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ARS VETERINARIA, Jaboticabal, SP, Vol. 18, nº 2, 125-129, 2002. ANTIMICROBIAL SUSCEPTIBILITY OF Escherichia coli
ISOLATED FROM MASTITIC BOVINE MILK
(SENSIBILIDADE ANTIMICROBIANA DE Escherichia coli V. BUERIS 1, M. G. P. CORRÊA1,2, J. M. MARIN3
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 fortheir 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.
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 eramsensíveis 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 estudadasmostrou resistência a todos os agentes antimicrobianos testados.
PALAVRAS-CHAVE: Escherichia coli. Mastite bovina. Resistência a antibióticos.
INTRODUCTION
wide (FRANKLIN, 1999). Antimicrobial therapy is usuallyused for E.coli mastitis even though its therapeutic value Bovine mastitis is an infectious disease caused by has not been proved in clinical trials (ERSKINE et al., 1991; various pathogens, which differ in virulence, frequency and persistence in the mammary gland. The proportion of There is an increasing concern about the problem mastitic infection due to Escherichia coli has increased in of antimicrobial resistance and about variability in antibi- recent years, mainly as a result of improved control pro- otic susceptibility patterns for bacteria isolated from cows, grams of mastitis caused by Gram-positive bacterial patho-gens (FANG & PYORALA, 1996; JONES, 1986).
so it is prudent periodically to monitor bacterial isolates The extensive use of antibiotics has led to a heavy from cows with mastitis in a study population for the anti- increase in antibiotic resistance in animal production world- biotic 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:
jmmarin@rge.fmrp.usp.br
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 Agar (Difco), five isolated colonies were inoculated into (LANGENEGGER et al., 1970: LANGONI et al., 1991; 5.0 mL Tryptic Soy Broth (Difco) and incubated for 2 to 4 MORENO et al., 1997) concerning the characteristics of hours at 37OC. Turbidity of this log-phase suspension was the bovine mastitis due to E.coli in Brazil and the fact that adjusted to 0.5 McFarland before inoculation on Muller an increased number of antibiotics has been used in Hinton agar plates (Difco). The following disks (CEFAR) antimicrobial therapy, we decided to investigate the were tested: amikacin (30μg), ampicillin (10μg), carbenicil- incidence of E.coli mastitis and its antibiotic resistance lin (100μg), cephalothin (30μg), streptomycin (10μg), gen- tamicin (10μg), nalidixic acid (30μg) and tetracycline (30μg).
After 16 to 18 h of incubation at 37 OC, zones of inhibitionaround all disks were measured using reflected light and MATERIAL AND METHODS
interpreted according to the NCCLS- National Committeefor Clinical Laboratory Standards (1999) recommenda- Samples and Bacteriological Examinations: Milk
samples (2,144) from cows in seven Brazilian states ( EspiritoSanto, Goias, Mato Grosso do Sul, Minas Gerais, Parana,Rio Grande do Sul, São Paulo), from March 1997 to August RESULTS AND DISCUSSION
1998, were collected aseptically. The teat ends were cleanedwith alcohol (70%), moistened swabs and allowed to dry.
Identification of bacteria that cause mastitis in cows After discarding the first few streams, the samples (2-4mL) and their antibiotic susceptibility patterns are necessary were collected into sterile 10mL glass flasks and submitted to select appropriate antibiotics for treatment.
to California Mastitis Test (CMT) according to the method E.coli were isolated from 8.5% of all milk samples.
proposed by SCHALM & NOORLANDER (1957) on a scale Among the 182 E.coli isolated (Table1), 141 strains had of 1-5 (KLASTRUP, 1975). CMT-positive samples were kept the serogroup identified, twelve O-serogroups could be refrigerated at about 4 oC and dispatched to the laboratory identified (77.5% of total). O-serogroup could not be without delay. Bacterial strains were isolated in determined with regard to 41 isolates.
MacConKey (Difco) and identified by standard Out of the 279 E.coli strains investigated by bacteriological methods (KONEMAN et al., 1997). The LINTON et al. (1979), 217 (77.1%) could be O-serotyped, tests performed included: Gram stain, triple sugar-iron agar, and 67 differents O-serotypes were found. This indicates phenylalanine deaminase, Simmons citrate, methyl red, that E.coli mastitis is not caused by a limited number of Voges-Proskauer, indole production, urease, ornithine and pathogenic strains. Many other authors similarly reported lysine decarboxylases, arginine dihydrolase, catalase, having found a wide range of serotypes in cattle (LINTON oxidase, nitrate redutase reaction, and fermentation of & ROBINSON, 1984; WILSON et al., 1992; MYAU et al., glucose, lactose, sucrose, arabinose, raffinose, rhamnose, trehalose, xylose, dulcitol, mannitol and inositol.
Verocytotoxin-producing E.coli (VTEC) is a Fermentation of carbohydrates was tested on phenol red naturally occurring organism in the gut flora of cattle broth base (Difco) supplemented with 1% of the appropriate (CHAPMAN et al., 1997), and it is recognized as the most carbohydrate. The results were read after 24 and 48 h of important cause of potentially life-threatening human diseases such as haemorrhagic colitis and haemolyticuraemic syndrome. However EPEC are an important cause O-serotyping technique: E.coli isolated on
of infant disease in the developing world (GOMES et al., MacConkey agar Petri dishes and biochemically confirmed 1989), and classical EPEC serogroups such as O111 and were submitted to slide-agglutination tests using O119 have been recognized since a long time as the most polyvalent and monovalent sera against classic important EPEC serogroups associated with infant diarrhea enteropathogenic E.coli (EPEC) serogroups O26, O55, O86, in Brazil (CAMPOS et al., 1994; ROSA et al., 1998), what O111, O114, O119, O125, O126, O127, O128, O142 and O158.
represent a reason of concern considering the great number Commercially available antisera from Probac, Brazil (São of O111 and O119 isolates from mastitic milk. These are preliminary results, and further serotyping work is neededto confirm that these are EPEC serotypes.
Sensitivity Testing: Antibiotic susceptibility testing
Outbreaks of foodborne illness caused by E.coli was performed using the Kirby-Bauer disk diffusion have been associated with consumption of milk, ice cream, method with Mueller Hinton agar plates (CRAIG, 1993).
kefir, cheese and other dairy products (PARK et al., 1973; After culturing each E.coli strain on Brain Heart Infusion GLATZ & BRUDVIG, 1980). JONES et al. (1967), in a study 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 mastitis remains uncertain. The results of several trials have three EPEC serotype isolates. Also ABBAR & KADDAR failed to show a beneficial effect of antibiotics compared (1991) reported the isolation of EPEC serotypes isolates with untreated controls or with administration of drugs from cheese kishfa and gaymerr from Iraq. In Brazil without activity against coliforms in vitro ( PYORALA & BARUFALDI et al. (1984) and more recently SILVA et al. SYVAJARVI, 1987; ERSKINE et al., 1991; VAN (2001) isolated E.coli strains from pasteurized milk of type EENANNAAM et al., 1995). The development of resis- B, and 46 (22.1%) of these strains were serologically tance to antimicrobials used in animal may present a haz- identified as EPEC. It is believed that the isolation of EPEC ard to humans when the resistant bacteria can cause dis- serogroups from milk represents a potential risk especially ease in humans and can be transmitted via contaminated SMITH et al. (1985) reported the variation of per- Fifty-seven of 141 (40%) E.coli isolates were tested centage of E.coli mastitis in bovine between 1.3% and for antibiotic susceptibility. The frequency and patterns 3.0%, while PAAPE et al. (1996) reported a low percentage of resistance to the antibiotics are shown in Table 2.
around 1.0%, indicating the correct management of the Bacterial resistance to antibiotics varies widely herd and prevention of the environmental contamination.
(BARROW & HILL, 1989; BEZEK, 1998) and depends on In Brazil, however, we found a constant increment in the country, use of drugs and infection control practice.
participation of E.coli in bovine mastitis, FERREIRA et al. However, the extensive use of antibiotics has led to a heavy (1981) 1.3%, LANGONI et al. (1991) 2.8%, MORENO et al. increase in antibiotic resistance in animal production (1997) 7.1%, and 8.5% (this work). The explanation com- monly used for this increasing incidence is reduced coccal The occurrence of multi-resistant strains may be a subclinical mastitis, brought about by a combination of response to the selective pressure caused by the abusive dry cow therapy and teat dipping, plus increased E.coli addition of antibiotics to animal rations. However the challenge related to unhygienic loose-housing conditions antibiotic sensitivity patterns observed were somewhat different from those observed by other Brazilian authors Therapeutic value of antibiotics to treat coliform (LANGONI et al., 1991; MORENO et al., 1997), who found Table 1 - Distribution and frequence of Escherichia coli
Table 2 - Patterns and phenotypes of drug resistance
O-serogroup isolated from bovine mastitis, in amongst 57 strains of Escherichia coli isolated from bovine mastitis in seven Brazilian states 4 1 2 2 .5 * Number of strains is given in parentheses.
Am- Amikacin, Ap- Ampicillin, Ca- Carbenicillin, Ce- Cephal- othin, 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.
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ACKNOWLEDGEMENTS
ERSKINE, R. J., TYLER, J. W., RIDDEL, M.G. Theory, use We would like to thank to Laboratorio Vitafort S.A and realities of efficacy treatment of acute coliform masti- for the mastitic bovine milk samples.
tis. Journal of the American Veterinary Medical Asso-
ciation, v. 198, nº 6, p. 980-4, 1991.
ARTIGO RECEBIDO: DEZEMBRO/2001
FANG, W., PYORALA, S. Mastitis-causing Escherichia
coli: serum sensitivity and susceptibility to selected anti-
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