J. UPPI. Bact. 1976,40,317-330 The Qistribution of O-antigen Types of Escherichia coli in Normal Calves, compared with Man, and their R plasmid Carriage KATHERINE HOWE AND A. H. LINTON Department of Bacteriology, The Medical School, University of Bristol, Bristol BS8 I TD, England Received 21 October 1975 and accepted 15January 1976 The distribution of O-antigen types of Escherichia coli in calves over a ten-month period has been determined. A total of 400 calves from separate farms located over a wide area of England and Wales have been surveyed. Of the 148 0-types recognized, 93 were found in calves, compared with 107 in a previous survey in man; 42 of these were common to both hosts. In calves 60% of isolates were resistant to at least one antibiotic. Of these, 71 belonged to ten O-types, 9 of which were found in man. It is concluded that calves form a potential reservoir of R plasmid carrying E. coli. LINTON, HOWE & OSBORNE (1975) published data on calves fed a milk-replacer diet, with or without antibiotic growth promoters, over a 3-month period. Numbers of E. coli in the flora of these calves were fairly constant from birth to 3 months of age. However, in calves weaned on to hay and concentrates, the numbers of E. coli fell with age (unpublished data). Many workers have reported the presence of R factor-bearing E. coli in domestic animals (e.g. Smith, 1966; van der Heever, 1972; Hariharan, Barnum & Mitchell, 1974; Loken, Wagner & Henke, 1971; Larsen & Larsen, 1972) and the influence of antibiotics in increasing the numbers of resistant organisms (e.g. GuinCe, 1971 ; Linton et al., 1975). Babcock, Berryhill & Marsh (1973) have demonstrated the presence of R factor-bearing E. coli on dressed beef. The question as to how regularly strains of animal origin can colonize man, remains unanswered. Several workers have attempted, with mixed success, to colonize the human gut with strains of animal origin (Smith, 1969; Cooke, Hettiaratchy & Buck, 1972). Others, such as Burton, Hirsh, Blenden & Zeigler (1974) demonstrated that transfer had occurred between a resistant strain of E. coli of animal origin (051) and human gut flora but only when the relevant antibiotic was being administered. There is much controversy over the extent to which non-pathogenic E. coli in the animal community act as a source of R factor-bearing strains for man. The most probable route by which this could occur is by the ingestion of meat foods con- taminated with R factor-bearing E. coli of animal origin. These E. coli could either colonize the human alimentary tract or transfer their R factors to resident Gram negative flora during passage through the gut. To assess the significance of this problem it is necessary to enquire into the incidence of R factor-bearing E. coli in meat animals, the load and frequency of excretion of E. Cali from these animals, the 13171
Transcript
J . UPPI. Bact. 1976,40,317-330
The Qistribution of O-antigen Types of Escherichia coli in Normal Calves, compared with Man, and their
R plasmid Carriage
KATHERINE HOWE AND A. H. LINTON
Department of Bacteriology, The Medical School, University of Bristol, Bristol BS8 I TD, England
Received 21 October 1975 and accepted 15January 1976
The distribution of O-antigen types of Escherichia coli in calves over a ten-month period has been determined. A total of 400 calves from separate farms located over a wide area of England and Wales have been surveyed. Of the 148 0-types recognized, 93 were found in calves, compared with 107 in a previous survey in man; 42 of these were common to both hosts. In calves 60% of isolates were resistant to at least one antibiotic. Of these, 71 belonged to ten O-types, 9 of which were found in man. It is concluded that calves form a potential reservoir of R plasmid carrying E. coli.
LINTON, HOWE & OSBORNE (1975) published data on calves fed a milk-replacer diet, with or without antibiotic growth promoters, over a 3-month period. Numbers of E. coli in the flora of these calves were fairly constant from birth to 3 months of age. However, in calves weaned on to hay and concentrates, the numbers of E. coli fell with age (unpublished data).
Many workers have reported the presence of R factor-bearing E. coli in domestic animals (e.g. Smith, 1966; van der Heever, 1972; Hariharan, Barnum & Mitchell, 1974; Loken, Wagner & Henke, 1971; Larsen & Larsen, 1972) and the influence of antibiotics in increasing the numbers of resistant organisms (e.g. GuinCe, 1971 ; Linton et al., 1975). Babcock, Berryhill & Marsh (1973) have demonstrated the presence of R factor-bearing E. coli on dressed beef. The question as to how regularly strains of animal origin can colonize man, remains unanswered.
Several workers have attempted, with mixed success, to colonize the human gut with strains of animal origin (Smith, 1969; Cooke, Hettiaratchy & Buck, 1972). Others, such as Burton, Hirsh, Blenden & Zeigler (1974) demonstrated that transfer had occurred between a resistant strain of E. coli of animal origin (051) and human gut flora but only when the relevant antibiotic was being administered.
There is much controversy over the extent to which non-pathogenic E. coli in the animal community act as a source of R factor-bearing strains for man. The most probable route by which this could occur is by the ingestion of meat foods con- taminated with R factor-bearing E. coli of animal origin. These E. coli could either colonize the human alimentary tract or transfer their R factors to resident Gram negative flora during passage through the gut. To assess the significance of this problem it is necessary to enquire into the incidence of R factor-bearing E. coli in meat animals, the load and frequency of excretion of E. Cali from these animals, the
13171
318 K. HOWE AND A. H. LINTON
degree of contamination of the carcasses at slaughter and how closely the serotypes in various animal hosts parallel those commonly found in healthy humans.
Smith (1961, 1965) and Smith & Crabb (1961) investigated the numbers of E. coli in a wide range of domestic and pet animals and found considerable variation amongst animal hosts. Whilst the faecal flora of all animals examined, irrespective of species, were closely similar in early life considerable differences occurred with age. For instance, faecal E. coli counts were highest in calves in the first weeks of life: by 6 months of age they had fallen 10 000-fold.
What constitutes a so-called ‘animal E. coli’ still remains unresolved. For R-factor ecology to be set on a sound basis it is necessary either to be able to distinguish ‘animal’ and ‘human’ strains or to be able to say with reasonable certainty that they are indistinguishable. As a contribution towards solving this problem, serotyping of strains from each source is a useful technique.
The serotyping of E. coli colonizing the healthy human gut have been determined by a number of workers (Wiedemann & Knothe, 1971 ; Bettelheim et al., 1974; Hartley et al., 1975). Parallel information for animal populations is lacking (Cooke, 1974; Bettelheim et al., 1974) and most veterinary workers have concentrated on establishing the serotypes of recognized enteropathogenic strains in various animal hosts (Ingram, 1964; Sojka, 1965). A few papers describe the distribution of E. coli serotypes in healthy animals (GuinCe, 1963; Ewing & Davis, 1961; Weidemann & Knothe, 1971; Ward et al., 1974; Popa & Decun, 1974; Hartley et al., 1975) but more information is needed to establish serotypes regularly present.
The present paper records a survey undertaken to establish the range of 0-antigen types of E. coli commonly found in healthy calves and to determine whether R-factors were randomly distributed throughout the organisms or confined to specific 0 types.
Materials and Methods
Source of strains
Thc opportunity to sample a large number of healthy calves from individual farms over a wide area of England and South Wales arose when animals were being moni- tored for salmonella excretion prior to entering a large beef rearing unit for progeny testing (Osborne, Linton & Pethiyagoda, 1974). The samples from these calves were collected at their farms of origin prior to vaccination against salmonella. In addition calves in Cumbria, Dyfed, Avon and Somerset were sampled by veterinary surgeons. The samples from Dyfed were collected by a veterinary surgeon in the normal course of work. Samples from Cumbria and from Avon and Somerset were collected by veterinary surgeons involved in work connected with local veterinary investigation centres, Penrith and Langford, respectively. The survey yielded a representative selection of isolates from a large area of England and South Wales.
Large alginate swabs, especially designed for rectal swabbing (Linton, Howe, Pethiyagoda & Osborne, 1974) were used and posted to the laboratory with a mini- mum of delay. Duplicate samples from any one farm were avoided on the assumption that similar strains may colonize all the animals reared or living in close proximity (Fein, Burton, Tsutakawa & Blenden, 1974).
All the animals sampled were normal and healthy and were in the range 0-8 weeks
E. COLZ 0-ANTIGEN TYPES IN CALVES 319
old. The diets would have been dependent on the age of the animal and its situation but within the usual pattern for beef rearing.
Samples were collected over a 10-month period as indicated in Table 1. A total of 400 calves were examined with the aim of investigating possible variations due to geographical location or seasonal changes.
Isolation and identijication of strains
Rectal swabs, on arrival at the laboratory, were each spread on to unsupplemented bile lactose agar plates and incubated overnight at 37". Ten discrete lactose fermenting colonies were taken from each sample for biochemical tests. Colonies producing indole in peptone water and acid and gas in MacConkey broth after overnight incubation in a water bath at 44" were considered to be E. coli faecal type 1 (Anon, 1956; Anon, 1969).
Isolates not strictly E. coli faecal type I were not considered. The purity of the isolates was checked by subculture on bile lactose agar. After being satisfactorily identified each isolate was stored on a nutrient agar slope at room temperature in the dark.
Determination of resistance patterns of strains
Resistance patterns to antibiotics were determined on lysed blood agar by a con- ventional technique using UI Oxoid multodiscs (Bauer, Kirby, Sherris & Turck, 1966). Antibiotic sensitivity discs included tetracycline (50 pg), streptomycin (25 pg), ampicil- lin (25 pg), sulphafurazole (500 pg), nalidixic acid (30 pg), nitrofurantoin (200 pg), colistin sulphate (10 pg) and chloramphenicol (50 pg). Two additional single discs impregnated with neomycin (30 pg) and furazolidone (200 pg), respectively, were also applied. Plates were incubated overnight at 37" before reading.
Scoring of strains
To avoid repetition of the same 0-type from a single source a system of scoring was implemented. The isolation of a specific E. coli 0-antigen type from a given sample was scored as one isolate. In addition isolates of similar 0-antigen type but with different antibiotic resistance patterns were also accepted for scoring from the same sample. Any further isolates of the same 0-antigen type and resistance pattern from a given sample were discarded as replicates. This exclusion procedure reduced 3822 isolates of E. coli which had been 0-antigen typed to 946 scored strains actually included in the analysis.
It was felt that whilst an analysis of the total number of isolates would give more information on the frequency of certain 0-types, the scored values would indicate more precisely the occurrence of each 0-type in the calf population.
0-antigen typing of isoIated E. coli
0-antigens were determined by methods modified from Wiedemann & Knothe (1969) using an agglutination technique adapted for use in the Cooke microtitre apparatus (Cooke Engineering & Co., Alexandria, Virginia, U.S.A.).
320 K. HOWE AND A. H. LINTON
Preparation of antisera
Antisera were prepared in rabbits against the standard E. coli strains 01 to 153, using the methods of Edwards & Ewing (1972). Undiluted sera were stored in 2 ml aliquots at - 20" using sodium azide as preservative. Stock dilutions of the sera were made in physiological saline containing 0.5% (w/v) phenol and stored at 4". The sera were arranged for use in 12 pools according to antigenic relationships, each pool containing specific antisera against 12 E. coli standard antigens. A 13th pool contained antisera against the more recently recognized E. coli 0-antigens.
Preparation of antigens
Discrete smooth colonies from a bile lactose agar plate were inoculated into 4ml Schlecht broth and incubated statically for 18 h at 37". The culture was boiled for 1 h in a steamer, allowed to cool and 18 ml of crystal violet/formalin/saline added to give a transmittance of the resulting homogenized suspension of 30 % at 650 nm in a Unicam SP600 series 2 spectrophotometer.
If typing with this preparation did not produce a clear result, an 18 h broth culture was autoclaved at 121" for 2 h to destroy masking antigens and, after centrifuging, was suspended in 9 ml of crystal violet/formalin/saline. Whilst 0 antigen-masking by B and L type K-antigens is destroyed by boiling, autoclaving is required to destroy the masking effect of A-type K-antigens. The antigens were stored at room temperature.
0-an tigen determination
Stage 1
0-antigen suspensions were tested against the 13 0-antisera pools and a 3 % saline control in U-form Cooke microtiter plates, 0.05 ml of antigen being added to 0.025 ml of each stock pool. The plates were sealed with adhesive tape and incubated for 18 h at 50'.
Stage 2
pool showing a positive reaction in Stage 1. The antigen suspension was tested against each individual serum component of the
Stagc 3
Although 0-antigens react specifically with their homologous antiserum, there are many reciprocal and non-reciprocal antigenic relationships among cultures of different 0-antigen groups. These relationships may cause positive reactions with several pools i n Stage 1 and several single sera in Stage 2, i.e. antigenic cross reactions. It is therefore necessary to titrate those single sera giving positive reactions in Stage 2 against the test antigen. Doubling dilutions of sera, beginning at 1 : 50 were made using a Titertek multidiluter (Flow Laboratories Ltd, Irvine, Ayrshire) and the titre determined after overnight incubation at 50".
From the pattern of reactions obtained in Stages 1 and 2 and the titres in Stage 3 it was possible to determine the 0-antigen of the strain under test. For a strain to be allocated to a particular 0-group with certainty it must give the same titre (not more
E. COLZ 0-ANTIGEN TYPES IN CALVES 32 1
than one well difference) with specific 0-antiserum as given by the standard E. coli strain which was used to prepare the serum.
If no reaction was obtained in Stage 1 with antigen from boiled bacteria, the test examination was repeated with an autoclaved antigen. The presence of K(A) antigen was inferred when a boiled antigen gave no reaction in any of the pools, whilst the autoclaved antigen gave a positive reaction. If all the pools were negative using autoclaved bacteria, or if the reactions in Stage 2 did not come up to titre with any sera in Stage 3 with both boiled and autoclaved antigens, the strain was designated non-typeable (NT).
Some strains agglutinated during growth in broth or during antigen preparation and could not be used to produce homogenous antigens. These strains gave positive reaction in all 13 Stage 1 pools and in the saline control. They could not, therefore, be 0-antigen types and were termed auto-agglutinable (A).
Certain strains were sent to Dr B. Rowe, Public Health Laboratory Service, Colin- dale Avenue, London for determination of K and H antigens.
R-factor transfer
A nalidixic acid-resistant laboratory strain, E. coli NXR was used as a standard recipient in R-factor transfer experiments. The standard test was carried out as previously described (Linton et al., 1975).
Of the 3822 E. coli isolated from 400 calves, 946 were scored and a breakdown of these is set out in Table 1 . The samples collected in connection with the beef progeny testing
TABLE 1 Sources and numbers of scored isolates of E. coli included in the survey
programme are divided into batches 1-5 according to the dates of collection. The other three groups are recorded by reference both to the collecting centre and the date of collection.
In Table 1 the numbers of calves and the total number of isolates from each source are given; these are followed by the number of independent strains selected for further analysis based on the method of scoring, e.g. in Batch 1 September/October 1974, of the 223 isolates obtained by culture of the rectal swabs only 64 were selected by the scoring procedure. The scored isolates are subdivided into the numbers of sensitive and resistant isolates together with their percentages. In summary, 378 (40 %) of all scored isolates were found to be sensitive to the full range of antibiotics used and 568 (60 %) were resistant to at least one of the antibacterials.
Since the calves associated with the beef progeny testing programme were sampled over a 10-month period it was possible to evaluate the influence of seasonal changes on the relative proportion of sensitive to resistant E. coli. The batches sampled during the winter showed only minor variations but a significant fall in the proportion of resistant E. coli occurred by summer. This was most probably associated with changes in husbandry practice as had been observed with patterns of salmonella excretion. (Heard, Jennett & Linton, 1972; Linton et al., 1974; Osborne et al., 1974). A similar trend for the E. coli flora is to be expected.
A more detailed examination of the 568 resistant isolates showed that multiple resistance was common. The percentages for each number of R determinants encoun- tered were: 1, 15.1 %; 2, 35.4%; 3,22*2%; 4, 18.6%; 5,7*7% and 6,0*9%. Altogether 7 R determinants were found for streptomycin, sulphonamide, tetracycline, ampicillin, chloramphenicol, neomycin and furazolidone. An analysis of the distribution of the various Rdeterminants among the resistant isolates from each group of calves is presented in Table 2. Using a punch card system the occurrence of each R determinant within each group of isolates were determined. The frequency of occurrence was expressed as a percentage of the number of isolates. Taking the totals for all groups the order of frequency was found to be : sulphonamide, 4941568 (87-0 %); streptomycin, 458/568 (80.6 %); tetracycline 264/568 (46.5%); chloramphenicol, 72/568 (12.7 %); neomycin, 23/568 (4.1 %) and furazolidone, 15/568 (2.6 %).
Each of the 568 antibiotic resistant isolates were tested for their ability to transfer their drug resistance to a recipient E. coli by a standard test (Linton et al., 1975). Of all the resistant strains, 242 (42.6 %) exhibited transfer of the whole or part of their drug resistance (Table 2). The number of transcipients within each group of isolates and the number of individual R determinants are shown separately. Expressing the total number of each R determinant which transferred as a percentage of the initial number in the donor strains the following figures were obtained: streptomycin, 38.6 %; tetracycline, 46.6 %; ampicillin, 24.0 x; chloramphenicol, 66.7 %; neomycin, 21.7 %; sulphonamide, 37.9 % and furazolidone, 0 %. From these calculations it would appear that chloramphenicol was more regularly transferred than any other R determinant, tetracycline, streptomycin and sulphonamide next in order, ampicillin and neomycin least and furazolidone not at all.
Tetracycline resistance is known only to occur on plasmids and the presence of this R determinant is a useful measure of potential plasmid carriage (Franklin, 1967). From Table 2 it can be calculated that 101 isolates demonstrated transfer of plasmid but did not contain tetracycline resistance. These added to the number which contained
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324 K. HOWE AND A. H. LINTON
tetracycline resistance gave 365 potential plasmid carriers, i.e. 64.3 of all resistant isolates. Therefore a large proportion of resistant calf E. coli are potentially capable of transferring drug resistance.
TABLE 3 Incidence of E. coli 0-antigen types from calves expressed as
Distribution of 0-antigen types of E. coli in calves
The 0-antigen types of the 946 scored isolates are presented in Table 3. All isolates were cultured on non-antibiotic containing media; it is valid therefore to compare directly the percentages of antibiotic resistant with sensitive isolates. Each value is a percentage of 946 scored isolates.
The number of different 0-types found in a single sample from an individual calf was variable ranging from 1 to 8 among the 10 colonies examined. Of the 400 calves examined 113 yielded a single 0-type, 134 two types; 74, three; 44, four; 18, five; 12 six; 7, four; and 1, eight. These findings are in contrast with those for human subjects in which it was rare for more than two 0-types to be isolated from a single specimen.
The highest percentage of isolates was found to belong to 0-type 8, one of the three 0-types which possess K(A) antigen (08,09,0101). These three 0-types accounted for 31.9% of all the scored isolates in calves and 37.2% of all typed resistant E. coli. A possible link between K(A) antigen and drug resistance has been surmised (Hartley et al., 1975). However, since 10.3 % of sensitive E. coli in calves belonged to 0-types 8 and 9 the association was not absolute. All isolates of 0101 were invariably K(A) positive and always resistant to antibiotics. The distribution of K(A) antigen and drug sensitivity or resistance among the three 0-types is shown in Table 4.
TABLE 4 The distribution of K(A) antigen and drug sensitivity or resistance among
0-antigen types 8, 9 and IOI
K(A) positive K(A) negative
0-type Resistant Sensitive Resistant Sensitive Total * h
r > I >
08 68 14 09 33 8
0101 19 0 Totals 120 22
54 14 150 7 3 51 0 0 19
61 17 220 - L Y
J
142 78
Discussion The number of E. coli from calves which could not be typed by this procedure was only 17.9 %. In contrast, Shooter et al. (1974) found that 64% of E. coli from abattoir sources could not be typed. They concluded that strains of animal origin may comprise 0 groups which are not normally encountered in human faeces. It is possible, however, that many of their isolates were from the abattoir environment and not strictly of animal origin. The results of Bettelheim et al. (1974) also suggested that non-typeable E. coli strains are abundant in the animal population but the distribution of specific E. coli 0-types was not given.
The calf isolates which could be 0-typed fell into 93 (62.8%) of the 148 possible types. Of the 93 0-types isolated, 34 were invariably sensitive to the antibiotics tested,
326 rc. HOWE AND A. H. LINTON
I3 were invariably antibiotic resistant whilst 46 included both sensitive and resistant isolates (Table 3). Indeed, all types which occurred at 2% level or over (with the exception of 101) included both sensitive and resistant isolates. From the available data there is no evidence that complex resistance patterns are regularly associated with any particular O-types.
Some of the O-types found in this survey were present in relatively few animals whilst others occurred more frequently; the data in Table 3 indicate that ten types 02, 08, 09, 011, 017, 021, 086, 0101 and 0123 were present in calves at levels of 2 % or higher. Within these O-types 71 of all antibiotic resistant isolates were found and it is of interest that, with one exception (026), all these O-types have been isolated from man (Hartley et al., 1975). We do not fully understand the reasons why certain O-types are more abundant in calves than others but it may be due to their ability to colonize the calf gut. In this context strains 08 and 026 have been implicated in calf disease (Sojka, 1965). The adhesive properties of K88 positive strains of E. coli enable attach- ment to brush borders of the intestinal tissue of piglets (Jones & Rutter, 1972). The bacteria then multiply and reach abnormally large numbers in the anterior small intestine. A similar situation exists with respect to enteric disease in calves in which an analagous plasmid K99 is considered of major importance (Smith & Linggood, 1972). However, recent work has shown that genetic factors controlling the nature of the gut wall in piglets are also involved (Rutter, Burrows, Sellwood & Gibbons, 1975). Pigs may be divided into two phenotypic groups-those termed ‘adhesive’ in which the K88 strain can attach and those termed ‘non-adhesive’ in which no adhesion occurs. The suggestion that certain serotypes are ‘strong’ has been made (Cooke, 1974). When certain E. coli O-types appeared in the faeces of infants they were likely to persist (Gage, Gunther & Spaulding, 1961) and a similar situation could occur in calves. Another possible reason for the abundance of specific O-types may be that a high proportion of isolates are drug resistant. The wide use of antibiotics in animal manage- ment might be the principal cause in selecting particular O-types which include a large proportion of resistant strains.
The calves included in this survey were located over a wide area of England and Wales. To assess the effect of geographical location on the occurrence of O-types of E. coli the findings for each calf were analysed by a punch card system. The calf record cards were divided into 4 areas and the distribution of O-types analysed. The 2 larger groups were located in S.W. England (Cornwall, Devon, Avon, Somerset, Gloucestershire, Monmouth, Dorset and Wiltshire) and the ‘Midlands’ (Derbyshire, Nottinghamshire, Leicestershire, Warwickshire, Staffordshire, Lincolnshire, Bedford- shire, Buckinghamshire and Northamptonshire). These were compared with each other and with the smaller groups located in Cumbria and Dyfed (Table 5). Despite the fact that the number of calves in each region was dissimilar, the distribution of the dominant O-types did not show significant differences and it is assumed therefore that the geographical location does not affect the distribution of O-types of R coli in the calf gut flora.
One important objective of this survey was to consider how closely the O-antigen types of E. coli from calves correlate with those found in man (Hartley et al., 1975). The use of O-typing alone as an epidemiological tool has been criticized on the grounds that it is incomplete and consequently not adequate. Accordingly 90 strains, chosen at random from human urinary tract infections (17), human faeces (17) and calf
6. COLZ 0-ANTIGEN TYPES IN CALVES 321
faeces (56) all belonging to 0-types 8 , 9 and 101, were sent to Dr B. Rowe at Colindale for K and H antigen typing. The same K and H antigens were found in certain strains of the same 0-types from each of the three sources of E. coli. In a large number of strains H antigens were not detected and, in many, the K antigens could not be
TABLE 5 The comparison between the distribution of dominant" 0-types of
* 0-types in which 5 or more scored strains were isolated from one or more of the 4 areas.
identified; in none of the 0101 strains tested from all sources could the K antigens be identified. This indicates that, within this limited investigation, K and H antigens associated with these 0-types were not specific to E. coli isolated from humans or calves. Whilst further subdivision of the 3 0-types was possible by this means neverthe- less it was concluded that 0-typing alone provided a very useful means of distinguishing strains of E. coli in a general survey.
328 K. HOWE AND A. H. LINTON
A wide scatter of 0-types have been found to occur in both calves (93) and man (107). Among those represented in each host some overlap was experienced; 42 0-types were common to both (Table 6). The figures indicate that there is no absolute
TABLE 6 Distribution of antibiotic sensitivity and resistance in E. coli 0-types
from man and calves
NO. of O-types found in No. of 0-types
r-A-, common to man Man Calves and calves
- -
No. of 0-types including 53 46 28
No. of 0-types sensitive only 42 34 14 No. of 0-types resistant only 12 13 0
resistant and sensitive isolates
demarcation between ‘animal’ E. coli and ‘human’ E. coli. Further work is likely to give support to this point of view. Surveys in pigs, poultry and lambs currently under investigation also show considerable overlap with 0-types of human origin.
The 0-types isolated from calves and man may be further divided on the basis of the distribution of antibiotic sensitivity and resistance. In calves, 60/93 (64.5 :<) 0-types included resistant isolates and 79/93 (84.9 %) sensitive isolates. A similar evaluation of the human data reveals 65/107 (60.7 %) resistant and 95/107 (88.8 %) sensitive strains. These figures are comparable suggesting a similar qualitative distribution of drug resistance and sensitivity throughout the 0-types isolated. Some 0-types were invariably resistant, others sensitive but a much larger proportion included both sensitive and resistant strains (Table 6).
Although a similar number of 0-types isolated from each host were found to include resistant isolates the actual proportion of resistant to sensitive isolates in calves (60 : 40) was much higher than that found in healthy human subjects. It is not possible to quote precise figures for the proportion of resistant to sensitive strains in man since the resistant isolates analysed in the work of Hartley (1975) and her colleagues were from a different number of subjects than those examined for the analysis of sensitive strains and included resistant strains isolated under the selective pressure of antibiotic-containing media. A direct numerical comparison is therefore not valid. However, it was rare to isolate resistant strains from man on non-selective media whereas in calves 60 % of all strains isolated without antibiotic selection pressure were antibiotic resistant. If studies in other animal hosts at present in progress reveal a similar pattern it could be argued that this is a reflection of the considerable pressure exerted by widespread use of antibiotics in animals over previous years. The observation that the lack of resistant E. coli in man is considerably less overall is a matter of some encouragement. It may indicate that the disputed flow of resistance factors from the ‘animal reservoir’ to man is rather limited but the level of resistance (60%) of the total E. coli flora in calves must constitute a potentially important
E. COLZ 0-ANTIGEN TYPES IN CALVES 329
source of R factors. Whichever view is accepted about the spread of R factors it is agreed that the medical use of antibiotics has the effect of selecting R+ bacteria. In order to maintain the status quo in man it is essential to restrict the use of antibiotics as much as possible.
The authors wish to acknowledge with gratitude the assistance of Mr A. D. Osborne, Mr A. Richardson, Mr T. A. B. Morgan and Mr Hugh Davies, Veterinary Surgeons who took many calf rectal swabs for this survey. We are also indebted to the Milk Marketing Board for the opportunity to use materials already being collected from calves in connection with a salmonella survey on their beef progeny testing programme. Our thanks are expressed to Dr B. Rowe for further typing some of our isolates, to Mrs H. Clements and her colleagues for their competent technical assistance and to Professor M. H. Richmond for constant encouragement and critical evaluation of the manuscript. One of us (K.H.) was in receipt of an M.R.C. Scholarship award which is gratefully acknowledged.
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