Download - A Longitudinal Study of Escherichia coli in Cows and Calves with Special Reference to the Distribution of O-antigen Types and Antibiotic Resistance

J . uppl. Buct. 1976 40, 331-340

A Longitudinal Study of Escherichiu coli in Cows and Calves with Special Reference to the Distribution of

0-antigen Types and Antibiotic Resistance

KATHERINE HOWE, A.H. LINTON

Department of Bacteriology, The Medical School, University of Bristol, Bristol 858 I TD, England

AND

A. D. OSBORNE

Department of Veterinary Medicine, Langford House, Langford, Bristol BS I8 7DU, England

Received 21 October 1975 and accepted I 5 lanuary 1976

The faecal excretion of E. coli from 6 adult cows was followed over a period of 105 days. E. coli were excreted in only 23.4 % of specimens and the patterns of excretion varied between animals. All isolates of E. coli were antibiotic sensitive and fell into 6 0-antigen types; very few were non-typeable. The 0-types excreted by each animal, compared with those from other cows in the same house, were traced with time. A similar but less intense study was undertaken in 8 calves. In contrast E. coli was excreted from all samples and included both antibiotic sensitive and resistant strains; 37 0-types were represented. The persistence of specific 0-types within each calf and their spread to other calves in the same house were studied. A comparison of the 0-types in the bovine species with those isolated from man has been made.

A RECENT SURVEY of calves over a wide area of England and Wales has established the occurrence of a large number of 0-antigen types of Escherichia coli in their faeces (Howe & Linton, 1976). The survey was based on the examination of single specimens taken from calves on 400 different farms. No information was obtained on the persistence of specific 0-types within an individual calf or on the frequency of cross- infection between calves. To gain this information a limited longitudinal study of calves located on a commercial farm was undertaken over a 5-week period. The results are reported in this paper.

The work on calves raised the question of the relative position in adult cattle. It is known that the alimentary flora of the bovine species changes with ruminal development and that the flora of faecal excreta from adult animals differs markedly both qualitatively and quantitatively from that of the young (Smith, 1961). Smith & Crabb (1961) also reported on the fluctuations in the numbers of the strains forming the total E. coli population in healthy calves and healthy cows as revealed by phage typing studies. Larsen & Larsen (1972) investigated the levels of drug resistant E. coli in healthy animals, and reported 68-8 % resistant E. coli in calves but only 2.7 in cows.

So far as we are aware no information has been published on the 0-antigen types of

V311

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

E. coli in the adult cow. To determine this, 6 cows were sampled, usually daily, over a period of 105 days. Presumptive coliform counts were determined on each sample and the frequency of occurrence of each 0-antigen type of E. coli was followed together with information on their sensitivity or resistance to antibiotics.

Materials and Methods

Animals, housing and diet

cows

The cows involved in this study were non-clinical, potential salmonella carriers bought for research purposes but otherwise normal. They were kept in a research animal building in separate rooms and fed mostly on hay and a small amount of concentrates. Four animals (386, 657, 35 and 96) had been “in residence” since October 1974, the other two (418, 556) were bought in during the course of study.

Calves

The 8 calves included in this study were raised as replacements for a dairy herd on a farm in Somerset. They were 3-8 weeks old during the period of sampling and fed on a mixed diet comprising milk substitute, hay and concentrates. They were kept in a building which housed a total of 24 calves and were penned singly, each pen being adjacent to its neighbour in the same row.

Sampling

Faecal material was collected from the rectum of the calves and cows in disposable polythene sleeves. Using the sleeve as a glove the sample was taken from the rectum, the hand withdrawn through the sleeve thereby turning the sleeve inside out with the sample inside. It was then tied to retain the sample.

Media

Total counts of faecal coli-aerogenes bacteria were made on standard MacConkey bile lactose agar. The numbers of drug-resistant coliforms were determined separately on similar medium incorporating individual antibacterial agents at the following final concentrations : ampicillin (25 pg/ml) ; tetracycline (25 pglml).

Presumptive coliform counts

Counts were made according to methods previously described (Linton, Howe & Osborne, 1975). After counting, 10 lactose fermenting colonies were picked at random from the unsupplemented MacConkey plates and where growth occurred, 10 colonies from each of the plates incorporating antibiotics. Single colonies were inoculated into nutrient broth and incubated at 37” for 2 h, the broth cultures being used for biochemical tests, antibiotic sensitivity tests and 0-antigen typing described previously (Howe & Linton, 1976).

A STUDY OF E. COLZ IN COWS AND CALVES 333

Adult cows

A total of 402 faecal samples from 6 cows were examined. Of these only 94 (23.4%) yielded coli-aerogenes organisms on MacConkey’s agar. Considerable differences in the patterns of excretion occurred amongst the cows. One cow yielded coliform organisms regularly from all samples, others yielded them less frequently and one on only two occasions (Table 1). Usually coliform organisms were excreted daily for a

TABLE 1 The frequency of isolation ofpresumptive coliform organisms from each of six cows

Cow Ref. No. 35 96 386 657 418 556 Total

No. of specimens examined 70 70 26 88 74 74 402 No. of specimens from which coli- 3 2 26 17 12 34 94

aerogenes organisms isolated % 4.3 2 .9 100 19.3 16-2 45.9 23.4 Average coli-aerogenes count/ g faeces 4.03 3.79 5.29 5.26 5-94 4.90

number of days and then could not be detected for a period being replaced by a non-lactose fermenting flora. Later excretion of coliforms, often of the same 0- antigen type of E. coli as those present earlier, would recur.

The presumptive coliform counts of samples taken on different days were fairly constant from the same cow but amongst cows varied by one or two logarithms. The average counts for each animal are included in Table 1.

Up to 10 coliform colonies were picked from culture plates from each animal sample. Of these 736 isolates were confirmed as E. coli by biochemical tests. These were 0-antigen typed and tested for antibiotic resistance; without exception the 736 strains were fully sensitive to the range of antibiotics used. Only 6 proved to be non-typeable; the remainder fell unequally into 6 0-antigen types (Table 2). In this

TABLE 2 The frequency of isolation of 0-types of E. coli from 6 cows

Cow reference numbers

35 96 368 657 418 556 I r >

0-type No. of faecal samples Total %

0 8 010 039 093 0105 0 1 3 3 NT

No. of faecal samples from which E. coli isolated

- - l o - - - 10 10.6 2 1 3 3.2

5 - - - 5 5.3 3 2 1 5 1 7 9 8 54 57.4

3 - 3 3 .2 31 31 33.0

3 3 3 .2

- -

- - - - - - - - - - - - - - 3 2 26 17 12 34 94


.................... .................... .................... ..................... ....................... ........................ .........................

..........................

Table the frequency of isolation of each 0-type from the 94 faecal samples yielding E. coli, both within and between cows, is indicated. From the 94 faecal samples, a single 0-type only was isolated from 81, 2 0-types from 11 and 3 from 2 samples. Type 093 was isolated from the highest number of samples (54/94 = 57-4 %) and from all cows. Next in order of frequency of isolation was type 0133 (31/94=33 %) but this type was found in one cow only; this was followed by type 08 (10/94= 10.6 %), also from one cow and the remainder were less frequently isolated. The manner in which the 0-types were distributed among the daily samples from each cow throughout the 88 days of the study are presented in Fig. 1 .

NO. 386

........ ........ ........ ........ ........ ......... ......... ......... ......... ...........

28

.. ..

I

.. .. .. .. .. ... ... -f.--.-_

....... .. ....... .. ....... .. ....... ..

No.418

....... ....... .. ........... .. ........... .. * .......... .... ........... .... . -. - - - - * - .- * * .... * * * - _ _ _ _ _ _ * --* * .......... - ... No. 657

Type 093 was isolated on at least two occasions froin all 6 cows and was the only 0-type to be isolated from 3 of them (Nos 35, 96 and 657). Since this 0-type was not isolated from the first sainple taken from the 2 cows (Nos 418 and 516) brought in part way through the period of the study, it was assumed that 093 was a strain ‘resident’ in the research animal building environment.

Reference to the data in Fig. 1 on the 2 cows (Nos 418 and 556) indicates that their E. coli 0-types changed suddenly almost immediately after admission to the experi- mental animal buildings to be replaced by strains present in other cows in the unit. One of these cows (No. 418) continued to excrete fairly regularly the replacement strain 0-type 093 whilst the other cow gradually lost this type and subsequently continued to excrete its ‘own’ type 0133. This would suggest that the ability of different 0-types to become established varies amongst cows.

Fig. 1 . Longitudinal distribution of the 6 0-types of E. coli in each of 6 adult cows. The 0-types isolated from each rectal sample (where possible up to 10 colonies) are shown. A dash indicates samples from which no E. coli were isolated. The observations on cow no. 386 terminated on 17 February, thc date of slaughter. A, 08: 0, 010: *, 039: 0, 093: V, 0105: IN, 0133: A, NT.


Calves

Eight calves were sampled five times at weekly intervals. In contrast to the findings with adult cows all 40 calf faecal samples yielded presumptive coliform organisms and in greater numbers than in the cows (Tables 1 and 3). The numbers isolated from calves

TABLE 3 Presumptive coliform counts (Log,,lg) from faecal samples taken from

each of 8 calves sampled weekly for 5 weeks on non-selective and selective media

Sample No.

1 2 3 4 5

1 2 3 4 5

Calf reference numbers A

I 7 1 2 3 4 5 6 7 8 Mean

Counts on bile lactose agar 8 .3 9.1 9 .4 7.9 8.3 6.7 8.8 8.9 8.4 5.9 5.2 7.9 4.8 6.7 7.9 6.1 7.6 6.5 7.9 6.2 8.1 7.1 6.9 7.8 8 . 1 7.8 7.5 8.1 7.8 8.1 7.3 7.2 7.2 6.7 7.7 7.5 4.9 6 . 4 7.1 6.7 6.1 5.4 6.4 7.2 6.3

Counts on bile lactose agar incorporating 25 pg/ml tetracycline 5.6 4.3 5.5 5.4 7.1 4.9 6.3 0 4.2 3.4 4.4 0 0 5.2 0 4.1 0 0 4.8 0 4 .6 0 0 0 4.9 4.3 0 0 5.9 0 0 4.2 0 0 0 3.9 3.7 0 4.0 4.0

Counts on bile lactose agar incorporating 25 pg/nil ampicillin 5.6 4.9 7 .7 6.2 4.9 4.4 6.1 0 3.7 0 0 0 0 4.4 0 4.6 4.7 3.9 0 4.3 4.5 0 4.9 0 5.8 0 4.8 0 5.9 0 4.9 3.7 0 0 3.9 5.1 0 0 0 0

O=less than 5000 E. coZi/g (i.e. Loglo value 3.7).

on antibiotic containing-media were less than those isolated on non-selective media but were nevertheless significantly high although not all samples yielded detectable counts. All samples yielded antibiotic sensitive coliforms on non-selective media but resistant coliforms were cultured on the same medium from 29/40 (72-5 %) of samples. Antibiotic resistant coliforms were isolated from a further 3 samples on selective media containing 25 pg/ml of either tetracycline or ampicillin.

Up to 10 colonies were picked from each culture plate of antibiotic containing bile lactose agar and of similar medium without antibiotics; 713 of these isolates were confirmed to be E. coli by biochemical tests. Of these, 380 were isolated on the non- selective medium and included 123 antibiotic resistant strains (32.4 %); the remainder (333) were isolated on the antibiotic-containing media. Each of the 713 isolates were 0-antigen typed and their antibiotic resistance patterns determined.

Of the 380 isolated on non-selective media, 63 (166%) were non-typeable; the rest were distributed throughout 28 0-types. Most of the 333 isolated on selective media also fell into many of the same 28 0-types but 71 of the isolates belonged to a further 9 0-types (01,02,07,011,045,050,070,0101 and 0138) of which the largest num-


ber (47) belonged to type 0 1. Combining the observations on both groups, a total of 37 0-types of E. coli were represented in the 8 calves; these are shown in Table 4. Of the 37 0-types, 10 were antibiotic sensitive, 16 resistant and 11, both sensitive and resistant.

TABLE 4 Frequency of isolation of each 0-antigen type of E. coli expressed as percentages of the 40 faecal samples taken on 5 occasions from 8 calves

Antibiotic Antibiotic Antibiotic Antibiotic 0-type resistant sensitive 0-type resistant sensitive

1 2 4 5 6 7 8

10 11 15 17 21 23 25 35 36 41 45 49 50 52

15.0 2.5 7 .5

12.5 32.5 5.0

37.5 2 . 5 7 . 5

2 . 5 5.0

-

20.0 7 . 5 - 2 . 5

2 . 5

2 . 5 2 . 5

-

-

- 32.5

20.0 -

- -

12.5 5.0

17.5 7 . 5 2 . 5

15.0 2 .5

5.0

-

-

-

55 64 68 70 88

101 107 108 113 119 123 128 136 138 145 153

NT

A

2 . 5

5.0 2 . 5

5.0

-

-

- 10.0 5.0 5 . 0 2 - 5

27.5

35.0

5 .0

-

7 .5 2 . 5 2 .5

7 . 5

2 . 5 5 .0 2 .5

15.0

-

-

- - 5.0

5 . 0 2 . 5

37.5

-

NT: not-typeable; A: autoagglutinable.

The frequency of isolation of each 0-type from all faecal samples from the 8 calves is also given. Certain 0-types are clearly more dominant than others in this group of calves.

To study the persistence and the epidenliology of the E. coli 0-types within each calf and within the group, weekly samples were examined. Ideally, daily sampling would have provided a better understanding of these problems but this was not practicable. The distribution of the 37 0-types of E. coli in each of the calves on a weekly basis is set out in Table 5. The information is divided on the basis of whether the strains were antibiotic sensitive or resistant. The data indicate the combined effects of cross-infection and persistence of each 0-type within the calves. The results are obviously influenced by intermittent excretion which must have occurred especially with the less dominant strains.

Cross-infection must have occurred between the calves during the period of study but it is difficult from the data to establish the routes of cross-infection. A more useful approach is to compare the frequency of isolation of the various 0-types over the whole period of study both within individual calves and between calves. Table 6 presents this analysis.

TABL

E 5 A

long

itudi

nal s

tudy

of t

he 0

-ant

igen

type

s in

8 c

alve

s ov

er a

5-w

eek

sam

plin

g pe

riod

Cal

f ref

eren

ce n

umbe

r r

h

> O

rder

of

1 2

3 4

5 6

7 8

sam

plin

g --J---,

,-----,

A

,---'--

in w

eeks

S

R

S R

S

R

S R

S

R S

R

SR

S

R

1 N

T

128

6 15

3 7

NT

8

A

11

50

128

2 6

123

6 12

3 23

11

9 N

T

66

15

8

145

23

NT

10

1

66

7 8 11

15

3 A

86

41

23

N

T

NT

8 8

23

11

68

101

138

NT1

53

36

8 11

9

17

8 12

8 N

T

108

88

N

T

17

17

NT

N

T

66

8

8

68

23

113

153

NT

6 13

6 25

6

88

8 23

52

25

12

8 55

15

3 13

6 N

T

NT

1 6 8 23

45

123

NT

35

ir

3 8

4

55

123

119

153

6 15

3 36

64

N

T 6

NT

15

3 10

8 8

NT

N

T

8 36

N

T

136

153

NT

2 4

88

49

1 8 25

NT

55

8

61

88

25

23

21

14

5 15

3 13

6 N

T

9

4 6

6

NT

N

T

23

5 36

23

15

3

36

4 6 8 15

3

23

107

6 cl c F E

153

NT

82

11

9 5 6

NT

5 8

1

119

5 10

23

53

NT

To

tals

9

16

25

65

15

36

36

5 49

6 23

23

23

66

11

9 12

8 25

8

61

25

55

21

68

N

T

70

15

12

27

14

14

28

9 18

27

7

15

4 13

32

17

9

13

17

12

22

29

W

W 4

S=an

tibio

tic s

ensi

tive;

R=a

ntib

iotic

res

ista

nt; N

T=no

n-ty

peab

le;

A=a

utoa

gglu

tinab

le;

Tot

als

S 84

; R 1

13; S

+R

197

.

TA

BL

E 6

The

nicn

iber

of

times

the

doin

inan

t 0-

type

s w

ere

isol

ated

from

all

calv

es o

n ea

ch s

ainp

ling

occa

sion

and

from

all

sam

ples

obt

aine

dfio

m e

ach

calf

Ord

er o

f sa

mpl

ing

(wee

ks)

Cal

f ref

eren

ce n

umbe

rs

0-ty

pe

1 5 6 8 23

36

55

119

123

128

136

1 15

3 N

T 1

--

~ - -~

iH

23

36

55

1 I9

123

128

136 I

Ant

ibio

tic

sens

itive

is

olat

es

I A

ntib

iotic

re

sist

ant

isol

ates

r 153 I NT 12

34

5

~~

--

-_

_

--

__

-

24

13

3

22

2-

2

12

-3

1

-1

12

1

-1

11

1

-2

1-

3

--

-_

_

--

--

_

-1

-

1-

1

-

43

43

-

-1

11

2

14

3

4-

33

6

42

21

1

3-

13

-

- I

--

--

-

--

-

--

--

_

--

-_

-

-3

1-

-

31

--

1

-1

1-

-

32

43

-

44

31

2

12

34

56

78

- -

-

-

-

- -

-

23

31

-1

-3

2

11

12

--

1

-2

1-

2-

-

2 -

12

11

--

-

I-

--

--

1

2

22

--

1

1-

-

-

-

-

-

-

-

-

-

-1

--

__

--

2

32

21

-1

4

I-

--

-

1-

3

11

21

--

--

1

22

4-

31

-

-1

23

22

32

1

-3

21

1-

-

--

--

I-

--

-

-

-

-

-

- -

-

21

--

-

I-

-

I]

--

-

21

-

I-

-

1 2

13

22

-2

-

31

12

22

12

-_

-_

No.

of

calv

es

from

whi

ch

each

0-t

ype

was

iso

late

d

0 0 6 6 4 4 3 4 0 0 1 1 7 3 4 6 7 5 1 0 0 3 4

~

2 6 8

Tot

al n

o.

of i

sola

tes

of e

ach

0-ty

pe

0 0 13 8 7 5 4 6 0 0 2 1 15

5 5 13

15 8 1 0 0 4 5 2 12

14


Of the 37 0-types approximately two-thirds were isolated from fewer than 4 samples, and often on only one occasion from single calves. Only the more dominant 0-types are listed in Table 6. The antibiotic sensitive strains are analysed separately from the resistant ones. In this Table numbers of samples from which each dominant 0-type was isolated both from all calves on each sampling occasion and from all samples from each calf, are given separately. From this analysis it may be seen that many 0-types were present in a large proportion of calves and on the majority of sampling occasions throughout the period of study. Whether these were invariably present in the calf gut but were not excreted or failed to be isolated on each occasion, or whether cross- infection between calves was continuously occurring could not be determined from the available data.

A total of 456 (64 %) of the 713 isolates of E. coli from the 8 calves were resistant to at least 1 antibacterial. These were isolated from 32 out of the 40 faecal samples and represented 25 0-antigen types and non-typeable strains. Many were multiply resistant and the percentages demonstrating 1 , 2, 3 , 4 or 5 resistant determinants were found to be 21.4, 35.9, 21.4, 17.9 and 3.6%, respectively.

Discussion The importance of cattle and calves as potential sources of E. coli for man will depend on the load and frequency of excretion of these organisms. From the longitudinal studies described it would appear that E. coli is not regularly present in the faeces of adult cows and, when it does occur, is present in relatively low numbers. This is in agreement with other workers (Smith, 1961 ; Larsen & Larsen, 1972). Long periods of time often occurred when no E. coli could be detected.

In contrast, the calves regularly excreted E. coli in high numbers. It is to be expected, and we have supporting evidence (Linton, unpublished), that the load of carcass contamination at slaughter, and hence a possible route to man, will be proportional to the numbers of E. coli in the rectum. Carcasses from calves are therefore more likely to be contaminated with E. coli than those from cows. The importance of this observation, in the context of the potential sources of drug resistant strains for man, is enhanced by the additional evidence that antibiotic resistance was found frequently in E. coli from the calves but on no occasion from the cows. The absence of drug resistant E. coli in adult cows has been frequently observed (Linton, unpublished). From these observations it may be concluded that adult cattle are unlikely to be a significant source of drug resistant E. coli for man.

The longitudinal study on cows revealed a limited number of E. coli 0-types over an 88-day study period. Of the 6 0-types found, about half were regularly found throughout the period and in each of the cows, suggesting a limited but constant association of these 0-types with the gut of the adult cow.

In calves the variety of 0-types was greater. About one-third of the 37 0-types encountered were regularly present throughout the 5-week study period and in a large proportion of the calves. These strains appeared to be good colonizers of the calf intestine.

Being a longitudinal survey, the numbers of animals included in the present study was small. However, it is of interest to compare the 0-antigen types isolated in the survey with those from other surveys on calves (Howe & Linton, 1976) and man

26


(Hartley et al., 1975). Not only were the presumptive coliform counts in cows invariably lower than those found in calves but the number of O-types isolated was limited to six. All were sensitive to antibiotics. The same six O-types have also been isolated from man (Hartley el al., 1975) but only 3 of them from calves, 1 in the present survey and 3 in the larger survey (Howe & Linton, 1976).

In the present longitudinal study 37 different O-antigen types of E. coli were isolated from calves. These included 10 antibiotic sensitive E. coli, 16 O-types of antibiotic resistant E. coli and 11 types which occurred as both sensitive and resistant strains. The larger proportion of these O-types have been encountered in a more extensive calf survey (Howe & Linton, 1976), but an additional 10 O-types were isolated from the present longitudinal study, They included antibiotic resistant E. coli of O-types 10, 52,70 and 138 and antibiotic sensitive E. coli of O-types 41,49, 55,64, 136, 153.

Taking both surveys together, a total of 103 O-types have now been isolated from calves of which antibiotic resistant E. coli were found in 64 O-types and sensitive strains in 86 O-types. Of the 103 O-types, 78 have also been isolated from human sources (Hartley et al., 1975).

The expert technical assistance of Mrs Helen Clenients and her colleagues is grate- fully acknowledged. This work was supported by grants from the Medical and Agricultural Research Councils.

References HARTLEY, C. L., HOWE, K., LINTON, A. H., LINTON, K. B. & RICHMOND, M. H. (1975). The

distribution of R plasmids among O-antigen types of Escherichia coli isolated from human and animal sources. Anti-microb. Ag. Chemofher. 8, 122.

HOWE, K. & LINTON, A. H. (1976). The distribution of O-antigen types of Escherichh coli in normal calves, compared with man, and their R plasmid carriage. J. uppl. Bad. 40, 317.

LARSEN, H. E. & LARSEN, J. L. (1972). Occurrence and distribution of drug resistant E. coli in faeces from cattle and swine. Nord. vet. Med. 24, 651.

LINTON, A. H., HOWE, K. & OSBORNE, A. D. (1975). The effects of feeding tetracycline, nitrovin and quindoxin on the drug-resistance of coli-aerogenes bacteria from calves and pigs. J . appl. Bact. 38,255.

SMITH, H. W. (1961). The development of the bacterial flora of the faeces of animals and man: the changes that occur during ageing. J. appl. Bact. 24, 235.

SMITH, H. W. & CRABB, W. E. (1961). The faecal bacterial flora of the faeces of animals and man: its development in the young. J. Path. Bact. 82, 53.