Incidence of antibiotic resistance and R factors among gram-negative bacteria isolated from the neonatal intestine

Incidence of antibiotic resistance and R factors among gram-negative bacteria isolated from the neonatal intestine

198 February, 1972 T h e ]ournal o[ P E D I A T R I C S Incidence of antibiotic resistance and R factors among gram-negative bacteria isolated from ...

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February, 1972 T h e ]ournal o[ P E D I A T R I C S

Incidence of antibiotic resistance and R factors among gram-negative bacteria isolated from the neonatal intestine Stool specimens were collected ]tom 119 infants in the well-baby nursery and 14 inJants in the high-risk nursery o[ University Hospital and examined [or the presence of antibiotic-resistant enteric bacteria. A total of I02 resistant strains were isolated from the two groups and examined for the presence of R factors. 0[ these organisms, 38 per cent were capable o[ transferring all or part of their antibiotic resistance to an antibiotic-sensitive, F-, derivative of Escherlchia coli K-12. The data demonstrated that a significant number of the strains were resistant to arapiciIlin, streptomycin, or tetracycline and, to a lesser extent, cephalothin or kanamycin.

Kay M. Dailey, M.S., Alton B. Sturtevant, Jr., Ph.D., and Thomas W. Feary, Ph.D.,*

B i r m i n g h a m , Ala.

D w a I N o the past 10 years, gram-negative enterobacteria have replaced S t a p h y l o c o c c u s aureus as the most frequent cause of serious infection in man, in both adults 1 and neonates. ~ T o further complicate this situation, several investigators, including D a t t a 3 in England and Medeiros and O'Brien ~ and Gunter and Feary ~ in the United States, have demonstrated that the majority of these enterobacterial isolates are multiply antibiotic resistant. Furthermore, it has been suggested that at least 50 per cent 3 of these From the Department of Microbiology, University of Alabama Medical Center. ~RelOrlnt address: De!~artment o~ M~croMology, University of Alabama in Birmingham, Birmingham, Ala. 35233.

VoI. 80, No. 2, pp. 198-203

clinically isolated bacteria are resistafit as a result of R factors. R factors are extrachromosomal genetic elements that not only render the organism resistant to certain antibiotics, but confer upon it the ability to transfer a copy of the resistance determinant (R factor) to a completely antibiotic-sensitive recipient organism. T h e principal feature distinguishing .infectious drug resistance (resistance mediated by R factors) from other forms of drug resistance, then, is that it can be transferred from an organism of the same or different species by cell to cell contact (conjugation). I n this manner, a completely drug-sensitive organism may acquire resistance to as many as

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eight different antimicrobial agents as a result of a single encounter with a multiply resistant organism harboring an R factor. In addition to becoming drug resistant, the recipient cell is converted into a competent donor of the acquired R factor. It is now known that R factors are readily exchanged between multiply drug-resistant and drugsensitive members of the Enterobacteriaceae, as well as other microbial pathogens such as Vibrio, Serratia marcescens, Pasteurella pestis, and Pseudomonas aeruginosa. 6 Antibiotic-resistant 7 and R factor-containingS, 8, 9 gram-negative organisms have been recovered from children in the past, but no one has studied newborn infants in an attempt to determine what levels of antibioticresistant enteric bacteria are attained during the first week of life and what proportion of these bacteria are resistant as a result of R factors. The purpose of this study was to assess the incidence of antibiotic resistance and R factors among enteric bacteria isolated from newborn infants prior to any known exposure to antibiotics. For comparative purposes, enteric isolates were also obtained from neonates receiving antibiotic therapy. MATERIALS AND METHODS

Fecal specimens. Fecal specimens were obtained from 119 newborn infants (less than five days of age) from the well-baby nursery and 14 from the high-risk nursery of the University Hospital, Birmingham, Alabama. Only babies in the high-risk nursery were receiving antibiotic therapy during the time of study. Isolation of resistant bacteria. A portion of stool material was suspended in T M buffer (1.21 Gm. of tris [hydroxymethyl] aminomethane, 8.75 Gin. of NaC1, and 2.47 Gin. of MgSO4 9 7H20, per liter of distilled water and adjusted to p H 7.t with hydrochloric acid). A portion was then spread over the entire surface of a MacConkey agar (Baltimore Biological Laboratories) plate with a sterile cotton-tipped swab. Sensidiscs were then dispensed onto the surface of the seeded plate. The following antibiotic discs

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were used : ampicillin, 10b~g; chloramphenicol, 5/,g; tetracycline, 5/,g; cephalothin, 30/xg; kanamycin, 30/,g; nalidixic acid, 5/,g; dihydrostreptomycin, 10/xg; and gentamicin, 10b~g. After incubation at 37 ~ C. for 18 to 24 hours, a portion of resistant growth was picked, suspended in T M buffer, and streaked onto a fresh MacConkey agar plate. After overnight incubation, a single wellisolated colony was picked and used to inoculate a Kligler iron agar (Bahimore Biological Laboratories) slant which served as stock after incubation. Each isolate was subsequently identified by the methods outlined by Edwards and Ewing. 1~ Antibiotic-susceptibility testing. Drug-resistance patterns of all isolates were determined by spreading a portion of a 3 to 4 hour broth culture of the organism to be tested onto brain-heart infusion agar (Difco). Sensidiscs of the same type and concentration as described above were dispensed onto the surface of the seeded plates, and the resistance pattern was determined after incubation at 37 ~ C. for 18 to 24 hours. Identification of R factors. Enteric isolates found to be resistant to one or more antibiotics were considered potential donors of R factors to either Escherichia coli WI-A25 or E. coli WI-A2 (lae)71 Both of these strains are F derivatives of E. coli K-12 which act as recipients of R factors from antibioticresistant donors in conjugation experiments) When the potential donor was lactose positive, WI-A2 (lag) was used; when lactose negative, the lactose-fermenting, WI-A2, recipient was employed. Both recipient strains are resistant to 250/*g per milliliter of sodium azide but are completely susceptible to all antibiotics employed in this study. Mating procedures were carried out by mixing 0.1 and 0.2 ml., respectively, of overnight broth cultures of the prospective donor and recipient in 2 ml. of sterile Brain Heart Infusion Broth (Difco). The mixtures were incubated for 18 to 24 hours as a stationary culture. A swabful of each mixtm'e was smeared onto MacConkey agar plates containing a single appropriate antibiotic and 250/,g of sodium azide per milliliter. In this

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Dailey, Sturtevant, and Feary

m a n n e r , 12 mixtures could be placed on each plate. T h e m e d i a used were selective for antibiotic-resistant recombinants of either W I - A 2 (la~) or W I - A 2 (lae) in t h a t growth of the prospective d o n o r was prevented by sodium azide a n d growth of the recipient was p r e v e n t e d by a n antibiotic. After incubation at 37 ~ C. for 48 hours, recombinants were picked a n d streaked to the same selective m e d i u m to ensure pure colony isolation. T h e antibiotic-resistance p a t t e r n of at least one r e c o m b i n a n t colony from each m a t i n g m i x t u r e was then determ i n e d as described above to ascertain w h e t h e r p a r t i a l o r complete transfer of resistance from d o n o r to recipient h a d taken place. Antibiotics. A p p r o p r i a t e concentrations of each antibiotic ~ used in selective m e d i a were p r e p a r e d in sterile distilled water, a n d stock solutions were m a i n t a i n e d at MO ~ C. RESULTS

A total of 82 resistant enteric strains were isolated from stool specimens o b t a i n e d f r o m 119 infants in the well-baby nursery a n d 20 strains from stool specimens from 14 neonates in the high-risk nursery. At least 38 p e r cent of the isolates were resistant as a result of R factors as d e m o n s t r a t e d by transfer of resistance to a totally susceptible recipient strain of E. coli. W e l l - b a b y nursery. F u r t h e r characterization of the 82 enteric b a c t e r i a recovered f r o m the well-baby nursery revealed t h a t 41 were E. coil, 16 were m e m b e r s of the K l e b siella-Enterobacter group, 9 were Serratia strains, a n d 16 belonged to the Proteus group. T h e drug-resistance p a t t e r n of each isolate was d e t e r m i n e d for seven different antibiotics. T h e drug-resistance patterns as well as the incidence of R factors a m o n g these isolates are shown in T a b l e I. A m o n g the E. coIi isolates, the resistance patterns D s - T e (17 p e r cent) a n d A m - D s - T e (24.4 p e r cent) accounted for a large portion of e'Chloramphenlcol was provided by Parke, Davis & Co.; ampicillin (Penbritin) was supplied by Ayerst Laboratories; and gentamlcin (Garamycin) was provided by Seherlng Corp.

The Journal of Pediatrics February 1972

T a b l e I. I n c i d e n c e a n d t r a n s f e r a b i l i t y of resistance p a t t e r n s a m o n g enteric isolates recovered from the w e l l - b a b y nursery TTans-

Resistance pattern


[erred resistante

Am Ds Na Cf Gm Ds, Te Am, Ds Am, Te Am, C, K Am, Ds, Te Am, Cf, Na Am, Ds, Na Am, C, Ds, Te Am, Ds, Na, Te Am, C, Cf, Ds, Na, Te Am, C, Cf, Ds, Gin, Te

I 4 1 1 1 7 3 4 1 10 1 1 3 t I 1

0 1 0 0 0 4 (3) r ( 1) (4) (I) (9) 0 0 1 0 0 0



6 (18)

Isolate E. coli


Am Cf Am, Am, Am, Am, Am, Am, Am,

C Cf Ds Gm K (3, Na Cf, Te

Total Serratia Cf Ds Am, Cf Ds, Cf Am, Cf, Ds Total Proteus t Am Te Am, Cf C, Te Am, C, Ds Am, Cf, Na Am, Cf, Te Am, Ds, Te C, Na, Te Total

6 i 1 3 1 1 1 I 1

0 0 0 (1) ( 1) 0 0 0 0







2 3 1

0 0 0



3 4 1 2 1 1 1 1 2

2 0 0


(1) 0 0 0 (I ) (1) 2 (3)

Abbreviations: Am = ampicillin, C = chloramphenicol, Cf = cephalothin, Ds = dihydrostreptomycin, Gin = gentainicin, K ~- kanamycin, Na = nalidixic acid, Te = tetracycline. *Nuinbers in parentheses indicate that only a portion of the resistance pattern was transferred. tIncluded P. mirab~lls,s P. rettgeri, 6 and P. vulgaris.~

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the patterns identified. Of the 41 E. coli strains examined, 76, 66, and 66 per cent, respectively, were resistant to streptomycin, ampicillin, or tetracycline. Sixteen KlebsiellaEnterobacter strains were studied, and it was demonstrated that the majority (94 per cent) was resistant to ampicillin. Of the 16 Proteus isolates employed in this study, 50 per cent were resistant to ampicillin. Although only 9 Serratia strains were examined, 90 per cent were found to be resistant to cephalothin. It should be noted that two E. coil and one Klebsiella-Enterobacter were shown to be resistant to gentamicin. W h e n each of the 82 isolates was examined for resistance transfer, it was found that 58,6, 12.5, 31.2, and 0 per cent of the E. coli, KlebsiellaEnterobacter, Proteus, and Serratia strains, respectively, contained demonstrable R factors. High-risk nursery. T w e n t y enteric isolates were recovered from 14 infants in the highrisk nursery. All 14 of the patients were receiving some type of antibiotic therapy at the time the specimens were obtained. U p o n further identification, 11 were shown to be E. coli, 4 Enterobacter, and 5 Proteus strains. The incidence and transferability of resistance patterns a m o n g this group is presented in Table I I . I n contrast to 46 per cent of the E. coli recovered from well infants being resistant to three or more antibiotics, 91 per cent of the E. coli recovered from this group exhibited multiple resistance. A m o n g the E. coli isolates 91, 91, 73, and 45 per cent, respectively, were resistent to ampicillin, tetracycline, streptomycin, or kanamycin. A m o n g t h e 20 isolates from this group, 54.5, 25, and 0 per cent of the E. coli, Enterobacter, and Proteus strains, respectively, harbored R factors. R factors identified. O f the 102 isolates studied, 39 transferred their resistance either totally or partially. Table I I I shows the 24 different R factors identified in this study. Of the 39 strains bearing R factors, 56, 54, and 36 per cent were resistant to streptomycin, tetracycline, or ampicillin, respectively. Three of the R factors conferred resistance to gentamicin. N o particular resistance pat-

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Table II. Incidence and transferability of resistance patterns among enteric isolates recovered from the high-risk nursery

Resistance pattern

Isolate E. coli

Ds, Te Am, Cf, Te Am, Ds, K Am, Ds, Te Am, C, Cf, Te Am, Ds, K, Te Am, Ds, Na, Te Am, C, Cf, Gm, Te Total


Proteus ~


1 1 1 1 1 4 1 1 I1

Am Cf Te Am, Gm

1 1 1 1



Am Am, Cf C, Te

1 1 3

Trans[erred resistanee

1 0 0 ( 1) 0 (4) 0 0 I (5) 0 0 0 ( 1) (1) 0 0 0

Total 5 For explanation of abbreviations see TaMe I. ~Induded P. mirabills,a P. rettgeri,~ and P. vutgaris3 tern seemed to predominate a m o n g the R factors identified. DISCUSSION Antibiotic-resistant enteric organisms were isolated from both "well" and "high-risk" neonates. A m o n g a total of 102 strains isolated, 39 or 38 per cent harbored R factors, whereas 57 per cent of the 52 E. coli isolates contained R factors. The data presented here are cgmparable to data obtained by 1V[oorhouse9 who found that 84 per cent of the antibiotlc-resistant enteric isolates recovered from infants under two years of age harbored R factors. I n a study involving children of all ages, Gunter and Feary ~ were able to demonstrate that 60 per cent of their resistant E. coli contained R factors. These data are also comparable to those obtained by Guinee and associates 8 who found that from 56 to 63 per cent of the resistant E. coti isolated from infants under six months old harbored R factors. I t is quite interesting to note that


Dailey, Sturtevant, and Feary

The Journal o[ Pediatrics February 1972

T a b l e l I I . S u m m a r y of R factors ~ Resistance pattern Am Cf Ds K Na Te Am, Ds Am, Te Ds, Te Gm, Na Na, Te Am, Cf, Ds Am, Cf, Te

Resistance No. pattern 2 Am, Ds, Gm 1 Cf, Na, Te 2 Ds, Gin, Na 1 Ds, K, Na 1 Ds, Na, Te 2 Am, Gin, K, Te 3 C, Cf, Ds, Te 3 Cf, Ds, K, Te 5 Cf, K, Na, Te I Am, C, Ds, Na, Te 1 Cf, Ds, K, Na, Te 2 1 Total

No. 1 1 1 3 2 1 1 1 1 1 1 39

For explanation of abbreviations see Table I. * F r o m all 102 isolates studied.

infants u n d e r five days of age, who have never left the hospital, excrete drug-resistant enteric b a c t e r i a with the same p r o p o r t i o n of R factors as d o infants t h a t have left the hospital environment, 5, s, ~ a n d indeed the same as adults, s, s, s, 11 T h e influence of antibiotic t h e r a p y on the incidence of m u l t i p l y resistant E. coli was confirmed with the isolates recovered from the high-risk nursery, where it was f o u n d t h a t 91 p e r cent of the E. coli were resistant to three or more antibiotics as opposed to 46 per cent from the well-baby nursery. S t u r t e v a n t a n d associates la f o u n d t h a t antibiotic t r e a t m e n t caused an increase in the incidence of m u l t i p l y resistant strains as well as an increase in the incidence of R factors. N o evidence of increase in the incidence of R factors was found in the present study (only 20 strains from antibiotic-treated infants were s t u d i e d ) . A survey by F a r m e d z of the bacterial flora of infants in a "high-risk" unit, where a b o u t 50 per cent of the infants were receiving antibiotics, showed a high colonization rate of the u p p e r respiratory tract with antibiotic-resistant, g r a m - n e g a t i v e bacteria, I n contrast, the colonization rate was low in the " r e g u l a r " nursery where antibiotics were rarely given. A n o t h e r study conducted in the same w a r d by S h a l l a r d a n d Williams is i n d i c a t e d a r a p i d rise in resistant enteric organisms d u r i n g antibiotic t h e r a p y

a n d a slow return to sensitive flora after antibiotic t h e r a p y h a d ended. M c C r a c k e n 7 recently r e p o r t e d on a startling increase in the incidence of kanamycin-resistant E. coli isolated from neonates. Results o b t a i n e d by S t u r t e v a n t a n d associates 14 confirm these results in adults. T h e d a t a presented in the present r e p o r t indicate that a large p r o p o r t i o n of the resistant enteric bacteria is resistant to a m p i cillin, streptomycin, a n d tetracycline. This situation could pose a p r o b l e m to the clinician in t h a t these ordinarily useful antibiotics would be ineffective in t r e a t m e n t of an infection due to a resistant enteric organism from the neonate's intestinal flora. Also of interest was the isolation of gentamicinresistant organisms that contained R factors which conferred the resistance, since gentamicin has only been available for widespread use at University H o s p i t a l for little more t h a n a year. T h e conclusions to be d r a w n from this investigation are t h a t antibiotic-resistant enteric organisms m a y be recovered from the feces of neonates who are less t h a n five days of age. A t least 38 p e r cent are resistant as a result of R factors. A large p r o p o r t i o n of the isolates are resistant to arnpicillin, streptomycin, or tetracycline. We thank Dr. George Cassady, Department of Pediatrics, and his staff for their cooperation in obtaining stool specimens. REFERENCES

1. Turck, M.: The problem of infections due to gram-negative organisms, Antlmlcrob. Agents Chemother. 1966, 1967, p. 265. 2. McCracken, G. H., and Shinefield, H. R.: Changes in the pattern of neonatal septicemia and meningitis, Am. J. Dis. Child. 112: 33, 1966. 3. Datta, N.: Drug resistance and R factors in the bowel bacteria of London patients before and after admission to hospital, Br. Med, J. 2: 407, 1969. 4. Medeiros, A. A., and O'Brien, T. F.: Contribution of R factors to the antibiotic resistance of hospital isolates of Serratia, Antimlcrob. Agents Chemother. 1968, 1969, p. 30. 5. Gunter, A. C., and Feary, T. W.: Infectious drug resistance among clinically isolated Escherichia coli, J. Bacteriol. 96: 1556, 1968. 6. Watanabe, T.: Infective heredity of multiple

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9. 10. 11.

drug resistance in bacteria, Bacteriol. Rev. 27: 87, 1963. McCracken, G. H.: Changing pattern of the antimicrobial susceptibilities of Escherichia coli in neonatal infections, J. P~mATR. 78: 942, 1971. Guinee, P., Uguet0, N., and van Leeuwen, N.: Escherichia eoli with resistance factors in vegetarians, babies, and nonvegetarians, Appl. Microbiol. 20: 531, 1970. Moorhouse, E. C.: Transferable drug resistance in enterobaeteria isolated from urban infants, Br. Med. J. 2: 405, 1969. Edwards, P. R., and Ewing, W. H.: Identification of Enterobacteraceae, Minneapolis, i962, Burgess Publishing Co. Sturtevant, A. B., Cassell, G. H., Bobo, R. A.,

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and Feary, T. W.: Effect of antibiotic treatment on the incidence of infectious drug resistance among intestinal lactose-fermenting bacteria isolated from burn patients, Infect. Immun. 3: 411, 1971. 12. Farmer, K.: The influence of the hospital environment and antibiotics on the bacterial flora of the upper respiratory tract of the newborn, N. Zeal. Med. J. 67: 541, 1968. 13. Shallard, M., and Williams, A.: Studies on gram-negative bacilli in a ward for newborn babies, Med. J. Aust. 2: 455, 1966. 14. Sturtevant, A. B., Bobo, R. A., and Feary, T. W.: Antibiotic resistance among clinically isolated Escheriehia eoli and KlebsiellaEnterobacter. To be published.