsule was not observed for E. coli strains isolated from blood cultures of septic newborn and adult patients without meningitis, from the urinary tract of pregnant females or children, or from stool cultures of healthy adults. Comparable to studies of capsular polysaccharides of pneumococci, meningococci, and H. influenza, the concentration and persistence of the Kl capsular polysaccharide in serum and C.S.F. of infants with meningitis were found to be directly correlated with the morbidity and mortality of this disease.12,17 These findings pose several questions regarding the pathogenesis of neonatal E. coli Kl meningitis. What is the prevalence of E. coli Kl strains in infants, children, and adults? What is the origin of the Kl strain in neonatal meningitis? Do other structural components, such as the somatic (0) and flagellar (H) antigens, of E. coli Kl strains from diseased neonates differ from those of Kl strains obtained from healthy newborns and adults? Are Kl strains isolated from C.S.F. and blood more encapsulated than those found in healthy individuals? Do the biochemical characteristics of Kl strains differ in health and disease? To answer these questions we have studied the prevalence, capsular content, fermentation reactions, and serotypes of E. coli strains isolated from blood, c.s.F., urine, and rectal cultures of diseased and healthy individuals of varying ages.
Materials and Methods Bacterial Strains E. coli strains were collected from the following sources: (1) urine cultures of patients at Parkland Memorial HosTABLE I-ASSOCIATION OF CAPSULAR Kl ANTIGEN WITH E. COLI STRAINS CAUSING DISEASE
1100
pregnant women at delivery (maternal culture), from nonpregnant females 16-31 years of age attending a familyplanning clinic, and from nursing personnel at Parkland Memorial Hospital. Rectal, aural, and nasopharyngeal cultures were obtained with sterile cotton swabs moistened with sterile phosphate-buffered saline. Rectal swab cultures obtained outside Dallas were mailed in Stuartt transport media.
This antiserum, containing B (strain B-ll). approximately 0-5 mg. of anti-K1 capsular polysaccharide antibody per ml., was mixed (1/10 v/v) with trypticase soy broth (T.s.B.) plus agarose at a final concentration of 1-5%. The cultures were streaked onto antiserum-agar and incubated overnight at 37°C. After initial examination for haloes of precipitation, the plates were incubated an additional 24 hours at 4°C and reinspected, using a high-intensity spotlight against a dark background. This second inspection occasionally revealed a previously undetected halo-producing colony. Analysis of 664 rectal swab cultures yielding E. coli Kl strains revealed pure or almost pure growth in 35%, mixed heavy growth (greater than 10 to a majority of E. coli Kl-colonies) in 31 %, and a mixed light growth (less than 10 halo-producing colonies) in 34% of the cultures. This is illustrated in the figure.
Antigenic Analysis
KI
E. coli were identified by routine bacteriological methods and serotyped by agglutination and immunoelectrophoresis at the W.H.O. Collaborative Centre for Reference and Research on Escherichia.4
coli strains, taken from overnight cultures on T.s.B. agar, were inoculated into 250 ml. volumes of sterile-filtered casein hydrolysate 20 g. per litre (Nutritional Biochemicals), D-glucose 2-5 g. per litre, dipotassium hydrogen phosphate 2-5 g. per litre, and sodium chloride 5 g. per litre. The flasks were incubated with agitation for 9 hours at 37 °C. The pH (range 6-5-7-5) and D-glucose were maintained by addition of 5 ml. 1’OM " tris " buffer at 4, 6, and 8 hours, and of 5 ml. 10% D-glucose at 6 hours. The optical density at 545 nm. was determined at 9 hours, and cultures with readings between 2-5 and 4-5 were then stored at -20°C for assay. K1 was measured in the whole cultures by radial immunodiffusion using equine antiserum raised against the crossreacting capsular antigen of meningococcus group B, with purified group-B polysaccharide as a reference standard.7.18 Use of meningococcal group-B antiserum facilitated Kl analysis because it avoids multiple precipitation reactions with non-capsular antigens observed with E. coli
pital
and Children’s Medical Center of Dallas; (2) rectal swabs from healthy newborns in Boston, Cincinnati, Cleveland, Dallas, Durham, Los Angeles, Madison, Pittsburgh, Salt Lake City, San Francisco, Montreal, Winni-peg, Mexico City, and Medellin, Colombia; (3) c.s.F. cultures of neonates with meningitis who were part of the Cooperative Neonatal Meningitis Study or cared for at other medical centres; and (4) rectal swabs obtained from
Antiserum-aga Technique for Detecting E. coli Kl E. coli Kl polysaccharide is immunochemically indistinguishable from meningococcal group-B polysaccharide.7 Equine meningococcal group B antiserum was prepared by intravenous injection of formaldehyde-fixed meningococcus
group
Capsular Content Single colonies of E.
antiserum.10 Cultures were also assayed for N-acetyl neuraminic acid, the monomeric unit of the Kl capsular polysaccharide.7 The casein hydrolysate medium had no detectable neuraminic acid. 100 Al. of culture fluid was incubated with 400 1. of 0-2M acetate buffer, pH 4-6, containing 0-025 units of neuraminidase (Clostridia, Worthington Biochemicals) and 10 Ag. of human serum albumin for 24 hours.19 200 jul. was assayed using purified N-acetyl neuraminic acid as a standard (Sigma, type IV).20 The results of these two assays are expressed as /g. per ml. per O.D.
unit.
Results
Prevalence
of E. coli Kl Strains in Rectal Cultures ofHealthy Infants Two methods of detecting E. coli Kl strains were studied. In the first rectal swabs were plated onto eosin/methylene-blue (E.M.B.) agar and different appearing E. coli colonies were picked and inoculated onto antiserum-agar. The second method involved direct inoculation of rectal swabs onto antiserum-agar. To date, only E. coli Kl yielded haloes. A distinctive thin precipitin band was observed surrounding an occasional Staph. aureus colony presumably due to secretion of protein A. Halo-producing organisms were confirmed as E. coli Kl by agglutination and immunoelectrophoresis.4 These two methods were compared in 22 rectal swab cultures obtained from 10 healthy neonates colonised with E. coli Kl. When plated directly on antiserum-agar, 11 swabs contained a mixed light growth, 6 a mixed heavy growth, and ’
E. coli Kl colonies identified by halo precipitation reaction in meningococcal group B antiserum-agar.
Photographs demonstrate mixed light, mixed heavy, and growth of E. coli Kl in rectal swab cultures from neonates.
pure
1101 TABLE II-PREVALENCB-RATES OF E. COLI Kl IN RECTAL SWABS OF NEWBORNS FROM VARIOUS NURSERY POPULATIONS
I
I
5 swab cultures a pure or almost pure growth of E. coli Kl. The identical swab from each infant was streaked concurrently on E.M.B. agar. When 10 E. coli colonies were randomly picked from E.M.B. and inoculated on antiserum-agar, the chance of selecting a Kl colony was 45 % for swabs containing a mixed light growth, 93% for those with a mixed heavy for swabs having pure or almost growth, and of coli Kl as determined by the antiE. growth pure concluded that the antimethod. We serum-agar is more sensitive in detecting serum-agar technique Kl strains. Table 11 shows the prevalence of E. coli Kl in rectal swabs from fourteen nursery populations. Colonisa-
100%
TABLB III-VARIATION IN PREVALENCE-RATES OF E. COLI Kl RECTAL COLONISATION OF PREMATURE INFANTS*
tion-rates varied from 7%
to 38% with a mean of The rates varied with time. For example, Kl strains were detected in only 1 of 100 E. coli strains from neonates in Mexico City in December, 1973, and May, 1974, but 7 of 20 (35%) strains obtained in October, 1974, possessed Kl antigen. Table ill shows the variation in the weekly prevalence-rates of E. coli Kl in the premature nursery at Parkland Memorial Hospital. Every week from Dec. 10, 1973, to July 15, 1974, rectal swabs were taken from all infants in the premature nursery. There were no changes in the routine of handling these babies. During the first 19 weeks, 10% to 32% (mean, 20%) of infants had detectable E. coli Kl. During the next 8 weeks, Kl prevalence rates were 0 to 11% (mean, 66 %). For the last 4 weeks of the study, Kl strains were found in 11 to 16% (mean, 13-5%) of rectal swabs. These data demonstrate the variability of E. coli Kl prevalence-rates in an individual nursery.
19 °/ .
by Neonates Cultures were performed on full-term babies admitted immediately after birth to an observation unit of Parkland Memorial Hospital where all personnel observed strict isolation techniques. Nasal, aural, and rectal swab cultures taken within the first 9 hours of life yielded E. coli Kl strains from 4 of 101 neonates. These strains were isolated from ear cultures only.
Acquisition of
E. coli KI
Babies were transferred from the observation unit to the general nursery by 12 hours, and were in contact with their mothers before 24 hours of age. 29 % of rectal swabs taken on the second day of life yielded Kl strains. 20 of 50 hospital personnel had Kl organisms on rectal culture; 13 of 20 had identical serotypes of E. coli Kl as the infants under their care.
*
Determined by antiserum-agar technique. t From Dec. 10, 1973, to July 15, 1974.
Rectal swabs were cultured from 97 mothers at delivery and from their offspring daily while in hospital. 44% of mothers and 36% of infants had E. coli Kl strains. Two-thirds of neonates born to E. coli Kl positive mothers had Kl strains while only 11 % of infants born to Kl negative mothers demonstrated Kl colonisation. Viewed differently, of 35 neonates with E. coli Kl, 83% of their mothers also had Kl organisms. Conversely, only 23% of 62 Kl negative babies had Kl positive mothers. 88 % of E. coli Kl strains cultivated from 51 mother-infant pairs had identical 0 and H serotypes. Among full-term babies who eventually acquired E. coli K1, 77% had positive rectal cultures by the second day and 91 % by the third day of life. Studies comparing the time of acquisition by infants born to mothers with or without E. coli Kl suggested that Kl organisms are acquired later in neonates born to noncolonised mothers. E.. coli Kl rectal colonisation of premature babies occurred later than observed for term infants. Of 71 premature infants who acquired Kl organisms, 45 % were colonised in the first week, 76% in the second week, and 90% by the fourth week of life. Full-term and premature babies with E. coli Kl organisms were indistinguishable clinically from Kl negative infants. The duration of hospital stay was not affected by the colonisation status.
1102 TABLE IV-E. COLI
KI IN RECTAL SWABS OF INFANTS, CHILDREN, AND ADULT WOMEN*
whose infants had E. coli K1 meningitis; 11 mothers had the same serotype of E. coli as that causing the meningitis (table vi). In an additional mother/infant pair (N72, table vi), E, coli 083: Kl : H12 was cultured from multiple sites of the infant and from the maternal urine, but a different serotype was isolated from the maternal rectal swab. In 8 of 12 infants from whom rectal swab cultures were obtained, the 0 and H antigens were identical for the rectal and c.s.F. E. coli Kl isolates.
Capsular Polysaccharide Content E. coli from c.s.F., blood, and stool
KI
of healthy and and blood and stool of adults were assayed for their Kl capsular content (table vn). N-acetyl neuraminic acid determination yielded higher values for capsular content than immunodiffusion. Non-Kl E. coli contain some thiobarbituric-acid reactive material, probably due to the 7-deoxyketose in the cell-wall lipopolysaccharide. Both methods, however, yielded the conclusion that the Kl capsular content was not different among strains isolated from the c.s.F. and blood of sick neonates nor from stools of healthy infants, children, and adults. sick
Determined by antiserum-agar technique.
Prevalence
ofE. coli Kl in Rectal Swabs off Infants, Children, and Adult Women Kl strains were frequently found in rectal. cultures of healthy infants, children, and adult women (table iv). 22-42% of infants and children were colonised with Kl organisms and the highest carriage-rates were in pregnant and non-pregnant women aged 16-31. There were no sex differences in Kl carriage-rates for infants and children.
E. coli Serotypes from Diseased and Healthy Infants Table v shows the 0 and H antigens of E. coli Kl strains isolated from infants with meningitis or septi-
csemia, from rectal cultures of healthy neonates and their mothers, and from urine of pregnant women with urinary-tract infection. 018, 07, and spontaneous agglutination due to deficient polysaccharide sidechains of the 0 antigens were observed most commonly among C.s.F. strains. 59% of all E. coli Kl isolates were non-motile. H7 and H6 antigens were found most frequently among C.S.F. isolates and in rectal swabs of prematures, respectively. Serotypes of E. coli Kl from Neonates with Meningitis and from their Mothers Rectal swab cultures
were
obtained from 17 mothers
TABLE V-0 AND H ANTIGENS FOUND IN E. COLI
*
neonates
Fermentation Reactions
Selected biochemical reactions of the E. coli strains studied. All organisms gave characteristic indol, Simmon’s citrate, urease, lysine/iron/agar, and triplesugar/iron (T.S.I.) agar reactions. Some Kl organisms were colourless or light pink on E.M.B. agar and only slowly fermented lactose in T.S.I. agar. These strains were tested for /3-galactosidase activity using the substrate o-nitrophenyl-&bgr;-D-galactopyranoside. Strains on and T.S.I. fermentation E.M.B. showing delayed agar were
and positive galactosidase activity are designated " slow " or lactose fermenters and this has been found due to deficiency of permease, a transport protein allowing lactose in low concentrations to enter the bacterial cell. Using these criteria, 52 of 207 (25 %) E. coli Kl strains from rectal cultures of
Kl STRAINS
" delayed "
CULTURED FROM DISEASED AND HEALTHY INDIVIDUALS
Number of strains studied shown in parentheses.
t Spontaneous agglutination.
1103 TABLE VI-SEROTYPES OF E. COLI Kl STRAINS FROM NEONATES WITH MENINGITIS
N.D. =No E. coli Kl detected *
TABLE
VII-Kl
Sp.
ag.
=
on antiserum-agar. Spontaneous agglutination.
positive infants. Thus, differences in the rate degree of exposure do not adequately explain the age relationship or the predominance of E. coli Kl meningitis in the neonatal period. Similar Kl prevalence rates in newborns have been reported by ?5rskov and Sørensen.21 These workers found that approximately 25 % of healthy babies had detectable faecal Kl strains and that recovery of these organisms was less common in breast-fed babies. This finding, together with the observation that colostrum frequently contains Kl antibodies, suggests a protective role for breast feeding.22 Because most infants in our study were not breast fed, we are unable to explain the variable prevalence-rates for E. coli Kl strains in
Kl and
CAPSULAR POLYSACCHARIDE CONTENT OF E. COLI
detected. mean±s.D.
N.D. = not *
individuals and 13 of 46 (28%) E. coli Kl strains from c.s.F. or blood of sick babies were classified as slow lactose fermenters. Only 7 of 70 (10%) of E. coli non-Kl strains from blood or c.s.F. of neonates fermented lactose slowly.
healthy
Discussion Our studies have revealed certain similarities and
N.A. =Not available.
a
major difference between neonatal E. coli Kl meningitis and purulent meningitis beyond the newborn period caused by meningococci, H. influenzae type b, and pneumococci. First, most of the pathogens are encapsulated with a polysaccharide. Second, a protective effect of anticapsular antibody can be demonstrated in animal models. Third, mortality and morbidity of meningitis are related to the concentration and persistence of capsular polysaccharides in c.s.F. and/or blood. However, in contrast to the low carrier-rates of encapsulated pneumococci, meningococci, and H. !M/!MCti2e type b, E. coli Kl organisms are detected in rectal swabs of 20-40% of individuals of all ages. Further, Kl strains are the predominant organism in
rectal swab cultures of neonates. Slow lactose fermentation was found more commonly among Kl strains than non-Kl E. coli. It is unknown whether this Kl-associated genetic factor is related to gastrointestinal carriage of this organism. However, slow lactose fermentation was also found in comparable strains isolated from c.s.F. Proteins on the surface outer membranes of encapsulated meningococci are related to the disease potential of these bacterial24 Only a few of at least eleven different outer-membrane proteins were found on strains isolated from c.s.F. whereas the other proteins were found on nasopharyngeal cultures of healthy individuals. The finding of outer-membrane proteins on E. coli 25 emphasises the need for further studies of invasive E. coli Kl strains. Our studies indicate that somatic antigens 018 and 07 are more frequent among c.s.F. strains than those found on rectal cultures of healthy individuals. Analyses of the Kl content of c.s.F. E. coli indicate that the degree of encapsulation cannot distinguish virulent from non-virulent organisms. Although these studies were confined to Kl determinations, similar findings for K antigen concentrations of blood and stool E. coli isolates from adults have been reported.10,26,27 ’
1104
We have shown that vertical transmission from to infant is the principal means of E. coli Kl infection of healthy neonates. There was a greater than 80% likelihood that the same serotype of E. coli Kl found in the neonate would be identified in the maternal rectal culture also. On the other hand, at least 10% of colonised full-term infants were born to Kl-negative mothers, and these babies acquired the Kl organisms later than did neonates born to Klpositive mothers. E. coli Kl strains in rectal swabs of nursery staff had identical 0 and H serotypes as Kl organisms obtained from babies under their care. Infant-to-infant cross-infection via the hands of nursery staff is likely to be common enough to maintain one or more E. coli serotypes within a nursery for 11 long time. For example, two serotypes (O1: Kl : H-and 018ac : Kl :H-) accounted for 52 % of all E. coli Kl strains obtained from healthy full-term babies for 6 months. During the same time period, two different E. coli serotypes (016 : K1: H6 and 07 : K1: H - ) colonised 57% of premature infants in a separate nursery of Parkland Memorial Hospital. The role of nursery staff in cross-infection is more convincingly demonstrated in the premature unit. Of 71 premature babies with E. coli Kl strains, 39 (55 %) acquired the organism after the first week of life. 50% of nursery staff attending to these infants had Kl strains with identical 0 and H antigens. Although maternal rectal cultures were not done routinely in this phase of the study, the mothers had virtually no contact with their babies from delivery to discharge from the nursery. Thus, acquisition of Kl strains primarily of two specific serotypes after the first week of life in infants without detectable Kl organisms in the first 7 days argues strongly for a non-maternal source of infection. Most babies who develop E. coli Kl disease during the first months of life obtain the pathogen from their mother. This route of infection was documented in approximately 70% of infants with meningitis. This percentage may actually have been greater because we did not select more than one halo-producing colony In addition, rectal on antiserum-agar for serotyping. cultures from sick neonates yielded organisms identical to those causing disease in approximately 65% of cases, suggesting that these organisms were most likely acquired by aspiration of infected material at delivery or by the faecal-oral route during the early days of life. Bloodstream invasion with subsequent involvement of the central nervous system probably occurred from the gastrointestinal tract. The high prevalence-rate of E. coli Kl of similar capsular content and fermentation reactions in diseased patients and among healthy infants, children, and adults suggests that host immune factors are critical in the pathogenesis of neonatal E. coli Kl meningitis. We thank the Cooperative Neonatal Meningitis Study Group participants 12 for providing rectal swab cultures from healthy mother
coli strains isolated from blood and c.s.F. of We also acknowledge the assistance of N. Threlkeld and N. Davis in these studies. L. D. S. and G. H. M. are supported by grants from the United Cerebral Palsy Foundation and the John A. Hartford Foundation Inc. Requests for reprints should be addressed to G. H. M., Department of Pediatrics, 5323 Harry Hines Blvd, Dallas, neonates and E. sick neonates.
Texas 75235. U.S.A.
PLASMA-LIPIDS AND GLUCOSE/INSULIN RELATIONSHIP IN NON-INSULINREQUIRING DIABETICS WITH AND WITHOUT RETINOPATHY A. H. KISSEBAH* E. M. KOHNER B. LEWIS
Y. K. SIDDIQ C. LOWY T. R. FRASER
Endocrine Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0HS
Serum-lipid concentrations and their relationship to blood-glucose and serum-insulin were examined in non-insulin-requiring diabetics, 62 with and 45 without retinopathy. The age, sex, body-weight, and duration of known diabetes was comparable in the two groups. All were treated by diet only or diet and oral hypoglycæmic agents. Patients with retinopathy had higher fasting serumtriglyceride and serum-cholesterol levels than those without. Compared with a non-diabetic population, significantly more diabetics with retinopathy had raised serum-lipids. The lipid concentrations did not correlate with body-weight, serum-thyroid-stimulating-hormone levels, renal involvement, or fasting blood-sugar. While the blood-sugar concentrations Summary
similar in the two groups the absolute insulin increment and the relative insulin response to a 50 g
were
*
Present address: London W2.
St.
Mary’s Hospital Medical School;
DR SARFF AND OTHERS: REFERENCES 1. Smith, T., Bryant, G. J. exp. Med. 1927, 46, 133. 2. Kauffman, F. The Bacteriology of Enterobacteriaceæ. Copenhagen 1966. 3. Ørskov, I., Ørskov, F., Jann, B., Jann, K. Nature, 1963, 200, 144 4. Ørskov, F., Ørskov, I., Jann, B., Jann, K. Acta path. microbiol scand. B, 1971, 79, 142. 5. Ørskov, F., Ørskov, I., Jann, B., Jann, K. ibid. 1971, 80, 905. 6. Heidelberger, M., Jann, B., Jann, K., Ørskov, F., Ørskov, I., Westphal, O. J. Bact. 1968, 95, 2415. 7. Kasper, D. L., Winkelhake, J. L., Zollinger, W. D., Brandt, B., Artenstein, M. S. J. Immun. 1973, 110, 262. 8. Schneerson, R., Bradshaw, M., Whisnant, J. K., Parke, J. C., Robbins, J. B. ibid. 1972, 108, 1551. 9. Robbins, J. B., Myerowitz, R. L., Whisnant, J. K., Argaman, M., Schneerson, R., Handzel, Z. T., Gotschlich, E. C. Infect. Immun. 1973, 6, 651. 10. Kaijser, B. J. infect. Dis. 1973, 121, 670. 11. Robbins, J. B., McCracken, G. H., Jr., Gotschlich, E. C., Ørskov, F., Ørskov, I., Hanson, L. New Engl. J. Med. 1974, 290, 1216. 12. McCracken, G. H., Sarff, L. D., Glode, M. P., Mize, S. G., Schiffer, M. S., Robbins, J. B., Gotschlich, E. C., Ørskov, F., Ørskov, I. and the Cooperative Neonatal Meningitis Study Group. Lancet, 1974, ii, 246. 13. Bukantz, S. E., de Gara, P. F., Bullowa, J. G. M. Archs intern. Med. 1942, 69, 191. 14. Alexander, H. E. J. Pediat. 1944, 25, 517. 15. Hoffman, T. A., Edwards, E. A. J. infect. Dis. 1972, 126, 636. 16. Dorff, G. J., Coonrod, J. D., Ryfel, M. W. Lancet, 1971, i, 578. 17. Ingram, D. L., Anderson, P., Smith, D. H.J. Pediat. 1972, 81, 1156. 18. Mancini, G., Carbonara, A. O., Heremans, J. F. Immunochemistry, 1965, 2, 235. 19. Liu, T. Y., Gotschlich, E. C., Dunne, F. T., Jonsenn, E. K. J. biol. Chem. 1971, 246, 4703. 20. Warren, L. ibid. 1959, 234, 1971. 21. Ørskov, F., Sørensen, K. B. Br. med. J. (in the press). 22. Hanson, L., Kaijser, B. Unpublished. 23. Frasch, C. E., Chapman, S. S. J. infect. Dis. 1973, 127, 149. 24. Gold, R., Winklehake, J. L., Mars, R. S., Artenstein, M. S. ibid 1971, 124, 593. 25. Schnitman, C. A. J. Bact. 1970, 104, 882. 26. Glynn, A. A., Brumfitt, W., Howard, C. J. Lancet, 1971, i, 514. 27. McCabe, W. R., Carling, P. C., Bruins, S., Greely, A. J. infect. Dis
1975, 131, 6.