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Neonatal escherichia coli septicemia—bacterial counts in blood
July 1974
128
The Journal o f P E D I A T R I C S
Neonatal Escherichia coli septicemia--bacterial counts & blood Bacterial counts in blood of E. coil (number of viable organisms per milliliter) are reportedfor 30 neonates who were cultured because of suspected septicemia. Fifteen of the infants died; 13 of the deaths occurred within 48 hours after cultures were obtained and appropriate antibiotic therapy had been initiated. Thirty-five positive cultures were obtained from the 30 neonates; in 11 (31%) of the cultures the colony counts were in excess of 1, O00/ml; in 5 (14 %), the counts ranged from 50 to 1,000/ml; in 11 (31%), from 5 to 49/ml; in 8 (23%), from 0 to 41ml. Seventy-three per cent (8/11) of the neonates with colony counts greater than 1,000/ml died.
D a l e E. D i e t z m a n , M . D . , * Seattle, Wash., G e r a l d W . F i s c h e r , M . D . , Honolulu, H a w a i i , and Fritz D. Sehoenknecht, M . D . , Seattle, W a s h .
CONCENTRATIONS of bacteria in blood (viable organisms per ml) have not been previously quantitated in neonatal septicemia. Because of special interests in blood culture methods at the University of Washington Hospital microbiology laboratory, blood culture specimens obtained in sufficient volume are cultured quantitatively on pour plates in addition to the routine broth methods. In this study we report concentrations of bacteria in blood of 30 neonates with Escherichia coil (E. coli) bacteremia or septicemia who were cultured because of suspected septicemia during an eight-year period from June, 1965, through May, 19.73. The majority of these neonates were premature infants. Their clinical records were reviewed, and their clinical courses were related to the bacterial counts in their blood. Bacteremias which were caused by organisms other than E. coli were far less common during this period, and such cases are not included in this report. MATERIALS
AND METHODS
Blood samples from infants in the University Hospital neonatal intensive-care unit who were suspected of havFrom the Children's Orthopedic Hospital and Medical Center andDepartment of Pediatrics, University of Washington School of Medicine; the Department of Pediatrics, Tripler Army Medical Center; and the Department of Microbiology and Laboratory Medicine, University of Washington School of Medicine. *Reprint address: The Children's Orthopedic Hospital and Medical Center, 4800Sand Point Way N,E,, Seattle, Wash. 98105.
Vol. 85, No. 1, pp. 128-130
ing septicemia were cultured; agar pour plates and standard broth culture methods were used. All infants with positive cultures for E. coli were included in the study, except when blood was taken from cord vessels, from indwelling catheters, or at postmortem examination, or the culture contained mixed organisms. The medical record and bacteriology reports of each case were retrospectively reviewed to note the clinical evidence of sepsis 1and to determine whether the patients had meningitis or a localized site of infection and whether they survived.
See related article, p. 117. The samples were drawn by the house physicians, after careful cleansing of the skin at the puncture site with iodine and alcohol solutions, 2 in sterile vacutainer tubes containing approximately 1.5 ml of 0.05% sodium polyanethol sulfonate (SPS, Liquoid) anticoagulant, 3 and transported to the laboratory immediately. The cultures were processed as soon as received. Pour plates were made by incorporating 1 or 0.5 ml of patient's blood into m e l t e d agar a n d w e r e i n c u b a t e d a e r o b i c a l l y a n d anaerobically at 35 ~ C. The remainder of the blood-Liquoid mixture, at least 1 ml, was inoculated into 100 ml of a trypticase soy yeast broth bottle and incubated at 35 ~ C. 4 Plate and broth cultures were examined and subcultured at regular intervals until positive, or up to ten days. Surface and deep colonies were counted and subcultured for identification and antibiotic susceptibility
Volume 85 Number 1
testing. The actual plate colony counts were corrected and expressed as colonies per milliliter, based on the quantity of blood cultured in the plate. Care was taken to document that the samples had been promptly cultured in the laboratory and did not reflect bacterial multiplication in vitro (the generation time of E. coli is about 20 to 30 minutes at room temperature). RESULTS Of the 30 infants with suspected septicemia who had blood cultures positive for E. coli, 26 were boys, and all but two were less than eight days of age. Seven had cultured-documented meningitis, four had enterocolitis, one had an open meningomyelocele, and one had a surgical wound infection. The remaining 17 infants did not have a localized focus of infection. Only one of the infants had apparent transient bacteremia. He was a premature infant cultured because of lethargy and poor feeding. His positive culture was obtained at age 14 days; growth occurred in the broth culture but not on either pour plate. Blood cultures obtained one day prior to and two days after (no antibiotics given during the interval) the positive culture showed no growth. Five of the 30 infants had a second positive blood culture--a total of 35 cultures from the 30 infants. The colony counts of the 35 cultures are summarized in Table I. The bacteremia levels varied greatly, ranging from less than five to more than 1,000 organisms per milliliter. Only one culture positive in broth was negative on both pour plates (0 colonies per milliliter). No sample was positive by pour plate culture and negative by broth culture. The groupings in Table I show that 23% (8/35) of the positive cultures had counts of less than five colonies per milliliter. Two of these eight cultures were obtained on the same day from a patient with meningitis who had received antibiotic therapy (ampicillin and k a n a m y c i n ; o r g a n i s m was " s e n s i t i v e " ) for more than 48 hours. Another culture was taken two days after initiation of antibiotic therapy. None of the other 28 patients had received antibiotics before their blood for culture was obtained. Approximately one third (31%) of all the pour plates had moderately low colony counts of five to 49 per milliliter. Notably, another one third (31%) had colony counts in excess of 1,000 per milliliter. There were five patients who each had two positive cultures. For three of them the two cultures were taken on the same day. The two colony counts for these three infants were, respectively: 2 and 2; ) 1,000 and 24; and 4 and 44. One patient had a count of 16 per milliliter, initially, and a repeat count of ) 1,000 the next day (he had not received antibiotics). The other patient initially had a count of 8 per milliliter, and the repeat count was 2 per milliliter after two days of antibiotic therapy.
Neonatal E. coli septicemia
129
Table I. Colony counts* in newborn infants with E. coli bacteremia and septicemia Colony count range (colonies per ml)
No. of cultures
0-4 5-49 50-199 200-1,000 ) 1,000 Total
8t 11 4 1 11, 35
Per cent
23 31 11 3 31
*There were 35 culturesfrom 30 differentpatients tThree cultures from two patientswere taken after 2 to 3 days of antibiotic therapy. None of the other 28 patients had receivedantibiotics. *These 11 cultureswerefrom 11 differentpatients;6 of them had meningitis; 2 had enterocolitis.Eight of the 11 infantsdied.
Table II. Association of initial colony counts with meningitis and death
Colony count per ml
No. of patients
Percentage q[ patients with meningitis
l~ercentage of patients who died
( 1,000 ) 1,000
t9" 11
5 (1/19) 60 (6/11)
37 (7/19) 73 (8/11)
*Twoof these patients had receivedantibiotics;one of them had meningitis and died.
Fifteen of the patients died; 13 of the deaths occurred within 48 hours after cultures had been obtained, and appropriate antibiotic therapy had been initiated. Six of 11 patients with colony counts of ) 1,000 per milliliter had meningitis (Table II). The patients with colony counts of ) 1,000 per milliliter had a case fatality rate of 73% (8/11), whereas patients with lower bacterial counts had a case fatality rate of 37% (7/19). Colony counts were compared with the birth weights of the infants; there was no clear relationship. Nearly all of the positive broth and pour plate cultures were reported as positive by the laboratory within 24 hours; all were positive within 48 hours. DISCUSSION In this study we have documented concentrations of E. coli in blood in 30 infants. Infections with E. coli were the predominant cause of neonatal septicemia during the study period. E. coli was ideal for this quantitative study as it is not fastidious and grows well in pour plates. Only one of 30 positive broth cultures did not show detectable growth on a pour plate, which indicates that the pour plate quantitation was quite sensitive. We c a n n o t be certain, however, whether cultures using larger volumes of blood such as 5 and 10 ml in both
130
Dietzman, Fischer, and Schoenknecht
broth and pour plate methods might have detected any additional cases ofbacteremia, as such volumes were not obtained. By documenting bacteremla counts we were able to estimate m i n i m u m volumes of blood which would have been required for detection of these cases of E. coil sepsis with the use of broth culture techniques. Although Franciosi and Favara 5reported that a single culture using 1 to 3 ml of blood was adequate to confirm sepsis in each of 28 cases of neonatal sepsis, they did not show whether culturing 1 ml of blood or less was as accurate as culturing 3 ml. Additionally, they did not document the counts of bacteria in these infants. Albers and his associates6reported persistent c o n c e n t r a t i o n s of bacteria in blood ranging from one to 126 organisms per milliliter in three a s y m p t o m a t i c neonates, b u t reported a c o u n t of 20 organisms per milliliter in only one infant with possible septicemia. In our study, eight cultures from seven patients had low counts of less than five organisms per milliliter. Of these eight cultures, three (from two patients) were taken after two to three days of antibiotic therapy, and another was from an infant with transient bacteremia. The other four infants with colony counts of less than five organisms per milliliter had clinical evidence of septicemia, but two of them had significant congenital anomalies (aplastic pituitary; rudimentary thymus and anomalies of fourth arch derivatives) which complicated their clinical evaluation. It could be questioned whether culturing of blood in volumes of less than 1 ml from these patients with low colony counts would have been sensitive enough to ensure detection of their bacteremia. The great majority of cases (77%; 23 of 30) of neonatal E. colibacteremia in this study, however, had colony counts of more than four organisms per milliliter. Culturing of as small amounts of blood as 0.2 to 0.5 ml would have been expected to give positive results in these cases. This estimate is supported by the experimental observations of Fischer and associates. Our study does not consider infections with more fastidious organisms, or infections in infants beyond the neonatal period,
The Journal of Pediatrics July 1974
Bacterial counts greater than L000 organisms per milliliter, such as occurred in one third of these infants, have not been previously documented in neonates. Such high bacterial counts probably reflect poor host defenses and related to the fulminant course of the infection. The observation that 13 of 15 deaths occurred within 48 hours of the time culture was taken further emphasizes the fulminant nature of these infections. Although there was a high mortality rate in the neonates found to have more than 1,000 organisms per milliliter of blood, there were deaths in infants with bacterial counts of less than 50 organisms per milliliter. For this reason, bacterial counts cannot be relied upon to give precise prognostic information in a single situation. We do not propose the general use of quantitative pour plate cultures, and they should never be considered a replacement for more sensitive broth cultures. We wish to thank the microbiology technicians of the University Hospital Clinical Microbiology Laboratory for their technical assistance. We gratefully acknowledge the stimulation, encouragement, and helpful suggestions provided by Drs. C. George Ray, John C. Sherris, David E. Woodrum, Errol Alden, and Alan W. Hodson. REFERENCES
1. Gotoff,S. P., and Behrman, R. E.: Neonatal septicemia, J. PEDIATR.76: 142, 1970. 2. Eitzman, D. V., and Smith, R. T.: The significance of blood cultures in the newborn period, Am. J. Dis. Child. 94: 601, 1957. 3. Rosener, R.: A quantitative evaluation of three blood culture systems, Am. J. Clin. Pathol. 57: 220, 1972. 4. Roome, A. P. C. H., and Tozer, R. A.: Effect of dilution on the growth of bacteria from blood cultures, J. Clin. Pathol. 21: 719, 1968. 5. Franciosi, R. A., and Favara, B, E.: A single blood culture for confirmation of the diagnosis of neonatal septicemia, Am. J. Clin. Pathol. 57: 215, 1972. 6. Albers, W. H., Tyler, C. W., and Boxerbaum, B.: Asymptomatic bacteremia in the newborn infant, J. PEDIATR.69: 193, 1966. 7. Fischer, G. W., Crumrine, M. H., and Jennings, P. B.: Experimental E. coil sepsis in rabbits, J. PEDIATR.85: 117, 1974.
128
The Journal o f P E D I A T R I C S
Neonatal Escherichia coli septicemia--bacterial counts & blood Bacterial counts in blood of E. coil (number of viable organisms per milliliter) are reportedfor 30 neonates who were cultured because of suspected septicemia. Fifteen of the infants died; 13 of the deaths occurred within 48 hours after cultures were obtained and appropriate antibiotic therapy had been initiated. Thirty-five positive cultures were obtained from the 30 neonates; in 11 (31%) of the cultures the colony counts were in excess of 1, O00/ml; in 5 (14 %), the counts ranged from 50 to 1,000/ml; in 11 (31%), from 5 to 49/ml; in 8 (23%), from 0 to 41ml. Seventy-three per cent (8/11) of the neonates with colony counts greater than 1,000/ml died.
D a l e E. D i e t z m a n , M . D . , * Seattle, Wash., G e r a l d W . F i s c h e r , M . D . , Honolulu, H a w a i i , and Fritz D. Sehoenknecht, M . D . , Seattle, W a s h .
CONCENTRATIONS of bacteria in blood (viable organisms per ml) have not been previously quantitated in neonatal septicemia. Because of special interests in blood culture methods at the University of Washington Hospital microbiology laboratory, blood culture specimens obtained in sufficient volume are cultured quantitatively on pour plates in addition to the routine broth methods. In this study we report concentrations of bacteria in blood of 30 neonates with Escherichia coil (E. coli) bacteremia or septicemia who were cultured because of suspected septicemia during an eight-year period from June, 1965, through May, 19.73. The majority of these neonates were premature infants. Their clinical records were reviewed, and their clinical courses were related to the bacterial counts in their blood. Bacteremias which were caused by organisms other than E. coli were far less common during this period, and such cases are not included in this report. MATERIALS
AND METHODS
Blood samples from infants in the University Hospital neonatal intensive-care unit who were suspected of havFrom the Children's Orthopedic Hospital and Medical Center andDepartment of Pediatrics, University of Washington School of Medicine; the Department of Pediatrics, Tripler Army Medical Center; and the Department of Microbiology and Laboratory Medicine, University of Washington School of Medicine. *Reprint address: The Children's Orthopedic Hospital and Medical Center, 4800Sand Point Way N,E,, Seattle, Wash. 98105.
Vol. 85, No. 1, pp. 128-130
ing septicemia were cultured; agar pour plates and standard broth culture methods were used. All infants with positive cultures for E. coli were included in the study, except when blood was taken from cord vessels, from indwelling catheters, or at postmortem examination, or the culture contained mixed organisms. The medical record and bacteriology reports of each case were retrospectively reviewed to note the clinical evidence of sepsis 1and to determine whether the patients had meningitis or a localized site of infection and whether they survived.
See related article, p. 117. The samples were drawn by the house physicians, after careful cleansing of the skin at the puncture site with iodine and alcohol solutions, 2 in sterile vacutainer tubes containing approximately 1.5 ml of 0.05% sodium polyanethol sulfonate (SPS, Liquoid) anticoagulant, 3 and transported to the laboratory immediately. The cultures were processed as soon as received. Pour plates were made by incorporating 1 or 0.5 ml of patient's blood into m e l t e d agar a n d w e r e i n c u b a t e d a e r o b i c a l l y a n d anaerobically at 35 ~ C. The remainder of the blood-Liquoid mixture, at least 1 ml, was inoculated into 100 ml of a trypticase soy yeast broth bottle and incubated at 35 ~ C. 4 Plate and broth cultures were examined and subcultured at regular intervals until positive, or up to ten days. Surface and deep colonies were counted and subcultured for identification and antibiotic susceptibility
Volume 85 Number 1
testing. The actual plate colony counts were corrected and expressed as colonies per milliliter, based on the quantity of blood cultured in the plate. Care was taken to document that the samples had been promptly cultured in the laboratory and did not reflect bacterial multiplication in vitro (the generation time of E. coli is about 20 to 30 minutes at room temperature). RESULTS Of the 30 infants with suspected septicemia who had blood cultures positive for E. coli, 26 were boys, and all but two were less than eight days of age. Seven had cultured-documented meningitis, four had enterocolitis, one had an open meningomyelocele, and one had a surgical wound infection. The remaining 17 infants did not have a localized focus of infection. Only one of the infants had apparent transient bacteremia. He was a premature infant cultured because of lethargy and poor feeding. His positive culture was obtained at age 14 days; growth occurred in the broth culture but not on either pour plate. Blood cultures obtained one day prior to and two days after (no antibiotics given during the interval) the positive culture showed no growth. Five of the 30 infants had a second positive blood culture--a total of 35 cultures from the 30 infants. The colony counts of the 35 cultures are summarized in Table I. The bacteremia levels varied greatly, ranging from less than five to more than 1,000 organisms per milliliter. Only one culture positive in broth was negative on both pour plates (0 colonies per milliliter). No sample was positive by pour plate culture and negative by broth culture. The groupings in Table I show that 23% (8/35) of the positive cultures had counts of less than five colonies per milliliter. Two of these eight cultures were obtained on the same day from a patient with meningitis who had received antibiotic therapy (ampicillin and k a n a m y c i n ; o r g a n i s m was " s e n s i t i v e " ) for more than 48 hours. Another culture was taken two days after initiation of antibiotic therapy. None of the other 28 patients had received antibiotics before their blood for culture was obtained. Approximately one third (31%) of all the pour plates had moderately low colony counts of five to 49 per milliliter. Notably, another one third (31%) had colony counts in excess of 1,000 per milliliter. There were five patients who each had two positive cultures. For three of them the two cultures were taken on the same day. The two colony counts for these three infants were, respectively: 2 and 2; ) 1,000 and 24; and 4 and 44. One patient had a count of 16 per milliliter, initially, and a repeat count of ) 1,000 the next day (he had not received antibiotics). The other patient initially had a count of 8 per milliliter, and the repeat count was 2 per milliliter after two days of antibiotic therapy.
Neonatal E. coli septicemia
129
Table I. Colony counts* in newborn infants with E. coli bacteremia and septicemia Colony count range (colonies per ml)
No. of cultures
0-4 5-49 50-199 200-1,000 ) 1,000 Total
8t 11 4 1 11, 35
Per cent
23 31 11 3 31
*There were 35 culturesfrom 30 differentpatients tThree cultures from two patientswere taken after 2 to 3 days of antibiotic therapy. None of the other 28 patients had receivedantibiotics. *These 11 cultureswerefrom 11 differentpatients;6 of them had meningitis; 2 had enterocolitis.Eight of the 11 infantsdied.
Table II. Association of initial colony counts with meningitis and death
Colony count per ml
No. of patients
Percentage q[ patients with meningitis
l~ercentage of patients who died
( 1,000 ) 1,000
t9" 11
5 (1/19) 60 (6/11)
37 (7/19) 73 (8/11)
*Twoof these patients had receivedantibiotics;one of them had meningitis and died.
Fifteen of the patients died; 13 of the deaths occurred within 48 hours after cultures had been obtained, and appropriate antibiotic therapy had been initiated. Six of 11 patients with colony counts of ) 1,000 per milliliter had meningitis (Table II). The patients with colony counts of ) 1,000 per milliliter had a case fatality rate of 73% (8/11), whereas patients with lower bacterial counts had a case fatality rate of 37% (7/19). Colony counts were compared with the birth weights of the infants; there was no clear relationship. Nearly all of the positive broth and pour plate cultures were reported as positive by the laboratory within 24 hours; all were positive within 48 hours. DISCUSSION In this study we have documented concentrations of E. coli in blood in 30 infants. Infections with E. coli were the predominant cause of neonatal septicemia during the study period. E. coli was ideal for this quantitative study as it is not fastidious and grows well in pour plates. Only one of 30 positive broth cultures did not show detectable growth on a pour plate, which indicates that the pour plate quantitation was quite sensitive. We c a n n o t be certain, however, whether cultures using larger volumes of blood such as 5 and 10 ml in both
130
Dietzman, Fischer, and Schoenknecht
broth and pour plate methods might have detected any additional cases ofbacteremia, as such volumes were not obtained. By documenting bacteremla counts we were able to estimate m i n i m u m volumes of blood which would have been required for detection of these cases of E. coil sepsis with the use of broth culture techniques. Although Franciosi and Favara 5reported that a single culture using 1 to 3 ml of blood was adequate to confirm sepsis in each of 28 cases of neonatal sepsis, they did not show whether culturing 1 ml of blood or less was as accurate as culturing 3 ml. Additionally, they did not document the counts of bacteria in these infants. Albers and his associates6reported persistent c o n c e n t r a t i o n s of bacteria in blood ranging from one to 126 organisms per milliliter in three a s y m p t o m a t i c neonates, b u t reported a c o u n t of 20 organisms per milliliter in only one infant with possible septicemia. In our study, eight cultures from seven patients had low counts of less than five organisms per milliliter. Of these eight cultures, three (from two patients) were taken after two to three days of antibiotic therapy, and another was from an infant with transient bacteremia. The other four infants with colony counts of less than five organisms per milliliter had clinical evidence of septicemia, but two of them had significant congenital anomalies (aplastic pituitary; rudimentary thymus and anomalies of fourth arch derivatives) which complicated their clinical evaluation. It could be questioned whether culturing of blood in volumes of less than 1 ml from these patients with low colony counts would have been sensitive enough to ensure detection of their bacteremia. The great majority of cases (77%; 23 of 30) of neonatal E. colibacteremia in this study, however, had colony counts of more than four organisms per milliliter. Culturing of as small amounts of blood as 0.2 to 0.5 ml would have been expected to give positive results in these cases. This estimate is supported by the experimental observations of Fischer and associates. Our study does not consider infections with more fastidious organisms, or infections in infants beyond the neonatal period,
The Journal of Pediatrics July 1974
Bacterial counts greater than L000 organisms per milliliter, such as occurred in one third of these infants, have not been previously documented in neonates. Such high bacterial counts probably reflect poor host defenses and related to the fulminant course of the infection. The observation that 13 of 15 deaths occurred within 48 hours of the time culture was taken further emphasizes the fulminant nature of these infections. Although there was a high mortality rate in the neonates found to have more than 1,000 organisms per milliliter of blood, there were deaths in infants with bacterial counts of less than 50 organisms per milliliter. For this reason, bacterial counts cannot be relied upon to give precise prognostic information in a single situation. We do not propose the general use of quantitative pour plate cultures, and they should never be considered a replacement for more sensitive broth cultures. We wish to thank the microbiology technicians of the University Hospital Clinical Microbiology Laboratory for their technical assistance. We gratefully acknowledge the stimulation, encouragement, and helpful suggestions provided by Drs. C. George Ray, John C. Sherris, David E. Woodrum, Errol Alden, and Alan W. Hodson. REFERENCES
1. Gotoff,S. P., and Behrman, R. E.: Neonatal septicemia, J. PEDIATR.76: 142, 1970. 2. Eitzman, D. V., and Smith, R. T.: The significance of blood cultures in the newborn period, Am. J. Dis. Child. 94: 601, 1957. 3. Rosener, R.: A quantitative evaluation of three blood culture systems, Am. J. Clin. Pathol. 57: 220, 1972. 4. Roome, A. P. C. H., and Tozer, R. A.: Effect of dilution on the growth of bacteria from blood cultures, J. Clin. Pathol. 21: 719, 1968. 5. Franciosi, R. A., and Favara, B, E.: A single blood culture for confirmation of the diagnosis of neonatal septicemia, Am. J. Clin. Pathol. 57: 215, 1972. 6. Albers, W. H., Tyler, C. W., and Boxerbaum, B.: Asymptomatic bacteremia in the newborn infant, J. PEDIATR.69: 193, 1966. 7. Fischer, G. W., Crumrine, M. H., and Jennings, P. B.: Experimental E. coil sepsis in rabbits, J. PEDIATR.85: 117, 1974.