9 Journal of Food Protection, ol., No., 00, Pages 9 9 Copyright q, International Association for Food Protection Enterococcus faecalis and Enterococcus faecium Isolates from Milk, Beef, and Chicken and heir Antibiotic Resistance W. CHINGWARU, S. F. MPUCHANE, AND B. A. GASHE* Department of Biological Sciences, University of Botswana, Gaborone, Private Bag 00, Gaborone, Botswana MS 0-8: Received 8 August 00/Accepted December 00 ABSRAC he occurrence and antibiotic resistance of enterococci, especially Enterococcus faecalis and Enterococcus faecium, in milk, beef, and chicken in Gaborone, Botswana, were studied. Enterococci were isolated from these sources with the use of bile esculin agar and identi ed with API 0 Strep kits. Antibiotic resistance was determined by the disk diffusion method. he antibiotics tested were vancomycin, teicoplanin, ampicillin, tetracycline, and cephalothin. Among the, enterococci isolated from the samples, E. faecalis (.%) and E. faecium (9.0%) were found to be the predominant species. Other enterococcal species made up % of the isolates. More than 9 and 9% of the E. faecalis and E. faecium isolates, respectively, were found to be resistant to ampicillin. Almost,., and.% of the E. faecalis isolates from milk, beef, and chicken, respectively, were also resistant to cephalothin. he percentages of E. faecium isolates that were found to be resistant to cephalothin were.8,.9, and.% for milk, beef, and chicken, respectively. Resistance to vancomycin was widespread. It was found that 8.8,.8, and.% of the E. faecalis isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. In contrast,.8,., and 0.% of the E. faecium isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. Isolates that were resistant to multiple drugs were found in relatively large numbers. Enterococci are found in a variety of environments. hey frequently occur in large numbers in dairy and food products (, ). hey also occur in humans and animals (, 8). Enterococci have been regarded as harmless commensals and have been used as indicators of fecal contamination (). Enterococci can survive for long periods on environmental surfaces and on the hands of health care workers (). Several investigators have found enterococci, particularly those that are resistant to multiple drugs, on various objects in a hospital environment, including on bed rails, night tables, curtains, bathroom sinks, toilet rings, electronic thermometers, and other patient care equipment (,, 9). Enterococci have recently been identi ed as secondary invaders in hospital-acquired infections (). Since the 980s, numbers of infections in hospitalized patients due to enterococci have increased tremendously (). Colonization with vancomycin-resistant enterococci (RE) in nonhospitalized patients has been on the increase in the United States and Europe (). In the United Kingdom, RE were isolated from raw sewage and from farm animals, including pigs, chickens, ducks, and turkeys, but no RE were recovered from cattle and sheep (). Some fermented traditional foods in Africa also harbor enterococci, although their resistance patterns are not known (). he development of the resistance of enterococci to vancomycin in animals is mainly attributed to the use of antibiotics, especially avoparcin (a glycopeptide antibiotic), in animal feeds as a growth promoter (9). In Europe, evidence suggests that foodborne RE may cause human coloniza- * Author for correspondence. el: --99; Fax: -809; E-mail: gasheba@mopipi.ub.bw. tion (, ). Some enterococci are resistant to commonly used antibiotics such as ampicillin, tetracycline, and cephalothin. However, such enterococci can be signi cantly reduced with vancomycin and teicoplanin therapy. A problem arises when these enterococci are also resistant to vancomycin and teicoplanin. here is little information available on the prevalence of drug-resistant enterococci in southern Africa. A signi - cant percentage of people who are immunocompromised as a result of acquired immunode ciency syndrome (AIDS) and those under intensive care in health care centers live in this region. In Botswana, unfortunately, no assessment of the prevalence of antibiotic-resistant enterococci among human immunode ciency virus AIDS patients has been carried out, but there is a generally high level of antibioticresistant enterococci among hospitalized patients. When patients are infected with drug-resistant enterococci, there may be a problem with the availability of or the selection of an appropriate drug for treatment. he appearance and increase in the incidence of vancomycin elsewhere has become a cause for great concern. RE could be coming partly from food animals, and this possibility has prompted much research on microbial isolates, mainly in the developed world. Reservoirs for antibiotic-resistant enterococci have not been completely determined. Animals, human food, and the inanimate environmental components have been suspected sources of some of the resistant clinical isolates (,,, 8, 9). Much information on the antibiotic resistance of enterococci isolated from clinical sources has accumulated (), but the levels of antibiotic resistance of enterococci isolated from foods have not yet been thoroughly docu-
9 CHINGWARU E AL. J. Food Prot., ol., No. ABLE. Enterococcal species from different sources Enterococcus species % of positive samples (no. positive/no. tested) Milk Beef Chicken E. faecalis E. faecium E. gallinarum E. avium E. durans E. hirae E. casseli avus E. mundtii.8 (/90) 8. (8/90).9 (/90). (0/90). (/90). (/90) 9. (9/90) 0. (/90). (/9) 8. (/9).9 (/9).0 (8/9). (/9). (/9) 0 (0/9) 0 (0/9).9 (0/8).9 (/8).0 (/8). (/8) 0 (0/8) 0 (0/8).9 (8/8) 0 (0/8) mented (). It has been realized that there is a need to assess the occurrence of drug resistance in animal products widely used for food in Botswana. his paper reports on enterococci, especially Enterococcus faecalis and Enterococcus faecium, isolated from milk, beef, and chicken and their patterns of susceptibility to ve of the antibiotics that are commonly directed against them. MAERIALS AND MEHODS Sampling period. his study was conducted between May 999 and July 000. Milk. Milk was either purchased from supermarkets or obtained from dairy farms located around Gaborone. A total of milk samples ( raw-milk samples and 8 pasteurized-milk samples) were obtained. Samples were transported to the laboratory in a cool box in 0 to 0 min. From each milk sample, ml was aseptically transferred into test tubes containing 9 ml of 0.% sterile peptone water (Mast Diagnostics, Merseyside, UK). Further dilution was carried out as deemed necessary. From the diluted samples, 0. ml was spread plated onto bile esculin agar (BEA; CM888). Plates were incubated at 8C for h (). Beef and chicken. Fifty-seven beef samples and 8 chicken samples were purchased from supermarkets around Gaborone. Portions ( g each) of minced and unprocessed beef or chicken samples were transferred into stomacher bags containing ml of sterile peptone water. he samples were homogenized with a stomacher (Seaward Stomacher 00, ekmar, Cincinnati, Ohio) for min. A 0.-ml portion of the diluted sample was spread plated onto BEA and incubated at 8C for h (0). Isolation. After meat and milk samples had been incubated on BEA at 8C for h, colonies showing brown halos were presumed to be enterococcus colonies (). A minimum of four colonies were picked, transferred onto fresh BEA, and incubated at 8C for h. Gram stains were carried out for each isolate to determine purity and reaction. Gram-positive cocci forming short chains in pairs were subcultured onto nutrient agar (CM) slants and incubated at 8C for h. he isolates were maintained at 8C with periodic subculturing. Identi cation. Pure cultures were streaked onto Columbia agar base (Oxoid, Basingstoke, UK) supplemented with % sheep blood and incubated at 8C for h. hese cultures were used to inoculate galleries of API 0 Strep (biomérieux, Paris, France) for the identi cation of enterococci. Readings were taken after h and after h as speci ed by the manufacturer. Species names were recorded for sample codes. Chemicals and media. All of the media and antibiotic disks used in the study were obtained from Oxoid. Antibiotic susceptibility testing. Each of the characterized Enterococcus species was inoculated in Mueller-Hinton broth (Oxoid). he asks were incubated at 8C on a Gallenkamp shaker (00 rpm) for h or slightly longer depending on the growth pattern for the Enterococcus species involved. A modi ed version of the method of Steinberg and Lehrer () was used to obtain an absorbance of at 00 nm. One milliliter of the cell suspension was transferred into to 0 ml of molten Mueller-Hinton agar, mixed thoroughly, and poured into plates. he mixture was then allowed to solidify in the plates. Antibiotic disks (ampicillin [0 mg], vancomycin [0 mg], teicoplanin [0 mg], tetracycline [0 mg], and cephalothin [0 mg]) were then placed onto the medium with sterile forceps. Care was taken to ensure that the disks were completely in contact with the agar. he plates were incubated at 8C for h. he diameters of the zones of inhibition were measured with a ruler as recommended by the NCCLS (8) and Willey et al. (0). Staphylococcus aureus ACC 9 was used as a control strain for the test. RESULS AND DISCUSSION Enterococcal diversity in the samples. A total of, enterococcal isolates were picked from the samples ( milk, beef, and 8 chicken samples). hese isolates comprised 90 isolates from milk, 9 from beef, and 8 from chicken. E. faecalis (.%) and E. faecium (9.0%) accounted for most of the isolates from the different samples (able ). he remaining species identi ed were Enterococcus casseli avus (.%), Enterococcus gallinarum (.%), Enterococcus avium (.%), Enterococcus durans (.%), Enterococcus hirae (.0%), and Enterococcus mundtii (0.%) (able ). A similar spectrum of enterococci had previously been isolated from animal and animal products elsewhere (8, 0). Eight species were isolated from milk, but only six and ve species were isolated from beef and chicken samples, respectively (able ). E. faecalis strains were isolated from.% of the beef samples. his percentage was much lower than that reported by Klein et al. (0) (8%). Enterococci occasionally isolated from animal products, such as E. casseli avus, are not part of the normal ora of cattle. In this study, E. durans and E. hirae, which are suspected to originate from the environment, were also isolated from beef (0). Similar enterococcal species diversity in poultry was reported by De
J. Food Prot., ol., No. ENEROCOCCI IN MILK, BEEF, AND CHICKEN 9 riese et al. (). De riese et al. (8) highlighted the nding that E. faecium and E. faecalis are commonly isolated from poultry and poultry products, while E. gallinarum and E. avium are rarely isolated from such products (able ). Enterococci are known to exist in the feces of animals and to persist in the environment for long periods. hus, the degree of contamination of milk, beef, or chicken by these bacteria generally depends on the maintenance of proper cleanliness and hygiene during milking or slaughter. Since enterococci can grow at refrigeration temperatures (8C) and at elevated temperatures (8C) and can withstand pasteurization temperatures it is possible to nd them in large numbers in milk. Antibiotic resistance of E. faecalis and E. faecium isolates. Almost all of the E. faecalis and E. faecium isolates from the milk, beef, and chicken samples were resistant to ampicillin (ables and ). Calia () and Lerner () have also reported high levels of ampicillin resistance for these species. arious researchers have noted an increase in the ampicillin resistance of these species since the early 990s (). However, hal et al. () reported that ampicillin-resistant enterococci were not present in either chickens or cows fed antibiotic-free feeds. he proportions of enterococci that were resistant to only one of the other antibiotics ranged from 8 to 9% (ables and ). Such large proportions of ampicillin-resistant enterococci leaves only the glycopeptides, vancomycin, and teicoplanin as drugs of choice for the treatment of enterococcal infections (). Klein et al. (0) isolated enterococci that were sensitive to most drugs, including ampicillin, from beef samples in Germany. hese researchers then recommended the use of ampicillin for patients with enterococcal infections originating from meat. On the basis of our ndings, the use of ampicillin for the treatment of E. faecalis or E. faecium infections in animals and in patients at health care facilities in Botswana may not produce the desired effect. It is necessary to consider the resistance pattern of the enterococci in question before administering any antibiotic (). In Botswana, cattle are left in the pasture and in a few cases are given feeds without antibiotics or growth promoters until they are slaughtered; hence, they should have harbored enterococci that were sensitive to the antibiotics used in the tests. However, these cattle might have received antibiotics for the treatment of diseases, e.g., mastitis. Chickens in Botswana regularly receive feeds supplemented with growth promoters. Although we were unable to obtain information regarding the extent of the use of antibiotics on dairy and poultry farms, we suspected their use to be common. he level of enterococcal resistance to cephalothin (a cephalosporin with a b-lactam ring like ampicillin), although not comparable to the level for ampicillin, was also high. As much as.,., and.% of the E. faecalis isolates from the milk, beef, and chicken samples, respectively, were resistant to cephalothin. In addition,.8,.9, and.9% of the E. faecium isolates from milk, beef, and chicken, respectively, were resistant to cephalothin. Levels of resistance to tetracycline was high for E. faecalis and E. faecium isolates from milk samples but were ABLE. Resistance patterns for Enterococcus faecalis isolates from different sources Source isolates Resistance pattern a Milk A e A A A e e e Ae A A e e Ae Ae Ate Ae Beef A e A Ae A A Ae Ae Ae Chicken 0 A e A A Ae resistant strains 00 8 8 0 0 0 9 00 0 % resistance 9. 8.8 8.8 8.... 0.. 0. 0. 0. 0.... 0. 0. 0.... 99..8..8.... 8... 9.. 8..9...0..8.9 a A, ampicillin;, vancomycin; e, teicoplanin;, tetracycline;, cephalothin. very low for isolates from beef and chicken samples (ables and ). etracycline inhibits protein synthesis, while the four remaining antibiotics are all cell wall directed. he mode of action of tetracycline may be a factor in the low levels of resistance of the enterococcal isolates from the
9 CHINGWARU E AL. J. Food Prot., ol., No. ABLE. Resistance patterns for Enterococcus faecium isolates from different sources Source isolates Resistance pattern a Milk A e A A A e Ae A A e Ae A Ae Beef A e A A A Ae A A Ae Ae Chicken A e A A A Ae A Ae Ae resistant strains 90 8 90 9 8 8 8 8 9 8 % resistance 9..8...8.9.9 9..9.8......9.. 9.. 9...9.....9.... 00 0..0 9......... a A, ampicillin;, vancomycin; e, teicoplanin;, tetracycline;, cephalothin. samples. It may also indicate less use of this antibiotic in animal husbandry. In general, it was E. faecium, rather than E. faecalis, that appeared to be more resistant to vancomycin, teicoplanin, tetracycline, and cephalothin in all samples (able ). he trend for resistance to teicoplanin was similar to that for resistance to vancomycin. Glycopeptide resistance levels for enterococcal isolates in this study were generally higher (.% of isolates from chicken were resistant to vancomycin and 8.% were resistant to teicoplanin, and.8% of isolates from beef were resistant to vancomycin and.% were resistant to teicoplanin) than those reported by Levy and Salyers () for enterococcal isolates from food in Denmark. hese investigators reported that 9% of their E. faecium isolates and none of their E. faecalis isolates from chicken exhibited resistance to vancomycin. None of their isolates from beef showed such resistance. Almost all of the samples contained ampicillin-resistant enterococci. Generally, fewer samples showed resistance to all of the antibiotics except ampicillin in pasteurized-milk and meat samples than in raw-milk samples. Enterococcal resistance levels in pasteurized-milk and meat samples ranged from. to.% for vancomycin, from 8 to.% for teicoplanin (although the level was.9% for three raw milk samples), from to.% for tetracycline, and from to.% for cephalothin. Enterococcal resistance levels in raw-milk samples ranged from to 8% for vancomycin, from 8 to % for teicoplanin, from 8 to % for tetracycline, and from to % for cephalothin. Robredo et al. () recovered strains of RE from.% of the chicken samples they obtained from supermarkets in Spain. Descheemaeker et al. () found all of the strains of E. faecium they isolated from fecal samples of poultry and other animals in Belgium to be glycopeptide resistant. However, Qadri et al. (0) found that % of enterococcal isolates from poultry samples contained RE. Of these RE isolates, % were E. faecium isolates and the rest (%) were E. gallinarum isolates. an den Braak et al. (9) reported that 9% of the poultry samples they collected in he Netherlands contained RE, and 9% of their RE isolates were identi ed as vancomycin-resistant E. faecium. he present study also revealed a predominance of vancomycin-resistant E. faecium in beef samples (.%) and in chicken samples (0.%). E. faecalis accounted for.8 and.% of the beef and chicken isolates, respectively. Such a predominance of vancomycin-resistant E. faecium and E. faecalis was also noted for milk samples. he presence of enterococci with extensive antibiotic resistance in beef and chicken samples could have been due to the contamination of the carcasses with environmental (especially fecal) enterococci during slaughter and processing. Multiresistance patterns of E. faecalis and E. faecium. he E. faecalis strains isolated from milk, beef, and chicken exhibited,, and 0 resistance patterns, respectively. Five (.%) of the E. faecalis isolates from milk and one of the isolates from beef exhibited resistance to all of the antibiotics used in the test (able ). he percentage of E. faecalis isolates that were resistant to four antibiotics in milk was.8%. here were few E. faecalis isolates from beef (.%) or chicken (.8%) that exhibited resistance to four or more antibiotics. he level of resistance increased as the number of antibiotics decreased. On the other hand, E. faecium exhibited 8,, and resistance patterns in milk, beef, and chicken samples, respectively. here were higher percentages of multiple-drug re-
J. Food Prot., ol., No. ENEROCOCCI IN MILK, BEEF, AND CHICKEN 9 sistance for E. faecium isolates from beef and chicken than for E. faecalis isolates from those sources. At least.% of the E. faecium isolates from milk were resistant to all antibiotics. However, well over.% were resistant to four or more antibiotics in milk. None of the E. faecium isolates from beef and chicken were resistant to all ve antibiotics. Of the E. faecium isolates from beef and chicken samples,. and %, respectively, were resistant to three or more antibiotics. Some of the E. faecalis and E. faecium isolates from the different samples were resistant to vancomycin alone or in combinations. CONCLUSIONS he antibiotics used in this study represented the major groups of antibiotics used in health care centers, namely, b-lactams (ampicillin), cephalosporins (cephalothin), glycopeptides (vancomycin and teicoplanin), and tetracyclines (tetracycline). he high levels of multiresistance of the enterococcal isolates has caused concern about the antibiotic resistance situation for enterococci from milk, beef, and chicken, the types of foods chie y consumed by people worldwide (,, ). We found very high levels of enterococcal resistance to the most important antibiotics (singly and in combination) used to control human enterococcal infections, especially glycopeptides (vancomycin and teicoplanin). Such resistance is likely to be passed from livestock to humans. It would be interesting to assess the antibiotic resistance situation for enterococcal isolates from health care institutions in Botswana. ACKNOWLEDGMENS We thank Mrs. A. 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