Journal of Applied Bacteriologv 1976, 41, 453-464

The Effect of Tetracycline on the Coliform Gut Flora of Broiler Chickens with Special Reference to

Antibiotic Resistance and 0-Serotypes of Escherichia coli

KATHERINE HOWE, A. H. LINTON

Department of Bacteriology, Medical School, University of Bristol, Bristol BS8 1 TD, England

AND

A. D. OSBORNE

Department of Veterinary Medicine, Langford, Bristol BS 18 7DU, England

Received 15 June 19 76 and accepted 9 August 1976

The effect of terramycin, administered prophylactically in drinking water, on the gut flora of broiler birds was investigated. Exposure to the antibiotic for only 24 h profoundly affected the counts of tetracycline-resistant strains and selected 0-serotypes carrying resistance determi- nants. Large numbers of Escherichia coli resistant to sulphonamides were found in treated and control birds and this is discussed in relation to the use of sulphaquinoxaline as a coccidiostat. Evidence of carcass contamination by antibiotic resistant E. coli found in the gut is presented.

INVESTIGATIONS to identify the potential reservoirs of antibiotic resistant Escherichia coli in food-animals for man showed that large numbers of resistant E . coli were regularly found in chickens (Linton, in press). This observation led us to test a large number of birds which were killed daily in a busy factory. Most of the E. coli cultured were resistant to sulphonamide and a significant proportion of strains resistant to tetracycline were also isolated from birds which had received terramycin prophylac- tically in their drinking water on two occasions, each of 24 h duration (unpublished data).

Because of the potential public health implications, we decided to investigate this ob- servation. It was not possible to monitor individual birds throughout their life in com- mercial units where often as many as 18000 birds run together. An attempt was made, therefore, to rear birds in the Veterinary School at Langford under conditions similar to those found commercially.

The effect of brief exposure to tetracycline on the coliform organisms in the gut flora was determined. The birds were reared to 63 days and droppings sampled regularly. All isolates of E. coli were 0-serotyped and the resistance patterns determined. Batches of food were also examined for antibiotic-resistant E. coli. From this study it was possible to observe which of the 0-serotypes found were good colonizers of the chicken gut and

14531

454 K. HOWE, A. H. LINTON AND A. D. OSBORNE

to analyse the various R determinants and their distribution within the various 0- serotypes. It was found that tetracycline resistance was strongly associated with a par- ticular 0-serotype in these birds. The implications of the use of tetracycline as a prophylactic are critically appraised. The high proportion of sulphonamide resistance and its relation to the use of sulphaquinoxaline as a coccidiostat in the feed is discussed.

Materials and Methods

Chickens

Every attempt was made to raise birds experimentally under conditions resembling those used commercially. Twelve newly hatched chicks (not more than 24 h) of a Ross breed were coilected from a batch being used to restock a commercial rearing house. The birds were transported on the same day to Langford in a cardboard box as used for delivery from the hatchery to the rearing farm. At Langford the birds were divided into two groups of six and housed in separate buildings for the duration of the experiment. Throughout the first 28 days of life each group was kept in a box under an infrared lamp. To facilitate sampling of single birds. each bird was caged separately from 28 days until the time of slaughter. Birds and feed were obtained from commercial sources and the ‘treated’ group was given terramycin on the same two days of life as was the commercial practice. Both groups of six birds were managed similarly although housed separately. All birds were slaughtered, along with others, on a normal slaughter line at a commercial factory.

Diet

Three commercially compounded foods, obtained from the rearing farm, were fed ad lib. to the birds. For the first 15 days ‘starter crumbs’ were fed, from day 16-36 ‘grower

TARLE 1 Additires to chicken fees’

Additives ~ ~- ~.

Vilamiris A :,I i.u./kg

GrowIh promorers Zinc bacitracin Payzone (R) (Nitrovin)? 1 3-5 dinitro-rrtoluamide Pancoxin (R) comprising::

parts/ lo6

Coccidiostats

parts/ 1 O6 I Amprolium Ethopabate Sulphaquinoxaline

Starter crumbs fed from

days 1-15 ~~

8000 1600

15

10 -

I25

Grower pellets fed from

days 1&36

8000 1600

15

10 10

100 5

60

Finisher pellets fed from

days 31-63

6000 1200

10

10 __

100 5

60

* Compounded by BOCM. Silcock. + Cyanamid.

Merck Sharp & Dohme.

E. COLI SEROTYPES IN CHICKEN ON TERRAMYCIN 455

pellets’ and from day 37-63 ‘finisher pellets’. The various additives incorporated in these feeds are listed in Table 1. Apart from the growth promoters, permitted by legisla- tion based on the Swam Report (Anon. 1969), it was noted that 60 parts/106 sulpha- quinoxaline was added as a coccidiostat to the second and third feeds.

Water was available ad lib. Following the practice adopted by some commercial systems terramycin was added to the drinking water (6 g/10 galls/lOOO chickdday) of one group of birds for 24 h on days 1 and 35 after arrival.

Sampling

Fresh droppings from the birds were collected for bacteriological examination. Eleven samples of pooled droppings were collected during the first 28 days from each group while the birds were running together. A further 11 samples from each of the 12 separately caged birds were collected during the period 28-63 days. At 63 days the birds were transported to the factory for slaughter under commercial conditions. After slaughtering and at the time of evisceration rectal contents from 10 of the 12 birds were collected. Under the pressure of commercial conditions (3000 birddh) it was not possi- ble to plan the order in which the birds were slaughtered and the rectal contents could not be recovered from all of the 12 birds. After washing, the abdominal cavities of the 12 birds were swabbed with a large alginate swab (Linton et al. 1974). This made possi- ble an assessment of carcass contamination by the birds’ own gut flora, based on a fairly comprehensive knowledge of 0-serotypes of E. coli demonstrated in the gut flora in life, at slaughter and from the factory environment.

Bacteriology

Droppings were collected in sterile pots. Decimal dilutions were prepared in saline (0.85%) from weighted amounts. Presumptive coliform counts were determined by the method previously described (Linton et al. 1975) on Bile Lactose Agar without salt (BLA-Oxoid Cm7b) and the same medium incorporating 25 pglml tetracycline (BLA + T). From each sample ten colonies typical of E. coli were picked from the BLA plates and two colonies from the BLA + T plates. Each isolate was confirmed as E. coli by an Eijkmann test and an indole test at 44 OC. Subsequently all confirmed isolates were 0- serotyped by a microtitre technique and their antibiotic resistance patterns determined (Howe & Linton 1976). The abdominal swabs were plated directly onto BLA and ten colonies typical of E. coli picked and identified in the same way.

Samples (20-30 g) of each of the three foods fed to the birds were added to 100 ml of single strength nutrient broth (Howells BE Joynson 1975). The suspension was plated onto BLA immediately and again after incubation at 37OC for 18-24 h. Colonies typical of E. coli were confvmed and their properties determined as above.

Results

Presumptive coliform counts

Counts were done on 22 occasions throughout their 63 days of life. The results for each group of birds are presented separately in Fig. 1. In this Figure the duration of feeding

45 6 K. HOWE, A. H. LINTON A N D A. D. OSBORNE

each of the three diets, the day on which the birds were caged separately and the two days on which terramycin was added to the drinking water of the 'treated' group, are indicated.

Caged together -Caged separately 4

Starter -Grower pellets+- Finisher p e l l e t s - t l c rumbs

\ -, I --.i

z

\, , , , \ I , , n !

I I I I I I I 1

70 4 1 0 10 20 30 40 50 60

Days of l i fe

Fig. I . Presumptive coliform counts (log,,/g) from droppings, on non-selective (BLA) and selec- tive (BLA + T) media, 22 sampling occasions. 0, BLA; 0, BLA + T. (a) Terramycin treated group: (b) control group.

Properties of Escherichia cob

Seven hundred and sixty-three isolates of E. coli were obtained from the tetracycline- treated group and 732 from the control group. The proportions of, the strains in each group resistant to at least one antibacterial agent were very high, 87.8% and 76.9% in the treated and untreated groups respectively. Of the eight antibacterial agents tested (sulphonamide, tetracycline, streptomycin, ampicillin, chloramphenicol, nitro furantoin, colistin sulphate and nalidixic acid) resistance was found to the first three only. The proportions of E. coli isolates from each group of birds resistant to these three agents are presented in Table 2. A similar high proportion of E. coli (over 90%) were found to be resistant to sulphonamide in both groups but only the terramycin-treated group showed a high proportion of strains resistant to this antibiotic. The incidence of each of the three R determinants among all the resistant E. coli from each group of birds and the percentage of strains containing one, two or three R determinants in different com-

E. COLI SEROTYPES IN CHICKEN ON TERRAMYCIN 457

E U a t

TABLE 2 Analysis of the antibiotic resistance properties of E. coli isolates from

each group of birds

I I I I I I

( b )

Tetracycline- treated group Control group

No. of isolates No. resistant to one or more agents % resistant to one or more agents % of isolates containing

R determinants for Sulphonamide (Su) Tetracycline (T) Streptomycin ( S ) % of isolates containing

each combination of R determinants

su sus SuT SuST

763 670

87.8

96.8 49.5

5 . 1

45.8 3.9

48.7 1 .5

732 563

76.9

92.5 9.5

22.3

68.4 20.1 9 . 4 1 . 1

binations are included in Table 2. The incidence of each R determinant on the various days of sampling is shown in Fig. 2.

As all 1498 isolates of E. coli were 0-serotyped, it was possible to relate the days on which each 0-serotype was isolated to the days on which the birds were receiving

1 1 I I 1 I 1 0 10 20 30 40 50 60

Days of l ife

Fig. 2. Percentage of each R determinant in antibiotic resistant E. coli isolated on each sampling occasion from all birds in the terramycin-treated and control groups. 0, Sulphonamide resistant; A , Tetracycline resistant; W, Streptomycin resistant. (a) Terramycin treated group; (b) control group.

45 8 K. HOWE, A. H. LINTON AND A. D. OSBORNE

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E. COLI SEROTYPES IN CHICKEN ON TERRAMYCIN 45 9

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460 K. HOWE, A. H. LINTON AND A. D. OSBORNE

TABLE 4 The diversity of numbers of diflerent 0-serotypes of antibiotic resistant E. coli isolated

from single bird faecal droppings

No. of 0-serotypes per faecal dropping

0 1 2 3 4 5

Tetracycline- No. of samples 2 16 18 20 8 2

Control birds No. of samples 3 18 21 13 9 2

A r \

__ .~ ~~

treated birds % 3.0 24.2 27.3 30.3 12.1 3 .0

YO 4.5 27.3 31.8 19.7 13.6 3.0

various additives in their diet or drinking water. These findings are presented in Table 3. All the E. coli strains referred to in this Table were isolated on antibiotic-free medium and therefore indicate the dominant E. coli serotypes in the gut flora. Strains both sen- sitive and resistant to antibiotics are included. No further 0-serotypes were isolated on BLA + T, where a selection pressure for tetracycline resistant E. coli was exerted. The first 1 1 samples consisted of pooled droppings from each group of six birds and the oc- currence of each 0-serotype from each sample is indicated by a single entry. Samples 12-22 were from individual birds and in Table 3 individual 0-serotypes from the same sample are recorded once only even if more than one colony of the ten picked at ran- dom were of the same 0-serotype. This way of presenting the information indicates the incidence of each 0-serotype in the two groups of birds through life. The number of 0- serotypes of E. coli isolated from individual droppings from single birds varied from one to five (Table 4).

The important question of relationship between antibiotic sensitivity or resistance, and 0-serotype was next considered. To ascertain this the R determinants of all anti-

TABLE 5 The distribution of antibiotic sensitive and resistant E. coli and their R determinants,

among the dominant 0-serotypes found in one or both groups of birds

Tetracycline-treated birds Control birds 3

No. of colonies No. of colonies containing -&

0-serotype sens. resist TR SuR SR

8 15 45 3 45 0 9 0 303 300 303 7

15 13 0 0 0 0 18 0 4 0 4 0 44 0 111 1 111 1 65 6 34 0 34 0 70 0 59 0 59 0 78 0 0 0 0 0 83 54 25 3 23 20 96 0 40 4 40 1

103 0 0 0 0 0 114 0 13 13 13 13

-~

r

No. of colonies - sens. resist

0 0 1 15 7 2

45 3 0 151 0 0 0 68

84 1 24 32 0 65 1 92 0 43

~

\

No. of colonies containing -

TR SuR SR

0 1 0 11 15 3 0 2 0 0 3 0 1 151 2 0 0 0 0 68 0 0 84 0 2 32 29 0 65 0 0 92 63

41 43 1

TR, SuR. SR-R determinants against tetracycline, sulphonamide and streptomycin respectively.

E. COLI SEROTYPES IN CHICKEN ON TERRAMYCIN 461

biotic resistant E. coli isolated on antibiotic-free medium (BLA) were tested for their 0-serotypes. The results, for 0-serotypes which were found in at least 5% of specimens from each group of birds, are presented in Table 5.

Escherichia coli contamination of the abdominal cavity afrer evisceration

Contamination of the abdominal cavity by E. coli had been observed frequently in birds slaughtered commercially. The relationship between the gut flora and the rectal contents at slaughter of the 12 birds in this study, and contamination of the abdominal cavity following slaughter and evisceration at the factory was found by comparing 0- serotypes found in droppings throughout life with those isolated from abdominal swabs taken immediately after evisceration. The figures for both groups of birds are presented in Table 3.

Escherichia coli isolated from the various feeds

Nine different E. coli 0-serotypes were isolated from the three feeds (Table 6). Representative strains chosen at random, of each antibiotic resistant 0-serotype, were shown to transfer their drug resistance to a recipient E. coli by a standard test (Linton et al. 1975). It is possible, therefore, that transfer of R-plasmids could have occurred in the chicken gut.

TABLE 6 E. coli 0-serotypes isolated from the various feeds

Resistant Sensitive

Starter crumbs

Grower pellets

Finisher pellets

08 Su 09 TSu 012 su,ssu 096 Su 09 Tsu

083 SSu

015 012

015 083 0128 N.T.

09 018 093 N.T.

Summarizing the findings in both groups of birds, 28 0-serotypes were isolated from at least one specimen throughout the life-span of the 12 birds (Table 3).

Discussion

Although birds in this study were kept under experimental conditions to make possible regular sampling and identification of specimens every effort was made to simulate the conditions that operate commercially.

The presumptive coliform counts from both groups of birds revealed that by the second day of life the number of coli-aerogenes organisms established reached 109/g of

462 K. HOWE, A. H. LINTON AND A. D. OSBORNE

droppings. Counts of this order persisted throughout most of their life. In the control group the difference between the tetracycline-resistant and the total presumptive coliforms counts fluctuated but generally the tetracycline-resistant counts were of the order of 10- to 100-fold lower than the total counts. The rapid colonization by antibiotic-resistant coli-aerogenes bacteria indicate that these organisms were present in the food or the environment. In the terramycin treated group the initial 24 h response to terramycin in the drinking water increased the proportion of tetracycline-resistant strains over and above that in the control group. In the treated group, the tetracycline- resistant presumptive coliform counts were virtually the same as the total counts over the first 40 days of life. There was a limited decline in numbers of coli-aerogenes organisms during the final 20 days of life subsequent to a second stimulation of tetracycline-resistant strains by terramycin given on day 35.

A marked drop in the presumptive coliform counts in the terramycin-treated group occurred on both occasions when the diet was changed; the ratio between the total count and the tetracycline-resistant count was not, however, altered. No obvious changes in the distribution of the 0-serotypes of E. coli were apparent on these two oc- casions.

The high proportion of sulphonamide-resistant E. coli may reflect the use of sulpha- quinoxaline as a coccidiostat in two of the three feeds. This was the conclusion reached by Heller & Smith (1973), but Renault (1 974) argued that the high levels of resistance found in birds in France were more likely to have arisen from the general bacterial con- tamination of the environment. He found high levels of sulphonamide resistance before the use of sulphaquinoxaline had been authorized in France. Subsequently a com- parative study using methylbenzoquate (a non-sulphonamide coccidiostat) and Pancoxin-plus, in 18 flocks, showed no clear difference between the two coccidiostats. Renault concluded that the use of sulphaquinoxaline at 60 parts/106 does not cause any increase in the proportion of E. coli strains resistant to sulphonamides. Whichever view is taken, coccidiostats which have been shown not to select for resistance against clinically useful antibacterial agents should be used.

In the terramycin-treated birds 49.5% of resistant E . coli were resistant to tetracycline compared with 9.5% in the untreated birds. These figures are highly signifi- cant because selection was achieved by exposure to terramycin in drinking water (about 13 g/100 1) on only two, widely separated days. It is suggested that the dubious advan- tage of using this antibiotic ‘prophylactically’ is far outweighed by the profound effect it has on the antibiotic resistance of the resident E. coli and it should be prohibited. The relatively high proportion of streptomycin-resistant E. coli in the untreated birds was due to the prevalence of strains resistant to both sulphonamide and strepto- mycin-known to be linked in the same plasmid (Heller & Smith 1 9 7 3 t a n d these appeared to colonize preferentially the untreated birds. Most of the strains with this resistance pattern (SSu) belonged to 0-serotype 103, a type which was completely absent from the treated group of birds. In the treated group, strains resistant to sulphon- amide and tetracycline (TSu) were prevalent (Table 2). The majority of tetracycline- resistant strains belonged to 0-serotype 9. This resistant serotype was isolated from both the starter crumbs and the grower pellets and, under the selection pressure of terramycin, appeared to be a good colonizer of the chicks in the treated group. This serotype was isolated only occasionally from the untreated group. As all isolates of 0-serotype 9 had the same resistance pattern (TSu) it might be expected that the presence of sulphaquinoxaline in the diet of the untreated birds would have also selected

E. COLI SEROTYPES IN CHICKEN ON TERRAMYCIN 463

this strain but this was not apparent. This observation suggests that terramycin exerted a stronger selection pressure for this particular O-serotype than did sulphaquinoxaline. The colonizing ability of strains, as well as superimposed selection pressures, together decide the composition of E. coli in the gut.

Certain O-serotypes persisted throughout the life (e.g. 09, 083 and 096). Others ap- peared early in life and disappeared (e.g. 015,018 and 0131) and others appeared late and persisted to the time of slaughter (e.g. 03, 08, 065, 070, 078, 0114). A similar ‘progression’ of O-serotypes was described by Craven & Barnum (197 1) in a pig unit.

A number of the O-serotypes which were present at slaughter were found in the ab- dominal cavity after evisceration. Of the O-serotypes isolated from the droppings, five out of eight in the treated group and fourteen out of nineteen in the untreated group were also isolated from the abdominal cavity after evisceration and washing. Presumably these were derived by direct or indirect contamination from the gut contents from the same or other birds. As the 12 birds were included along with others on a commercial slaughter line it is conceivable that the nine O-serotypes not found in the gut contents of the same birds were derived from others on the slaughter line. Six of the nine additional O-serotypes were isolated from swabs of the abdominal cavity of 10 birds randomly selected from those slaughtered prior to the 12. This is presumptive evidence that con- tamination occurred along the slaughter line.

Some O-serotypes included antibiotic sensitive and resistant strains; others only sen- sitive or resistant ones. Many of the 28 O-serotypes were isolated on relatively few oc- casions but 10 (03,08, 09,018,044,077,083,096,0103, 01 14) were isolated from at least 5% of single bird droppings from one or both groups of birds. These O-serotypes were presumably good gut colonizers for these birds under the influence of the diet and environment to which they were exposed.

Up to five antibiotic resistant O-serotypes were found among the 10 colonies picked from single faecal droppings (Table 4). Similar diversity of O-serotypes was found in calves and pigs; in man rarely more than one antibiotic resistant O-serotype was found in single faecal samples (Linton 1976). The greater the number of O-serotypes in the reservoir the greater the chance that strains able to colonize the human gut will be pre- sent. Poultry carry a large and diverse antibiotic resistant E. coli flora and consequently constitute a potentially important reservoir from which cross-infection may occur to man.

Very few of our isolates were non-typeable (37 out of 1498, i.e. 2%) and we also found a low percentage (9%) in a wide survey involving many different flocks (un- published data). In contrast, in a study of E. coli isolated from a poultry packing sta- tion, Shooter et al. (1974) found that a high proportion of isolates were non-typeable and concluded that the serotype distribution of E. coli in poultry is different from that in man. In the present study a total of 28 O-serotypes were isolated from the gut con- tents of the 12 birds. Some of the dominant O-serotypes were found also in man, e.g. 04,05,08,09,018,021,0131 (Hartley et al. 1975; Linton 1976), but others were found in large numbers only in poultry, e.g. 044, 070,096 and 01 14. There is strong evidence, therefore, of an overlap between O-serotypes colonizing the guts of chicken and man.

We wish to record our sincere thanks to the staff of the Poultry Packing Station who were very co-operative in allowing us to use their services. We also thank Mrs Helen Clements and her colleagues for excellent technical assistance. The work was supported by a grant from the Medical Research Council.

464 K. HOWE, A. H. LINTON AND A. D. OSBORNE

References

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CRAVEN, J. A. & BARNUM, D. A. 1971 Distribution of porcine faecal coliflora throughout a barn. Canadian Journal of Comparative Medicine 35,274-278.

HARTLEY, C. L., HOW, K., LINTON, A. H., LINTON, K. B. & RICHMOND, M. H. 1975 Distribu- tion of R plasmids among the 0-antigen types of Escherichia coli isolated from human and animal sources. Antimicrobial Agents and Chemotherapy 8, 122-13 1.

HEUER, E. D. & SMITH, H. W. 1973 The incidence of antibiotic resistance and other charac- teristics amongst Escherichia coli strains causing fatal infection in chickens: the utilization of these characteristics to study the epidemiology of the infection. Journal of H-vgiene, Cambridge 71,771-781.

HOW, K. & LINTON, A. H. 1976 The distribution of 0-antigen types of Escherichia coli in nor- mal calves. compared with man, and their R plasmid carriage. Journal of Applied Bacteri-

HOWELLS, C. H. L. & JOYNSON. D. H. M. 1975 Possible role of animal feeding stuffs in spread of antibiotic-resistant intestinal coliforms. Lancet i, 156-157.

LINTON, A. H. 1977 Antibiotics and Antibiosis in Agriculture. Proceedings of the Uniilersit?, of Nottingham. School of Agrigzclture, in press.

LINTON, A. H., HOWE, K. & OSRORNE, A. D. 1975 The effects of feeding tetracycline, nitrovin and quindoxin on the drug resistance coli-aerogenes bacteria from calves and pigs. Journal of Applied Bacteriology 38,255-275.

LINTON, A. H., HOW, K., PETHIYAGODA, S. & OSBORNE, A. D. 1974 Epidemiology of Salmonella infection in calves (1): Its relation to their husbandry and management. Veterinanl Record 94,581-585.

RENAULT, L. 1974 Resistance to sulphonamides of E. coli of broilers. Veterinary Record 95,302 (Correspondence).

SHOOTER. R. A., COOKE, E. M., O'FARRELL, S.. BETTELHEIM, K. A., CHANDLER, M. E. & BUSHROD, F. M. 1974 The isolation of Escherichia coli from a poultry packing station and an abattoir. Journal of Hygiene, Cambridge 13,245-247.

OlOgJs 40,3 17-330.