ARS VETERINARIA, Jaboticabal, SP, Vol. 18, nº 2, 125-129, 2002.
ISSN 0102-6380
ANTIMICROBIAL SUSCEPTIBILITY OF Escherichia coli
ISOLATED FROM MASTITIC BOVINE MILK
(SENSIBILIDADE ANTIMICROBIANA DE Escherichia coli
ISOLADA DE LEITE BOVINO MASTÍTICO)
V. BUERIS 1, M. G. P. CORRÊA1,2, J. M. MARIN3
SUMMARY
A total of 182 Escherichia coli strains isolated from 2,144 samples of mastitic bovine milk were tested for O serogroups.
Fifty-seven strains with classical enteropathogenic E.coli (EPEC) serogroups O26, O55, O111, and O119 were examined for
their susceptibility to eight antimicrobial agents. All were susceptible to gentamicin, but resistance to tetracycline (24.5%),
nalidixic acid (17.5%), carbenicillin (15.7%), ampicillin (7.0%), amikacin (7.0%), cephalothin (2.0%) and streptomycin (1.7%)
was observed. None of the E.coli strains studied showed resistance to all antimicrobial agents tested.
KEY-WORDS: Escherichia coli. Bovine mastitis. Antibiotic resistance.
RESUMO
Um total de 182 cepas de Escherichia coli foram isoladas de 2.144 amostras de leite mastítico, e os sorogrupos
analisados. Cinqüenta e sete cepas apresentando os sorogrupos clássicos de E.coli enteropatogênica (EPEC) O26, O55,
O111 e O119 foram examinados para verificar a sua sensibilidade a oito agentes antimicrobianos. Todas as cepas eram
sensíveis a gentamicina, mas apresentaram resistência a tetraciclina (24,5%), ácido nalidíxico (17,5%), carbenicilina (15,7%),
ampicilina (7,0%), amicacina (7,0%), cefalotina (2,05) e estreptomicina (1,7%). Nenhuma das cepas de E.coli estudadas
mostrou resistência a todos os agentes antimicrobianos testados.
PALAVRAS-CHAVE: Escherichia coli. Mastite bovina. Resistência a antibióticos.
INTRODUCTION
Bovine mastitis is an infectious disease caused by
various pathogens, which differ in virulence, frequency
and persistence in the mammary gland. The proportion of
mastitic infection due to Escherichia coli has increased in
recent years, mainly as a result of improved control programs of mastitis caused by Gram-positive bacterial pathogens (FANG & PYORALA, 1996; JONES, 1986).
The extensive use of antibiotics has led to a heavy
increase in antibiotic resistance in animal production world-
wide (FRANKLIN, 1999). Antimicrobial therapy is usually
used for E.coli mastitis even though its therapeutic value
has not been proved in clinical trials (ERSKINE et al., 1991;
PYORALA et al., 1994).
There is an increasing concern about the problem
of antimicrobial resistance and about variability in antibiotic susceptibility patterns for bacteria isolated from cows,
so it is prudent periodically to monitor bacterial isolates
from cows with mastitis in a study population for the antibiotic susceptibility (BEZEK, 1998).
1 Pós-Graduanda, Área de Microbiologia, Faculdade de Ciências Agrárias e Veterinárias - FCAV-Unesp - Campus Jaboticabal
2 Microbiologista do Laboratório Vitafort S.A.
3 Professor Doutor, Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia - Universidade de
São Paulo - Campus Ribeirão Preto - Corresponding author: José Moacir Marin - Departamento de Morfologia, Estomatologia
e Fisiologia - Laboratório de Genética - Avenida do Café s/nº - 14040-904 - Ribeirão Preto – São Paulo – Brasil. E-mail:
[email protected]
125
BUERIS, V., CORRÊA, M. G. P., MARIN, J. M. Antimicrobial susceptibility of Escherichia coli isolated from mastitic bovine milk. / Sensibilidade
antimicrobiana de Escherichia coli isolada de leite bovino mastítico. Ars Veterinaria, Jaboticabal, SP, Vol. 18, nº 2, 125-129, 2002.
Because of the lack of information available
(LANGENEGGER et al., 1970: LANGONI et al., 1991;
MORENO et al., 1997) concerning the characteristics of
the bovine mastitis due to E.coli in Brazil and the fact that
an increased number of antibiotics has been used in
antimicrobial therapy, we decided to investigate the
incidence of E.coli mastitis and its antibiotic resistance
spectra.
MATERIAL AND METHODS
Samples and Bacteriological Examinations: Milk
samples (2,144) from cows in seven Brazilian states ( Espirito
Santo, Goias, Mato Grosso do Sul, Minas Gerais, Parana,
Rio Grande do Sul, São Paulo), from March 1997 to August
1998, were collected aseptically. The teat ends were cleaned
with alcohol (70%), moistened swabs and allowed to dry.
After discarding the first few streams, the samples (2-4mL)
were collected into sterile 10mL glass flasks and submitted
to California Mastitis Test (CMT) according to the method
proposed by SCHALM & NOORLANDER (1957) on a scale
of 1-5 (KLASTRUP, 1975). CMT-positive samples were kept
refrigerated at about 4 oC and dispatched to the laboratory
without delay. Bacterial strains were isolated in
MacConKey (Difco) and identified by standard
bacteriological methods (KONEMAN et al., 1997). The
tests performed included: Gram stain, triple sugar-iron agar,
phenylalanine deaminase, Simmons citrate, methyl red,
Voges-Proskauer, indole production, urease, ornithine and
lysine decarboxylases, arginine dihydrolase, catalase,
oxidase, nitrate redutase reaction, and fermentation of
glucose, lactose, sucrose, arabinose, raffinose, rhamnose,
trehalose, xylose, dulcitol, mannitol and inositol.
Fermentation of carbohydrates was tested on phenol red
broth base (Difco) supplemented with 1% of the appropriate
carbohydrate. The results were read after 24 and 48 h of
incubation at 37o C.
O-serotyping technique: E.coli isolated on
MacConkey agar Petri dishes and biochemically confirmed
were submitted to slide-agglutination tests using
polyvalent and monovalent sera against classic
enteropathogenic E.coli (EPEC) serogroups O26, O55, O86,
O111, O114, O119, O125, O126, O127, O128, O142 and O158.
Commercially available antisera from Probac, Brazil (São
Paulo) were used.
Sensitivity Testing: Antibiotic susceptibility testing
was performed using the Kirby-Bauer disk diffusion
method with Mueller Hinton agar plates (CRAIG, 1993).
After culturing each E.coli strain on Brain Heart Infusion
Agar (Difco), five isolated colonies were inoculated into
5.0 mL Tryptic Soy Broth (Difco) and incubated for 2 to 4
hours at 37OC. Turbidity of this log-phase suspension was
adjusted to 0.5 McFarland before inoculation on Muller
Hinton agar plates (Difco). The following disks (CEFAR)
were tested: amikacin (30μg), ampicillin (10μg), carbenicillin (100μg), cephalothin (30μg), streptomycin (10μg), gentamicin (10μg), nalidixic acid (30μg) and tetracycline (30μg).
After 16 to 18 h of incubation at 37 OC, zones of inhibition
around all disks were measured using reflected light and
interpreted according to the NCCLS- National Committee
for Clinical Laboratory Standards (1999) recommendations.
RESULTS AND DISCUSSION
Identification of bacteria that cause mastitis in cows
and their antibiotic susceptibility patterns are necessary
to select appropriate antibiotics for treatment.
E.coli were isolated from 8.5% of all milk samples.
Among the 182 E.coli isolated (Table1), 141 strains had
the serogroup identified, twelve O-serogroups could be
identified (77.5% of total). O-serogroup could not be
determined with regard to 41 isolates.
Out of the 279 E.coli strains investigated by
LINTON et al. (1979), 217 (77.1%) could be O-serotyped,
and 67 differents O-serotypes were found. This indicates
that E.coli mastitis is not caused by a limited number of
pathogenic strains. Many other authors similarly reported
having found a wide range of serotypes in cattle (LINTON
& ROBINSON, 1984; WILSON et al., 1992; MYAU et al.,
1998).
Verocytotoxin-producing E.coli (VTEC) is a
naturally occurring organism in the gut flora of cattle
(CHAPMAN et al., 1997), and it is recognized as the most
important cause of potentially life-threatening human
diseases such as haemorrhagic colitis and haemolytic
uraemic syndrome. However EPEC are an important cause
of infant disease in the developing world (GOMES et al.,
1989), and classical EPEC serogroups such as O111 and
O119 have been recognized since a long time as the most
important EPEC serogroups associated with infant diarrhea
in Brazil (CAMPOS et al., 1994; ROSA et al., 1998), what
represent a reason of concern considering the great number
of O111 and O119 isolates from mastitic milk. These are
preliminary results, and further serotyping work is needed
to confirm that these are EPEC serotypes.
Outbreaks of foodborne illness caused by E.coli
have been associated with consumption of milk, ice cream,
kefir, cheese and other dairy products (PARK et al., 1973;
GLATZ & BRUDVIG, 1980). JONES et al. (1967), in a study
126
BUERIS, V., CORRÊA, M. G. P., MARIN, J. M. Antimicrobial susceptibility of Escherichia coli isolated from mastitic bovine milk. / Sensibilidade
antimicrobiana de Escherichia coli isolada de leite bovino mastítico. Ars Veterinaria, Jaboticabal, SP, Vol. 18, nº 2, 125-129, 2002.
of coliforms in Canadian pasteurized dairy products, found
three EPEC serotype isolates. Also ABBAR & KADDAR
(1991) reported the isolation of EPEC serotypes isolates
from cheese kishfa and gaymerr from Iraq. In Brazil
BARUFALDI et al. (1984) and more recently SILVA et al.
(2001) isolated E.coli strains from pasteurized milk of type
B, and 46 (22.1%) of these strains were serologically
identified as EPEC. It is believed that the isolation of EPEC
serogroups from milk represents a potential risk especially
for children.
SMITH et al. (1985) reported the variation of percentage of E.coli mastitis in bovine between 1.3% and
3.0%, while PAAPE et al. (1996) reported a low percentage
around 1.0%, indicating the correct management of the
herd and prevention of the environmental contamination.
In Brazil, however, we found a constant increment in the
participation of E.coli in bovine mastitis, FERREIRA et al.
(1981) 1.3%, LANGONI et al. (1991) 2.8%, MORENO et al.
(1997) 7.1%, and 8.5% (this work). The explanation commonly used for this increasing incidence is reduced coccal
subclinical mastitis, brought about by a combination of
dry cow therapy and teat dipping, plus increased E.coli
challenge related to unhygienic loose-housing conditions
(BRAMLEY & DODD, 1984).
Therapeutic value of antibiotics to treat coliform
mastitis remains uncertain. The results of several trials have
failed to show a beneficial effect of antibiotics compared
with untreated controls or with administration of drugs
without activity against coliforms in vitro ( PYORALA &
SYVAJARVI, 1987; ERSKINE et al., 1991; VAN
EENANNAAM et al., 1995). The development of resistance to antimicrobials used in animal may present a hazard to humans when the resistant bacteria can cause disease in humans and can be transmitted via contaminated
food (McKELLAR, 1998).
Fifty-seven of 141 (40%) E.coli isolates were tested
for antibiotic susceptibility. The frequency and patterns
of resistance to the antibiotics are shown in Table 2.
Bacterial resistance to antibiotics varies widely
(BARROW & HILL, 1989; BEZEK, 1998) and depends on
country, use of drugs and infection control practice.
However, the extensive use of antibiotics has led to a heavy
increase in antibiotic resistance in animal production
worldwide (McKELLAR, 1998).
The occurrence of multi-resistant strains may be a
response to the selective pressure caused by the abusive
addition of antibiotics to animal rations. However the
antibiotic sensitivity patterns observed were somewhat
different from those observed by other Brazilian authors
(LANGONI et al., 1991; MORENO et al., 1997), who found
Table 1 - Distribution and frequence of Escherichia coli
O-serogroup isolated from bovine mastitis, in
seven Brazilian states (1997-1998).
Table 2 - Patterns and phenotypes of drug resistance
amongst 57 strains of Escherichia coli isolated
from bovine mastitis in seven Brazilian states
(1997-1998).
FREQ U EN CE
PATTERNS
PHENOTYPES
8 .8
Resistant to none (31) *
Sensitive (31)
16
8 .8
Resistant to 1 drug (12)
Ap (1)
125
14
7 .7
Ca (2)
158
14
7 .7
Na (3)
119
13
7 .2
Te (6)
55
12
6 .6
86
12
6 .6
127
11
6 .0
142
10
5 .5
114
09
5 .0
126
07
3 .8
128
07
3 .8
OTHER O-
41
2 2 .5
o
O -S E R O G R O U P S
N
111
16
26
%
Resistant to 2 drugs (10)
Na - St (1)
Na - Ce (2)
Am - Ca (3)
Na - Te (4)
Resistant to 3 drugs (4)
SERO G RO U PS
127
Ap - Ca - Te (1)
Am - Ca - Te (3)
* Number of strains is given in parentheses.
Am- Amikacin, Ap- Ampicillin, Ca- Carbenicillin, Ce- Cephalothin, Ge- Gentamicin, Na- Nalidixic acid, St- Streptomycin,
Te- Tetracycline.
BUERIS, V., CORRÊA, M. G. P., MARIN, J. M. Antimicrobial susceptibility of Escherichia coli isolated from mastitic bovine milk. / Sensibilidade
antimicrobiana de Escherichia coli isolada de leite bovino mastítico. Ars Veterinaria, Jaboticabal, SP, Vol. 18, nº 2, 125-129, 2002.
an increased incidence of resistance in E.coli population,
including a more extended multidrug resistance.
In this study, 54.4% of the strains tested were sensitive to all antimicrobial agents analyzed and only four
strains showed resistance to three antibiotics among the
eight tested, so the resistance patterns were not so
upsetting. However the constant monitoring of resistance
patterns of clinical isolates is necessary to chose suitable
antibiotics for therapeutic use, in order to avoid the selection of multidrug-resistant strains.
ACKNOWLEDGEMENTS
We would like to thank to Laboratorio Vitafort S.A
for the mastitic bovine milk samples.
ARTIGO RECEBIDO: DEZEMBRO/2001
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