Alterations in Gastrointestinal Microflora During Antimicrobial Therapy for Necrotizing Enterocolitis

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ARTICLES (CONTINUED)

Alterations

in Gastrointestinal

M icroflora

Dun ng

Antim icrobial

Therapy

for Necrotizing

Enterocolitis

Martin J. Bell, M.D., Penelope G. Shackelford, M.D., Ralph D. Feigin, M.D.,

Jessie L. Ternberg, M.D., and Thomas Brotherton, M(ASCP)

IrOSli liii’ i)epartiiu’nt.s of Surgery, and Pediatrie.s. I)itisiou.s of Pediatric Ssrgertj (flU! Infcetioti.s I)i.sease.s.

‘4%’(I.SIli?IgtOPl (Tnirersitij School of .1edieine, and St 11)01.5 (:llil(lrc’li s hospital, St Ioiii.s

ABSTRACT. Changes in the gastrointestinal microflora were noted in 22 infants during coml)ined topical and I)areIteral antimicrobial therapy for necrotizing enterocolitis (NEC). Gastric and fecal cultures obtained during therapy showed significantly decreased Cram-negative aerobic organisms,

Illost of which were Enterobacteriaceae, when compared with pretreatment cultures. Menibers of this bacterial family have been implicated in the pathogenesis of NEC in many reports. The data presented here show that the number of organisms retrieved can be reduced with this method of antinhicrol)ial therapy. Pcdi(ltri(’s 63:425-428, 1979, gust

roin-testiflal inicroflora, necrotizing enterocolitis,

aminoglyco-sides.

The role of the gastrointestinal microorganisms

in the pathophysiology of necrotizing

enterocoli-tis (NEC) remains unclear. One broadly accepted

explanation of the disease mechanism is that

tissue invasion by bacteria occurs following

ischernic damage to the protective mucosal

barrier of the gut.’ This explanation gives a

passive and secondary role to the enteric bacteria

in the disorder.

Accumulating experience with NEC suggests

that the enteric organisms may play a more direct role in the causation of the disease. For example, it has been shown that certain Enterobacteriaceae are found with significantly increased incidence

in the gastrointestinal tract of infants with NEC.2

Furthermore, it has been suggested that topical

aminoglycoside antibiotic therapy for NEC may

reduce the incidence of intestinal perforation.’

Prophylactic inclusion of aminoglycoside

anti-biotic in the feeding of high-risk neonates has

been shown to reduce significantly the incidence

of NEC.’5

The purpose of this study was to delineate the

response of the gastrointestinal microflora in

infants with NEC to treatment with combined

parenteral and topical antibiotics.

MATERIAL AND METHODS

During a 32-month period ending in July 1977,

22 infants who received a specific antimicrobial

regimen for the treatment of NEC were studied.

All patients received clindamycin (30 mg/kg/

day) and gentamicin (5 to 7.5 mg/kg/day)

intra-venously and topical gentamicin delivered by

gastric gavage (10 to 15 mg/kg/day in six aliquots

delivered every four hours).

There were 13 male and nine female infants.

Fourteen infants were black and eight were

white. The mean birth weight was 1,660 gm with

a range of

760

to 3,620 gm. Three infants weighed

more than 2,500 gm. The mean gestational age

was 32 weeks, with a range of 26 to 40 weeks.

Pulmonary disorders were present in 16 infants,

15 of whom had a birth weight of less than 2,000

gm. Septic complications of labor and delivery,

including premature rupture of membranes,

amnionitis, and maternal or infant fever, were

present in ten. Six infants were twins. One child

underwent exchange transfusion, one had

congen-ital heart disease, and one had duodenal atresia.

The mean age at onset of symptoms was 14

days, with a range of 1 to 75 days; onset was not

related to the birth weight or gestational age of

the infant. All infants were originally classified stage 2, although two progressed to stage 3 during

treatment.” Nineteen infants survived, including

Received March 30; revision accepted for publication August 2, 1978.

ADDRESS FOR REPRINTS: (M.J.B.) Division of Pediatric Surgery, St Louis Children’s Hospital, 500 South Kingshigh-way, St Louis, MO 63110.

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14 aerobic

0 anaerobic

120 hr, 15 patients)

f

2 aerobic

1

0 anaerobic

426 ANTIMICROBIAL THERAPY FOR NEC

TABLE I

GASTRIC MICROFLORA

Pretreatment (22 patients)

30 isolates

14 Gram-negative

16 aerobic

16 Gram-positive

1 l

0 anaerobic 0 yeast

First posttreatment samples (48 to 72 hr, 20 patients) 5 isolates

0 Grain-negative

I

aerobic

5 Gram-positive

I

t..0 anaerobic

0 yeast

Second posttreatment samples (96 to

8isolates

2 Gram-negative

I aerobic

5 Gram-positive

I

0 anaerobic 1 yeast

I

7 Klebsieila pneunzoniae

.J

4 Escherichia coli

I

2 Enterobacter

l 1 Citrobacter

9 Staphylococcus epi(lerni

i-dis

3 group D enterococcus

2 Staph aureus

1 gamma streptococcus 1 alpha streptococcus

_.1.

4 Staph epiderinidis I group D enterococcus

,1

1E coli t_1 Acinetobacter

_J_

4 Staph epiderinidis 1 1 Staph aureus

the two who ultimately required surgery. The

three deaths were due to pulmonary disease.

Gastric aspirate and fecal swab samples were

obtained at the time of diagnosis before antibiotic

treatment for NEC was begun and at intervals of

48 to 72 and 96 to 120 hours after treatment was

started. Specimens were processed to identify all

aerobic organisms. Anaerobic organisms were

identified according to the methods described and

used at the Virginia Polytechnic Institute.7 No

quantitative culture studies were performed.

Bacterial species were identified and tabulated,

and the changes in the types of organisms

retrieved before and during treatment were

subjected to statistical analysis using two-by-two

tables for paired observation.

RESULTS

Gastric Microflora

Pretreatment gastric samples were obtained

from 22 infants and yielded 30 isolates. There

were 14 Gram-negative aerobic isolates; all were

members of the Enterobacteriaceae family,

predominately Klebsiella pneumoniae and

Es-cherichia coli. This group consisted of 46% of all

pretreatment gastric isolates. The remaining

organisms (16) were Gram-positive aerobes. No

anaerobes or yeast were grown. After 48 to 72

hours of antibiotic therapy, five isolates were

obtained from 20 patients; none of the isolates

was a Gram-negative aerobe. After 96 to 120

hours of antibiotic therapy, eight isolates were

obtained from 15 patients; two of the isolates

were Gram-negative aerobes (Table I). The

de-crease in the number of patients sampled in the

first and second posttreatment groups is

account-ed for by surgical intervention in two patients,

death of one patient, and termination of therapy

or inadequate collection in four patients.

The reduction in the number of Gram-negative

aerobic organisms recovered from the

pretreat-ment sample to the first posttreatment sample

was significant (P < .001). This reduction was

maintained in the second posttreatment sample.

Gram-positive aerobic organisms were also

signif-icantly reduced (P < .04) during the course of

antibiotic therapy.

Fecal Microflora

Pretreatment fecal samples yielded 63 isolates

from 22 patients. Thirty of these isolates were

aerobic Gram-negative organisms-47% of all

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13 Kiebsiella pneumoniae 10 Escherichia coli

5 Enterobacter 1 Citrobacter 1 Pseudomonas

J

9 Bacteroides

- 1 Veillonella

f

13 Staphylococcus epidermidis 1 7 group D enterococcus

I

1 Clostridium

.‘ 1 Arachniapropionica

1 1 Propionibacteriu;n Pretreatment (22 patients)

63 isolates

30 aerobic

40 Gram-negative

10 anaerobic

I

20 aerobic

23 Gram-positive

#{188} anaerobic

0 yeast

First posttreatment samples (48 to 72 hr, 20 patients) 46 isolates

I

18 aerobic 24 Grain-negative

‘ 6 anaerobic

I

17aerobic

20 Gram-positive ..

1..3 anaerobic

2 yeast

Second posttreatinent samples (96 to 120 hr, 16 patients)

24 isolates

I

7 aerobic

9 Gram-negative

l

2 anaerobic

. . I 10 aerobic

1 1 Gram-positive

l

1 anaerobic

4 yeast

TABLE II

FECAL MICROFLORA

I

13 K pneumoniae

1 6 Bacteroides

I

1 1 Staph epidermidis

1.

6 group D enterococcus

f

2 Clostridiun

1,, 1 Prapionibacterium

I

4Ecoli

2 K pneurnoniae I_ 1 Enterobacter

2 Bacteroides

.1 9 Staph epidermidis

l_

1 group D enterococcus 1 Clostridium

pretreatment fecal isolates. Twenty-nine of these were Enterobacteriaceae, again predominantely

K

pneumoniae and E coli. Ten anaerobic

Gram-negative isolates were obtained, nine of which

were Bacteroides species. Twenty-three

Gram-positive isolates were obtained, of which 20 were

aerobic. There were no yeasts. After 48 to 72

hours of antibiotic therapy, the

Enterobacteria-ceae composed 39% of all isolates, and after 96 to 120 hours, 29% of all isolates (Table II).

The reduction in aerobic Gram-negative

orga-nisms from pretreatment to the first posttreat-ment sample was significant (P < .02). The total

reduction of Gram-negative aerobic organisms

from pretreatment to the second posttreatment

sample was significant at

P

< .003. Also noted

was a significant reduction

(P

< .02) in

Gram-negative anaerobic organisms from pretreatment

to posttreatment samples. Although there were

increases in the number of yeasts retrieved and in the relative numbers of Gram-positive organisms,

these changes were not significant.

DISCUSSION

Therapeutic modification of the

gastrointesti-nal microflora has its broadest application in

surgery of the colon. Survival of ischemic bowel

with the use of topical aminoglycoside in

experi-mental animals has been documented by Cohn’s;

his monograph summarizes much of the literature

about the effect of various antibiotics on the

bowel flora. Other uses for this type of therapy

include the treatment of various types of bacterial

enteritis with orally administered

aminoglyco-sides. ‘#{176}

Pharmacologic studies of gentamicin have

shown that when it is administered orally, only

small amounts are excreted in the urine, which

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428 ANTIMICROBIAL THERAPY FOR NEC

indicates minimal gastrointestinal absorption of

the drug.” Parenterally administered gentamicin

is excreted in the bile in minimal amounts and is

not otherwise excreted by the gastrointestinal

tract.12 A review of the literature has revealed no

study of the effect of parenterally administered

gentamicin on the gastrointestinal flora. From

these pharmacologic studies it seems that the

contribution of parenterally administered

gen-tamicin to the alterations in the gastrointestinal

flora observed in this study probably was

mini-mal. The decrease in the number of

Gram-negative aerobic organisms retrieved probably

was in large part the effect of the topically

administered aminoglycoside.

Parenterally administered clindamycin has, on

the other hand, been noted to affect the

gastroin-testinal microflora, as indicated by the reports of

complications associated with its use.’3 In our

study, the reduction in the gastric Gram-positive

aerobes and the fecal Gram-negative anaerobes

may be attributable to the effects of clindamycin.

We observed no evidence of pseudomembranous

enterocolitis or significant overgrowth of

staphy-lococci or yeasts.

The administration of parenteral antibiotics to

infants with NEC is readily justified on the basis

of the frequent occurrence of coincident

septi-cemia. The combination of clindamycin and

gentamicin offers a wide spectrum of

antimicro-bial activity against the enteric flora. However,

the choice of antibiotics should be dictated by

bacterial sensitivities based on institutional

surveillance. The known efficacy of gentamicin

against the Enterobacteriaceae makes it the

preferred agent for topical therapy for infants

with NEC. Sensitivity studies in our institution

reveal that 100% of the isolates tested in this

family of organisms remain sensitive to

gentami-cm.

Two members of the Enterobacteriaceae

fami-ly, E coli and

K

pneuinoniae, have been closely

associated with the pathogenesis of NEC in a

number of studies.’’ The data presented in our

study establish the fact that the method of

antimi-crobial therapy described for infants with

estab-lished NEC reduces significantly the retrieval

rate of these organisms.

REFERENCES

1. Lloyd J: The etiology of gastrointestinal perforation in the newborn. I Pediatr Surg 4:77, 1969.

2. Bell MJ, Feigin RD, Ternberg JL, Brotherton T: Evalu-ation of gastrointestinal microflora in necrotizing enterocolitis. I Pediatr 92:589, 1978.

3. Bell M, Kosloske A, Benton C, Martin L: Neonatal necrotizing enterocolitis: Prevention of perforation.

I Pediatr Surg 8:601, 1973.

4. Egan E, Mantilla G, Nelson R, Eitzman D: A prospec-tive controlled trial of oral kanamycin in the

prevention of neonatal necrotizing enterocolitis. I

Pediatr 89:467, 1976.

5. Grylack L, Scanlon JW: Prevention of necrotizing enterocolitis with gentamicin. Lancet 2:506, 1977. 6. Bell MJ, Ternberg JL, Feigin RD, et al: Neonatal

necrotizing enterocolitis: Therapeutic decisions based upon clinical staging. Ann Surg 187:1,

1978.

7. Holdeman LV, Moore WEC (eds): Anaerobe Laboratory

Manual. Blacksburg, Va, Anaerobe Laboratory,

Virginia Polytechnic Institute and State University,

1973.

8. Cohn I Jr: Intestinal Antisepsis. Springfield, Ill, Charles C Thomas, 1968, pp 5-11.

9. Escarzaga E: Gentamicin: Laboratory and clinical ex-perience in Mexico. I Infect Ds 124(suppl):443, 1971.

10. Nunnery AW, Riley HD: Gentamicin: Clinical and laboratory studies in infants and children. I Infect Dis 124(suppl):460, 1971.

11. Nunnery AW, Riley HD: Gentamicin: Pharmacologic observations in newborns and infants. I Infect Dis 124(suppl):402, 1971.

12. Riff U, Jackson GG: Pharmacology of gentamicin in man. I Infect Dis 124(suppl):98, 1971.

13. Tedesco FJ, Barton RW, Alpers PH: Clindamycin asso-ciated colitis-a prospective study. Ann Intern Med 81:429, 1974.

14. Frantz ID III, L’Heureux PL, Engel RR, Hunt CE:

Necrotizing enterocolitis. I Pediatr 86:259, 1975. 15. Hill HR, Hunt CE, Matsen JE: Nosocomial colonization

with Klebsiella, type 26 in a neonatal intensive care

unit, associated with an outbreak of sepsis, menin-gitis, and necrotizing enterocolitis. I Pediatr 85:415, 1974.

16. Speer ME, Taber LH, Yow MD, et al: Fulminant neonatal sepsis and necrotizing enterocolitis asso-ciated with a non-enteropathogenic strain of

Escherichia coli. I Pediutr 89:91, 1976.

17. Stanley MD, Nuss DM Jr, DeLeinos RA: Relationship between intestinal colonization with specific bacte-na and the development of NEC, abstracted. Pedi-atr Res 11:543, 1977.

18. Yeager AS, McNabb MB, Sullivan DW, et al: Cluster of cases of NEC associated with E. coli 085,

abstracted. Pediatr Res 1 1:545, 1977.

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1979;63;425

Pediatrics

Brotherton

Martin J. Bell, Penelope G. Shackelford, Ralph D. Feigin, Jessie L. Ternberg and Thomas

Necrotizing Enterocolitis