Toxigenic bacterial diarrhea: Nursery outbreak involving multiple bacterial strains

December 1976 The Journal o f P E D I A T R I C S

885

Toxigenic bacterial diarrhea: Nursery outbreak involving multiple bacterial strains An outbreak of watery diarrhea occurred in 14 of 15 infants in a special care unit over a four-day period Using the CHO cell assay for enterotoxin, we found that 11 of these patients had toxigenic bacteria in their stools. These bacteria comprised nine different serotypes of three species of organisms: Escherichia coli, Klebsiella, and Citrobacter. None of the three serotypes of E. coli were classic enteropathogenic serotypes. Rectal swab specimens from all 15 infants were examined for the presence of viruses by electron microscopic and cell culture techniques as well as by studies in suckling mice. None had parvovirus- or reovirus-like agents and two had adenoviruses. No other viral agents were detected. Of 38 bacterial strains isolated from ten control infants without diarrhea, three Klebsiella strains from two individuals were found to be toxigenic. Analysis of a total of 136 enteric isolates showed that toxigenicity as measured by the CHO assay was strongly associated with strains isolated during acute diarrheal illness when compared with strains isolated in convalescence (p < 10 -~) or with strains from control infants without diarrhea (p < 10-5). This study raises the possibility of an outbreak of disease caused by a transmissible plasmid responsible for a cholera-like enterotoxin production in several enteric bacterial strains.

Richard L. Guerrant, M.D.,* Michael D. Dickens, M.D., Richard P. Wenzel, M.D., and Albert Z. Kapikian, M.D., Charlottesville, Va., a n d B e t h e s d a , M d .

ACUTE INFECTIOUS GASTROENTERITIS, particularly in its epidemic form, has long been a major problem among newborn infants throughout the world. 1-:~ In the past these outbreaks have occasionally been associated with certain serotypes of Escherichia coli, the "classical enteropathogenic serotypes. ''4"~ More recently, however, infantile diarrhea has been associated in some instances with E. coli strains of various types, including many nontypable strains, that are capable of producing an From the Departments of Medicine and Pediatrics; University of Virginia School of Medicine, and National Institute of Allergy and Infectious Diseases, National Institutes of Health. Supported by a contract (NO1-A 1-42548) with the National Institutes of Health. Dr. Guerrant is a George Morris Piersol Teaching and Research Scholar of the American College of Physicians. *Reprint address: Box 251, Division of Infectious Diseases, University of Virginia School of Medicine, Charlottesville, VA 22901.

enterotoxin in vitro. 7, 8 The capability of some E. coli to produce a heat-labile, cholera-like enterotoxin has recently been shown to be transmissible between different strains of E. coli by a plasmid?' The importance of this transmissibility in patients, however, remains to be demonstrated. Our understanding of the importance of enterotoxi-

See related article, p. 892.

Abbreviations used Tox+: plasmid coding for heat-labile enterotoxin production CHO: Chinese hamster ovary LF: lactose-fermenting cyclic AMP: cyclic 3',5'-adenosine monophosphate SD: standard deviation FCS: fetal calf serum CPE: cytopathic effect

VoL 89, No. 6, pp. 885-891

886

Guerrant et al.

The Journal of Pediatrics December 1976

Table I. Underlying illnesses among patient and control groups Controls Patients with diarrhea Jan. 1974

Underlying illnesses

Prematurity _+ respiratory distress Congenital anomalies (CNS, cardiac, GU) Gastrointestinal anomalies Fever, suspected sepsis (meningitis, pneumonia, amnionitis) ABO incompatability Endocrine (secondary hypoparathyroidism) None (normal term infant) Totals

5

Nursery Jan. 1974 i

NBSCU March 1974

NBSCU Dec.Jan. 1975

4

1

20 12 4 5

5

41

4

2 1 l l

I

I

5 14

5

NBSCU = Newborn Special Care Unit; CNS = central nervous system; GU = genital-urinary.

genic bacteria in infants and in children has been limited by the need for a convenient assay for detecting enterotoxin. Based on the ability of cholera and E. coli enterotoxins to activate adenylate cyclase, the cyclic A M P associated elongation of Chinese hamster ovary cells to a "stretched," bipolar form has been employed as a simple morphologic tissue culture assay for these heat-labile toxins. 1~ Using this assay we have studied all lactosefermenting enteric isolates from the stools of all 15 patients in a newborn intensive care unit during an outbreak of watery diarrhea involving 14 of these patients. Identical studies were made of the stools of ten of these patients during convalescence and from ten control patients. In addition to bacteria, certain viruses have been associated with infantile diarrhea as well. 12 Although searches for entero- and adenoviruses have often revealed as many isolates from control subjects as from patients,':' recent immune or conventional electron microscopic studies have revealed parvovirus- and reovirus-like particles in association with diarrheal illnesses. 1'-1~ Rectal swab specimens from 15 infants in this study were examined for the presence of viruses by electron microscopic and cell culture techniques as well as by studies in suckling mice. T M

METHODS Clinical studies. All 15 patients in three rooms in a newborn special care unit were examined by one of the authors (M.D.D.), and a careful record was kept of the frequency and nature of each patient's stools, severity of dehydration, dietary and intravenous intake, antibiotics, and clinical course in the hospital. Stool specimens were obtained from all 15 patients at the peak of the outbreak on January 7, 1974. These specimens were examined promptly for fecal leukoeytes with methylene blue stain;

they were also cultured by standard techniques for Salmonella, Shigella, staphylococci, and all L F organisms, 1'~ and portions were saved for electron microscopic and other studies for viruses. Ten of the patients were re-examined during convalescence at one to ten weeks after recovery from the diarrheal illness. At that time, fecal specimens were obtained and cultured as above. The stools of ten age-matched control patients were also cultured for enterotoxin-producing LF isolates. Five of the control patients were studied at the same time as this outbreak from the adjacent newborn nursery where there was no diarrhea; five additional control patients were in the special care nursery at a subsequent date when diarrhea was no longer a problem. Only one of these patients had received antibiotics (methicillin and kanamycin) for suspected sepsis two weeks earlier. In order to establish a baseline for interepidemic infant bowel habits, continuous surveillance was m a d e for eight weeks in this intensive care nursery at the same season one year later (December, 1974, to January, 1975), when diarrhea was not a problem. Weekly visits were m a d e by nurse-epidemiologists to the intensive care nursery where a record was made of the range of daily numbers and character of stools, and the physicians' and nurses' notes were reviewed for indications of clinically significant diarrheal illness2 ~ in contrast to infants usually admitted to the regular nursery, infants admitted to the special care unit are often severely ill. Diagnoses among patients and controls are listed in Table I. Bacteriology. In addition to the standard stool e x a m i nations for conventional bacterial pathogens mentioned above, one to four isolates were selected at r a n d o m from each representative colony type on the eosin methylene blue agar plates, cloned for purity, and inoculated into 4 ml of trypticase soy broth with 0.6% yeast extract in 50 ml

Volume 89 Number 6

Erlenmeyer flasks. After 48 hours incubation at 37 ~ C, these cultures were centrifuged at 10,000 • g for 30 minutes, and the culture supernatants were passed through a 0.22 >m filter (Millipore Corporation, Bedford, Mass.). Sterile culture filtrates thus obtained were then studied in the CHO cell assay. No non-LF organisms were cultured from these patients. At least one LF isolate of each colony type from each patient during illness was serotyped at the Enterobacteriology Branch, Bureau of Laboratories, Center for Disease Control, Atlanta, Ga., by Ms. Connie Riddle and Ms. Mary Alyce Asbury. CHO assay for enterotoxin. The details for preparing the Chinese hamster ovary cell line (CHO-K 1) for studies of morphologic changes have been described elsewhere, l' Briefly, the ceils are grown on F-12 medimn with 10% fetal calf serum, are passaged by trypsinization, and are plated into eight-chamber culture slides (Lab Tek Products, Naperville, Ill.) or into 96-well fiat-bottom microtiter plates (Cooke Laboratory Products, Alexandria, Va.) at approximately 5,000 cells per 0.25 ml F-12 with 1% FCS and 0.05 mM methyl isobutyl xanthine (Aldrich Chemical Co., Milwaukee, Wis.). Promptly after the CHO ceils are added to the multichamber culture slides, 20 btl of the crude culture filtrate from each LF isolate are added to the tissue culture medium. Twentyfour hours later the number of cells per 100 that had become spindle shaped and had lost knoblike projections was enumerated by phase contrast microscopy, or by light microscopy of Giemsa-stained preparations. Each study was done in duplicate and was repeated with freshly prepared filtrates on a different day to give at least 400 cells counted per value for the percentage of cells elongated for each isolate. Viral studies. Suspensions from rectal swabs immersed in brain-heart infusion broth were examined for the presence of viruses by immune electron microscopy. A sample (0.8 ml) of the stool suspension was incubated with 0.2 ml of a 1:5 dilution of immune human serum globulin (convalescent sera were not available), for one hour at room temperature prior to further preparation for electron microscopy as previously described? .... Specimens were examined by immune electron microscopy to enable the detection of 27 nm parvovirus-like particles 1~ andto detect 70 nm human reovirus-like agents. 1~At least one rectal swab suspension derived from each patient was inoculated into roller tube cultures of WI 38, HEp-2, human embryonic kidney, and Rhesus monkey kidney cells. The inoculated suspensions were adsorbed to the cell cultures for four hours at approximately 35 ~ C, then washed and refed with maintenance medium which consisted of one-half Eagles' medium and one-half

Toxigenic bacterial diarrhea

887

medium '199 plus glutamine and appropriate antibiotics. The maintenance medium was free of serum except for that used in WI 38 cultures which contained 2% fetal calf serum. In certain instances in which the rectal swab suspensions appeared to contain much particulate matter, suspensions were clarified at 1,500 RPM for ten minutes in the refrigerated centrifuge prior to inoculation as above. On initial passage cultures were observed for cytopathic effect for 17 days; in addition, monkey kidney cultures were hemadsorbed twice with 0.4% guinea pig erythrocytes. Harvests from cultures with a suggestion of CPE were repassaged and observed for approximately one to two weeks. Each rectal swab suspension or clarified suspension was inoculated both intracerebrally and intraperitoneally into two litters of one-day-old general purpose (NIH) mice which were then observed for 21 days. Ifa pattern of illness or death was observed, mousebrain suspensions were passaged as above to additional suckling mice. RESULTS Over a four-day period from January 2 through January 6 of 1974, 14 of the 15 infants in this special care unit developed mild watery diarrhea, defined as increased frequency with a watery consistency of the stools. In each case, the diarrhea was thought to be of clinical significance by the physicians in care of the patients. The average number of stools per patient per day is shown for the first 19 days of January, 1974, in Fig. 1. Baseline surveillance for eight weeks in this nursery during the same season the following year revealed the average number ofst0ols per day to be 3.3 _+ 1.7 (SD) per patient. Over this latter period of surveillance, diarrhea was felt to be a transient problem in only four of the 41 patients hospitalized in this nursery from December 6, 1974, to January 30, 1975. Thus, "diarrhea" was defined in this study as _> 7 stools per day (_> 2 SD above mean for controls) or the physician's or nurse's diagnosis of "diarrhea" on the basis of stool character and volume plus a frequency of _> 5 stools per day (~> l SD above mean). The average duration of illness during the outbreak of diarrhea in January, 1974, was four days, with a range of one to 14 days. Three infants had recurrent diarrhea over seven to 11 days, and one required subsequent readmission for diarrhea in the month after the original illness. Only five of the 14 patients with diarrhea required intravenous therapy during the course of their illnesses. The age range was from two to 85 days. No child received a hyperosmotic diet, and none subsequently evidenced protein allergy or primary lactose intolerance. Seven of the 14 patients with diarrhea were receiving antibiotics (a

888

Guerrant et al.

The Journal of Pediatrics December 1976

0

6 m

0. e-

5

"r. 0

,n 0 0

3

2

4

6

8

10

12

JANUARY, 1974

14

16

18

Average+_S.D. Dec.1974-Jan.197 S

Fig. !. Average number of stools per patient per day for all patients in the newborn special care unit from January 1 through January 19, 1974, and for the control period one year later.

penicillin and an aminoglycoside) for suspected infection at the time of the outbreak. The enteric bacterial isolate(s) from each of these patients were resistant to one or more of the antibiotics they were receiving; however, no predominant pattern of antibiotic resistance was noted. The methylene blue examination of the stool in each case revealed no leukocytes; conventional pathogens, including Salmonella, Shigella, classic enteropathogenic E. coli serotypes, or staphylococci were not isolated from any of the infants. Three to six LF coliform isolates when present were examined from each patient for toxin production in the CHO assay. Based on prior experience with the CHO cell assay, both with strains of known enterotoxigenicity and controls, 1~' as well as from a controlled study of similar diarrheal illness among children in Brazil, a range of CHO cell responses with culture filtrates of E. coli from control children without diarrhea was observed. Filtrates of 60 LF isolates from 20 infants and children in Brazil without any gastrointestinal illness gave a m e a n of 5.4 __ 2.0% (SD) of CHO cells elongated. -'~ On the basis of these data, only strains giving CHO responses greater than 4 SD above the m e a n (i.e., _> 13.5%) were considered definitely positive for toxin. By these criteria, 11 of the 14 patients with diarrhea had at least one toxigenic isolate, and 33 of the 48 isolates tested from these patients were toxigenic (Table II). These toxigenic isolates, however, comprised a total of nine different serotypes of three species of organisms (Table III). There were three different serotypes of toxigenic E. coli (none of which were classic enteropathogenic serotypes), two toxigenic Citrobacter species, and four Klebsiella serotypes. Three patients were found to have more than one toxigenic

species simultaneously isolated. One patient (W. O.) carried E. coli serotype 012:H41 into convalescence, at which time it was nontoxigenic. Likewise, Klebsiella type 21 persisted in another patient (J. A.), and it was again nontoxigenic during convalescence. Three different colonies of E. eoli from the acute illness specimen of one patient (R. I.) were serotyped. All three were 012:H41, two of which were toxigenic and one of which was not. The proportions of infants with toxigenic isolates and the n u m b e r of toxigenic isolates among those (1) with acute diarrhea, (2) followed into convalescence at one to ten weeks after recovery, and (3) those control infants with no diarrhea are listed in Table IV. In addition to examination of isolates among patients with acute diarrhea, 50 isolates were tested from ten patients studied in convalescence. Five of the 50 isolates (from four convalescent patients) were toxigenic. From the ten control patients with no diarrhea, 38 isolates were tested, and only three Klebsiella were obtained from two infants that appeared to be toxigenic. In contrast to 79% (11 of 14) of the patients with acute diarrhea who had toxigenic bacteria, only four of ten patients in convalescence and two of ten control infants had one or more toxigenic organisms (p = 0.067 and p < 0.007, respectively). Differences in the total numbers of toxigenic organisms between the g r o u p with acute illness and those in convalescence or control infants with no diarrhea were highly significant (p < 10 -~ in both instances). Table IV summarizes the percentage of isolates per patient found to be toxigenic in the CHO cell assay. When three to five clones of E. coli and one to three clones of Klebsiella or Citrobacter were tested per patient, toxigenic isolates were the predominant or only isolates in ten of the

Volume 89 Number 6

Toxigenic bacterial diarrhea

0C* I

EcO75:H15

KI3

"

K2!

Room i

J.o

EcO83:NM

T

NEWBORN

3

[

I ST

(No Diarrhea)

COO

I

Room 2

T

Room

Table

.All

FO EcO83:NM

I ~

K21

\

r

J -o J L,o SPECIAL

CARE UNIT

EcO12:H41

Km

889

Jan. 7, 1974

II. N u m b e r of infants with toxigenic isolates and the n u m b e r of toxigenic isolates in those with acute diarrhea, those convalescent from diarrhea, and the control infants with n o d i a r r h e a No. toxigenic isolates

No. infants with toxigenic isolates Clinical group

Total No. Infants

Total isolates tested

Acute diarrhea (n = 14) Convalescent diarrhea (n = 10) Control (no diarrhea) (n = 10)

11/ 14"

33/48t

4/10

5/50

2/10

3/38

*Different from convalescent patients (p = 0.067 by Fischer's exact test) and control infants (p = 0.007 by Fischer's exact test). J'Different from isolates from convalescent and control groups (X~ = 35.6 and 32.3, respectively; p < 10 ~ in both comparisons.

EcOI2:H41

EcO12:H41 K18

Fig. 2. Location of patients' isolettes in three rooms in the special care unit and their enterotoxigenic bacteria during illness. K = Klebsiellapneumoniae; E = Escherichia colt; C. div. = Citrobaeter diversus," C. freund. = Citrobacter freundii. *Patients with adenoviruses isolated from feces.

14 patients with acute diarrhea. This is in distinct contrast to these patients in convalescence a n d to control infants, w h e n toxigenic isolates were p r e d o m i n a n t in only one of 20 instances. The location o f these infants' isolettes a n d their enterotoxigenic b a c t e r i a d u r i n g illness are d i a g r a m m e d in Fig. 2. Identical serotypes from different patients were f o u n d only from infants in the same room. All nine patients in two rooms h a d d i a r r h e a a n d toxigenic organisms, while only t w o of the five patients with d i a r r h e a in the third room had toxigenic organisms isolated. All 15 patients in the nursery at the time of the o u t b r e a k (14 with acute diarrhea, one without) h a d a d e q u a t e stool suspensions from swabs e x a m i n e d for the presence o f viruses by i m m u n e electron microscopy. N o n e o f these 15 patients' specimens taken during the p e a k of the d i a r r h e a o u t b r e a k were f o u n d to be positive for reovirus- or parvovirus-like agents. Two patients (C. L. a n d F. O.) were f o u n d by electron microscopy to be s h e d d i n g a d e n o virus in rectal swab specimens o b t a i n e d at the time o f illness. These two patients h a d diarrhea, were b o t h in room 1 (Fig. 2), a n d both also had toxigenic bacterial isolates from their stools. N o viruses were detected by cell culture or suckling m o u s e studies with rectal swab spec-

Table III. Toxigenic isolates in l l of 14 patients with diarrhea E. colt

Patients F. O. C.R. C. L. J.A. P.I. R.A. H, U. S.T. R.I. W. O. R.O.

I

Citrobacter

Klebsiella

Ec. 083:NM

K21 K 60 K21 K13

C. diversus

Ec. 075ah:Hl5 C. freundii Ee. Ec. Ec. Ec.

012:H41 012:H41 012:H41 083:NM

K NT* K 18

*NT = not typed.

Table IV. F r e q u e n c y of toxin-producing bacteria a m o n g patient, convalescent, a n d control groups % o f isolates tested from each infant that were toxigenic

1,2412,4 1,o741 9 No. o f infants Acute diarrhea (n = 14) Convalescent diarrhea (n = t0) Control (no diarrhea) (n = t0)

,oo

3

0

l

2

2

6*

8

1

1

0

0

0

8

0

1

0

0

l*

*Include qne control infant and four infants with diarrhea who only had one or two organisms (Klebsiella or Cttrobacter) for testing.

890

Guerrant et al.

imens from 14 of the 15 patients. Specimens from one patient (C. L.) (who also shed adenovirus by electron microscopy) induced a questionable cytopathic effect which, unlike others, persisted beyond a second passage in monkey kidney and human embryonic kidney cells. In addition, one of two litters of suckling mice inoculated with this suspension developed illness or died. Passage into two additional litters induced illness in one of the two litters; on third passage into two litters, no illness developed. DISCUSSION The etiology of epidemic diarrhea in special care nurseries is largely unknown. It has been attributed to several etiologic agents, including viruses, classic enteropathogenic E. colt of a few recognized serot)pes, and less often to other bacteria such as salmonellae, shigellae, klebsiellae, and pseudomonas. Because of the demonstration of enterotoxin production by strains of E. colt other than the classic enteropathogenic serotypes 7 and the demonstration of classic serotypes among control patients, 2..... the emphasis has shifted from serotyping organisms for pathogenicity to detection of the enterotoxin itself. Severe nosocomial gastroenteritis has been reported in 37 infants in a premature intensive care nursery over five winter months in a county hospital in Arizona. ~ These illnesses lasted several days despite antibiotic therapy and were fatal in four cases. They were associated with an enterotoxigenic E. colt, strain 0142. Although the mode of spread was not certain, this organism was isolated from air samples and from the hands of personnel. Certainly, it appears that such special care units with infants who are exposed to the hospital bacterial flora and often to antibiotics, but who have had little prior antigen exposure, may be uniquely susceptible to such nosocomial infections. In assaying for the heat-labile, cholera-like toxin in tissue culture, this study demonstrates the association of toxigenicity in multiple strains with acute diarrhea when compared with isolates from these patients in convalescence, or when compared with isolates from control individuals with no history of diarrhea. Although this association of toxigenicity does not prove an etiologic relationship, the absence of fecal leukocytes is against any invasive bacterial process; in addition the presence of the reovirus-like agent of infantile gastroenteritis or the parvovirus-like agent of epidemic viral gastroenteritis could not be demonstrated. There were no Salmonella or Shigella isolated from any of these patients, and neither staphylococci nor heat-stable Clostridia were found. Adenoviruses have been found in rectal swab suspensions derived from two patients. The role of adenoviruses in enteric infections, however, remains unclear,

The Journal of Pediatrics December 1976

since adenoviruses have been found among control individuals without diarrhea as well. 13 In a recent study adenoviruses did not appear to be important causes of diarrhea in children. TM At least two types of enterotoxin have been described from E. colt. In addition to the heat-labile enterotoxin that appears to act much like cholera toxin, a heat-stable toxin has been described in animals~,' and in some patients with diarrhea, x~ -'~ The CHO cell assay specifically detects the heat-labile, cholera-like toxin and not the heat-stable toxin; it does not respond to identically prepared culture filtrates of nontoxigenic control organisms? ~ ~1 Although the CHO cell assay is more sensitive than rabbit ileal loops to purified cholera toxin, CHO cell elongation responses to culture filtrates of toxigenic E. colt parallel rabbit ileal loop secretory responses. '-'' While this study did not employ the suckling mouse assay for the heatstable toxin, 26it does demonstrate the potential role of the cholera-like, heat-labile toxin in an outbreak of diarrhea in infants. The production of this heat-labile, cholera-like toxin has been associated with a plasmid that is transferable between strains of E. colt much like R-factor antibiotic resistance?' The multiplicity of strains in this epidemic, the inability to demonstrate a single serotype throughout the nursery, and the disappearance of toxigenicity, despite persistence of identical strains in convalescence in some instances, all raise the possibility that this outbreak may have been related to the spread of toxigenicity by a plasmid. Escherichia colt that produce the heat-labile toxin have been associated with diarrheal illness and were not found among ten infants who had not had recent diarrhea in this study nor among 20 control children without diarrhea in Brazil. '-'1 Toxigenic Klebsiella are less restricted to patients with diarrheal illness in this study. Although i~atients with no enteric symptoms had toxigenic klebsiellae isolated from their stools, their association with acute disease is significant (p < 0.05). Others, too, have noted the association of Klebsiella species with infantile diarrhea. ~'~In addition, it has recently been shown that Klcbsiella species from patients with tropical sprue may be enterotoxigenic in animal models2" An unexpected difficulty that we have encountered has been the relative instability of the enterotoxin-producing capability (both in rabbit loop and CHO cell studies) when these organisms are maintained and passed at room temperature on agar slants. This experience is reminiscent of the extreme lability of in vitro recipient strains of the tox § plasmid, 3~ and of the in vivo experience demonstrated by two patients in this study who carried the same organism into convalescence, at which time it was nontox-

Volume 89 Number 6

igenic. Because of this observation, however, this study must be considered p r e l i m i n a r y a n d will have to be confirmed by additional in vitro as well as p a t i e n t experience. Careful preservation o f the o r g a n i s m s by lyophilization or freezing a n d a n a t t e m p t to o b t a i n paired sera for study o f b o t h anti-viral a n d antitoxic i m m u n i t y which can n o w be d e m o n s t r a t e d TM :~0-3~s h o u l d s t r e n g t h e n subsequent studies o f the etiology of this i m p o r t a n t problem.

Toxigenic bacterial diarrhea

15.

16. 17. 18.

The authors gratefully acknowledge the help of Mr. C. A. Osterman and Ms. K. J. Hunting in the epidemiologic surveillance, and the technical and secretarial assistance of H. D. James, Jr., Ms. A. L. Vaughn, J. Skaar, S. H. Shen, J. Sandridge, L. Halterman, and D. Miller.

19.

REFERENCES

20.

1. Gordon JE, Guyana MA, Ascoli W, and Scrimshaw N: Acute diarrheal disease in less developed countries. 2. Patterns of epidemiological behavior in rural Guatemalan villages, Bull WHO 31:9, 1964. 2. Moffett HL, Shulenberger HK, and Burkholder ER: Epidemiology and etiology of severe infantile diarrhea, J PEDIATR 72:1, 1968. 3. Drachman RH: Acute infectious gastroenteritis, Pediatr Clin North Am 21:771, 1974. 4. South MA: Enteropathogenic Escherichia coil disease: New developments and perspectives, J PEDIATR 79:1, 1971. 5. Jacobs SI, Holzel A, Wolman B, Keen JH, Miller V, Taylor J, and Gross R J: Outbreak of infantile gastroenteritis caused by Escherichia coli 0114, Arch Dis Child 45:656, 1970. 6. Rogers KB: The spread of infantile gastroenteritis in a cubicled ward, J Hyg 49:140, 1951. 7. Gorbach SL, and Khurana CM: Toxigenic Escherichia coli: A cause of infantile diarrhea in Chicago, N Engl J Med 287:791, 1972. 8. Boyer KM, Petersen N J, Frazaneh I, Pattison CP, Hart MC, and Maynard JE: An outbreak of gastroenteritis due to E. coli 0142 in a neonatal nursery, J PEDIATR 86:919, 1975. 9. Skerman EJ, Formal SB, and Falkow S: Plasmid-associated enterotoxin production in a strain of Escherichia coli isolated from humans, Infect Immun 5:622, 1972. 10. Guerrant RL, Brunton LL, Schnaitman TC, Rebhun LI, and Gilman AG: Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Escherichia coli, Infect Immun 10:320, 1974. 11. Eichenwald HF, Ababio BA, Arky AM, and Hartman AP: Epidemic diarrhea in premature and older infants caused by Echo virus type 18, JAMA 166:1563, 1958. 12. Klein JO, Lerner AM, and Finland M: Acute gastroenteritis associated with Echo virus, type 11, Am J Med Sci 240:749, 1960. 13. Yow MD, Melnick JL, Blattner RJ, Stephenson WB, Robinson NM, and Burkhardt MA: The association of viruses and bacteria with infantile diarrhea, Am J Epidemiol 92:33, 1970. 14. Kapikian AZ, Wyatt RG, Dolin R, Thornhill TS, Kalica AR, and Chanock RM: Visualization by immune electron microscopy of a 27-nm particle associated with acute

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infectious nonbacterial gastroenteritis, J Virol 10:1075, 1972. Kapikian AZ, Kim HW, Rodriguez WJ, Cline WL, Parrott RH, and Chanock RM: Reovirus-like agent in stools: Association with infantile diarrhea and development of serologic tests, Science 185:1049, 1974. Editorial: Rotaviruses of man and animals, Lancet 1:257, 1975. Editorial: Virus of infantile gastroenteritis, Br Med J 3:555, 1975. Kapikian AZ, Kim HW, Wyant RG, Cline WL, Arrobio JO, Brandt CD, Rodriguez WJ, Sack DA, Chanock RM, and Parrott RH: Human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children, N Engl J Med 294:965, 1976. Edwards PR and Ewing WH: Identification of enterobacteriaceae, Minneapolis, 1962, Burgess Publishing Company. Wenzel RP, Osterman CA, Hunting K J, and Gwaltney JM, Jr: Hospital-acquired infections. I. Surveillance in a university hospital, Am J Epidemiol 103:251, 1976. Guerrant RL, Moore RA, Kirschenfeld PM, and Sande MA: Role of toxigenic and invasive bacteria in acute diarrhea of childhood, N Engl J Med 293:567, 1975. Cooper ML, Walters EW, and Keller HM: E. coli associated with infantile diarrhea, Ann NY Acad Sci 66:78, 1956. Kessner DM, Shaughnessy JH, Googins J, Rasmussen CM, Rose N J, Marshall AL, Andelman SL, Hall JB, and Rosenbloom PJ: An extensive community outbreak of diarrhea due to enteropathogenic E. coli 0111:B4, Am J Hyg 76:27, 1962. Center for Disease Control: Nosocomial gastroenteritis, Morbidity and Mortality Weekly Report 22:225, 1973. Smith HW and Halls S: Studies on Escherichia coli enterotoxin, J Pathol Bact 93:531, 1967. Dean AG, Ching YC, Williams RG, and Harden LB: Test for Escherichia coli enterotoxin using infant mice: Application in a study of diarrhea in children in Honolulu, J infect Dis 125:407, 1972. Sack DA, Wells JG, Merson MH, Sack RB, and Morris GK: Diarrhoea associated with heat-stable enterotoxin-producing strains of Escherichia coli, Lancet 2:239, 1975. Olarte J, Ferguson WW, Henderson ND, and Torregrosa L: Klebsiella strains isolated from diarrheal infants, Am J Dis Child 101:763, 1961. Klipstein FA, Holdeman LV, Corcino J J, and Moore WEC: Enterotoxigenie intestinal bacteria in tropical sprue, Ann Intern Med 79:632, 1973, Richardson SH, McKensie B, Dares E, Evans DJ Jr, and Evans DG: Transfer of plasmids controlling vascular permeability factor (PF) in Escherichia coil, Proc 10th Joint Conf US-Japan Cooperative Medical Science Program, Cholera Panel, 37, Kyoto, 1974. Donta ST, Sack DA, Wallace RB, Dupont HL, and Sack RB: Tissue-culture assay of antibodies to heat-labile Escherichia coli enterotoxins, N Engl J Med 291:117-121, 1974. Guerrant RL: Characterization and clinical use of the Chinese hamster ovary (CHO) cell assay for enterotoxins and antitoxin, in Fukumi H, and Ohashi M, editors: Symposium on cholera, U.S.-Japan Cooperative Medical Science Program, Kyoto, 1974.