Food Control 18 (2007) 306311 www.elsevier.

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Antimicrobial resistance of Salmonella spp. and Escherichia coli isolated from table eggs
A. Adesiyun
a

a,

, N. OYah a, N. Seepersadsingh a, S. Rodrigo a, V. Lashley b, L. Musai

School of Veterinary Medicine, Faculty of Medical Sciences, University of the West Indies, St. Augustine, Trinidad and Tobago b Poultry Surveillance Unit, Ministry of Agriculture, Land and Marine Resources, Port of Spain, Trinidad and Tobago Received 25 May 2005; received in revised form 18 October 2005; accepted 22 October 2005

Abstract The antimicrobial sensitivity of Salmonella spp. and Escherichia coli isolated from the shells and contents of table eggs sampled from sale outlets in Trinidad was determined using the disc diVusion method. The phage types of S. Enteritidis isolates, the phenotypic characteristics of E. coli isolates and the presence of O157 strain were also investigated. Of a total of 74 isolates of Salmonella tested, 17 (22.9%) exhibited resistance to one or more of the seven antimicrobial agents used compared with 104 (88.1%) of 118 E. coli isolates. The diVerence was statistically signiWcant (P < 0.05; X2). For both microorganisms, resistance was relatively high to streptomycin (54.2%) and tetracycline (35.9%) but low to gentamicin (11.5%) and sulphamethoxazole/trimethoprim (9.4%). Only 1 (1.4%) isolate of Salmonella was multi-resistant while 55 (46.6%) of E. coli isolates were resistant to three or more antimicrobial agents. The frequency of resistance to antimicrobial agents amongst both bacteria was not signiWcantly (P > 0.05; X2) aVected by the location of isolation on the egg (shell or content) or source of eggs (farms, shopping malls or other retailers). Eight (19.5%) of 41 S. Enteritidis isolates tested were resistant compared to 4 (26.7%) of 15 isolates of S. Ohio. All S. Enteritidis isolates belonged to phage type 1 (PT1) and all E. coli isolates were non-haemolytic, non-mucoid, sorbitol fermenters and non-O157 strains. It was concluded that the relatively high resistance amongst the bacteria tested could pose therapeutic problems in consumers, particularly in egg-borne salmonellosis or colibacillosis. 2005 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial resistance; Phage type; Salmonella; E. coli; Table eggs

1. Introduction Table eggs which constitute several dishes or foods consumed and are considered cheap sources of protein, have served as vehicles for numerous enteropathogens (Hangombe, Sharma, Skjerve, & Tuchili, 1999). Salmonella spp., particularly S. Enteritidis have been most frequently associated with table eggs (Nygard et al., 2004). Table eggs contaminated by bacterial pathogens and consumed raw or improperly cooked have been responsible for many epidemics of gastroenteritis worldwide (Nunes et al., 2003; Nygard et al., 2004). It has been recommended that table

Corresponding author. Tel.: +1 868 645 4481; fax: +1 868 645 7428. E-mail address: abiodunadesiyun@hotmail.com (A. Adesiyun).

eggs destined for human consumption be properly cooked or pasteurized prior to consumption (Davies & Breslin, 2003; Indar, Baccus-Taylor, Commissiong, Prabhakar, & Reid, 1998; Indar-Harrinauth et al., 2001). Enteric pathogens such as Salmonella, E. coli and other pathogens isolated from table eggs have been characterized as to their serotypes, phage types, toxigenicity and antibiotic sensitivity (CDC, 1996; Ibeh & Izuagbe, 1986; IndarHarrinauth et al., 2001; Radkowski, 2001). Salmonella Enteritidis phage type 4 (PT4) has been most frequently associated with egg-borne salmonellosis in many epidemics worldwide although other phage types have also been implicated (Berghold, Kornschober, & Weber, 2003; IndarHarrinauth et al., 2001; Kowalczyk-Pecka, Wernicki, & Puchalski, 2003; Mare, Van der Walt, & Dicks, 2000; Nygard et al., 2004). Pulse-Weld gel electrophoresis (PFGE)

0956-7135/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.10.013

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has been used to determine the relatedness of S. Enteritidis isolates (Cardinale et al., 2005). It has been demonstrated that E. coli O157 is capable of colonizing the caeca of layers and subsequently contaminate their eggs (Berry, Doyle, & Schoeni, 1985). In Trinidad and Tobago, eggnog containing Salmonellacontaminated eggs resulted in an outbreak of salmonellosis amongst inmates of a mental hospital, with several mortalities (Hyatali, Quamina, Mohess, & Foster, 1992). Transovarial transfer of Samonella in table eggs at poultry farms was recently demonstrated by Indar et al. (1998). Adesiyun et al. (2005b) determined the microbial health risk of table eggs in Trinidad while in another study (Adesiyun et al., 2005a) the prevalence of macrolides, tetracycline, beta-lactam and sulphonamides in table eggs sampled from sale outlets across the island emphasizing the risk they pose to consumers. In view of the fact that table eggs constitute many foods or drinks consumed locally, some raw or only partially cooked, the study was conducted to determine the antimicrobial sensitivity of Salmonella spp. and E. coli isolates from table eggs in Trinidad, to investigate the phage types of S. Enteritidis and Wnally to detect the presence of O157 strains amongst E. coli isolates. 2. Materials and methods 2.1. Source and collection of table eggs Table eggs from which the enteric pathogens studied originated from poultry farms, shopping malls and other retailers in Trinidad. The sampling protocol and number of eggs collected from each source were earlier described (Adesiyun et al., 2005b). Overall, a total of 46 egg composite samples, made of 25 eggs per composite were sampled from 23 poultry farms following two visits, usually 4 weeks apart, to each farm. From 14 shopping malls studied, each visited twice, a total of 31 egg sample composites, with 6 eggs pooled to constitute a composite while from 102 other retailers visited once, a total of 107 composite egg samples, with 6 eggs per composite, were studied (Adesiyun et al., 2005b). Overall, for the three sources studied a total of 1978 pooled table eggs sample were cultured for bacteria. 2.2. Swab sampling of eggs For 25 pooled eggs sampled from each farm following each visit, a sterile swab each soaked in saline was applied to the surface of each egg shell and the 25 swabs were then dipped into 20 ml of saline. For 6 pooled eggs from each outlet of either the shopping malls or supermarkets, each egg shell surface was similarly swabbed with a sterile swab and then dipped in 6 ml of saline. The contents of the saline were mixed thoroughly using a vortex mixer (Henry Treemner, USA) and subsequently inoculated into appropriate bacteriological media.

2.3. Sampling of egg contents Each egg in the pool of 25 (farms) or 6 (malls and other retailers) was dipped in 95% ethanol for 5 min after which the pointed end of each was Xamed for 510 s. with a Bunsen burner after which a sterile scalpel blade was used to make a small hole on the shell through which the contents were emptied into a stomacher bag. The egg contents (yolk and albumen) in each pool were then blended for 30 s at normal speed in a Stomacher 400 (Seaward, London, United Kingdom) after which the mixture was used to inoculate appropriate bacteriological media. 2.4. Isolation and identiWcation of bacteria To isolate Salmonella 10 ml of egg contents or 1 ml of saline containing swabs were added to 90 ml of lactose broth and incubated at 37 C overnight as pre-enrichment. Enrichment, isolation and identiWcation of Salmonella followed standard methods (FAO, 1992). Salmonella polyvalent antiserum A-I & Vi (Difco, Michigan, USA) was used to initially serologically identify Salmonella by slide agglutination test. To detect E. coli, swabs of egg shells and egg contents were used to inoculate eosin methylene blue (EMB) and identiWed using standard methods (Macfaddin, 1977). 2.5. Determination of the antimicrobial sensitivity of Salmonella and E. coli isolates To determine the antimicrobial sensitivity of the isolates, the NCCLS (2002) protocol was used with the following antimicrobial agents on discs (Difco, Michigan, USA) and concentrations: kanamycin (30 mcg), streptomycin (10 mcg), nalidixic acid (30 mcg), gentamicin (10 mcg), tetracycline (30 mcg), sulphamethoxazole/trimethoprim (23.25/1.75 mcg) and enroXoxacin (5 mcg). The tests were performed on Mueller Hinton agar (Difo, Michigan, USA) followed by incubation aerobically at 35 C for 24 h. The zone of inhibition were interpreted as recommended by the disc manufacturer and the NCCLS (2002). 2.6. Characterization of bacteria All Salmonella isolates were kindly conWrmed and serotyped by the Caribbean Epidemiology Centre (CAREC), Port of Spain, the regional reference centre for Salmonella. Representatives (19) of Salmonella Enteritidis isolates from each source were kindly phage typed by CAREC and the Veterinary Laboratories Agency, Weybridge, United Kingdom (Ohare et al., 2004). To determine phenotypic characteristics of all E. coli isolates, they were inoculated onto blood agar and sorbitol MacConkey agar and incubated at 37 C overnight. The ability of E. coli isolates to produce mucoid and haemolytic colonies was determined on blood agar while the ability to utilize sorbitol was determined on sorbitol MacConkey agar. Isolates of E. coli were screened

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A. Adesiyun et al. / Food Control 18 (2007) 306311 Table 2 Frequency of resistance to antimicrobial agents by serotypes of Salmonella Serotypes of Salmonella Enteritidis Ohio Mbandaka Javiana Braenderup Group C1 Georgia Caracas Total
a

for O157 strain by the use of E. coli O157 antiserum (Difco, Michigan, USA) by the slide agglutination test. 2.7. Statistical analysis The diVerences in the frequency of sensitivity to the various antimicrobial agents by the isolates of Salmonella and E. coli, source of eggs and location in eggs were compared and determined to be statistically signiWcant using the chisquare tests using the Statistical Package for Social Sciences (SPSS) version 10. All statistical analyses were two-tailed and interpreted at the 5% level of signiWcance. 3. Results 3.1. Frequency of antimicrobial sensitivity amongst isolates of Salmonella and E. coli Of a total of 74 isolates of Salmonella spp. tested, 17 (22.9%) were resistant to one or more of the seven antimicrobial agents compared with 104 (88.1%) of 118 isolates of E. coli (Table 1). The diVerence was statistically signiWcant (P < 0.05; X2). For both bacteria, resistance was comparatively high to streptomycin (54.2%) and tetracycline (35.9%) but low to gentamicin (11.5%) and sulphamethoxazole/ trimethoprim (9.4%). 3.2. Frequency of resistance patterns amongst isolates Only 1 (1.4%) isolate of Salmonella spp. was multi-resistant, i.e. resistant to 3 or more antimicrobial agents compared with as many as 55 (46.6%) of 118 isolates of E. coli tested. The predominant resistance patterns amongst E. coli isolates were: S-TE (20 isolates), S (17 isolates), S-TE-K (13 isolates), SXT-ENR-S-NA-TE-K (7 isolates) and SXT-STE (5 isolates). 3.3. Distribution of resistance amongst Salmonella serotypes Amongst the eight serotypes of Salmonella tested, 6 (66.7%) exhibited resistance to antimicrobial agents. Eight (19.5%) of 41 isolates of S. Enteritidis were resistant while 4 (26.7%) of 15 S. Ohio isolates exhibited resistance (Table 2). The resistance patterns observed were as follows: for S. Enteritidis: NA (4 isolates), S (2 isolates), K (1 isolate) and

No. of isolates tested 41 15 6 5 3 2 1 1 74

No. (%) resistancea 8 (19.5) 4 (26.7) 0 (0.0) 2 (40.0) 1 (33.3) 0 (0.0) 1 (25.0) 1 (25.0) 17 (22.9)

Resistant to one or more antimicrobial agents.

CN-S-K (1 isolate); S. Ohio: S (3 isolates) and NA (1 isolate); S. Javiana (2 isolates); S. Braenderup: S (1 isolate); S. Georgia: S (1 isolate) and S. Caracas (1 isolate). 3.4. Relationship between frequency of resistance amongst Salmonella and E. coli isolates and location of isolation from eggs The diVerences in the frequency of resistance to antimicrobial agents amongst the isolates of bacteria recovered from the shells and contents of table eggs were not statistically signiWcant (P > 0.05; X2) as shown in Table 3. 3.5. Distribution of resistant strains of Salmonella and E. coli by source of egg For both bacteria, the frequency of resistance to antimicrobial agents was highest Amongst isolates from shopping malls but the diVerence was not statistically signiWcant (P > 0.05; X2) compared to isolates from either layer farms or other retailers (Table 4). 3.6. Phage type of S. Enteritidis isolates All 19 isolates of S. Enteritidis representive of all sources belonged to phage type 1 (PT1). 3.7. Phenotypic characteristics of E. coli All 118 isolates of E. coli were non-haemolytic, nonmucoid, sorbitol-fermenters and non-O157 strains.

Table 1 Frequency of resistance to antimicrobial agents amongst isolates of Salmonella and Escherichia coli Type of bacteria Salmonella spp. E. coli Total
a b

No. of isolates testeda 74 118 192

No. (%) of isolate resistantb 17 (22.9) 104 (88.1) 121 (63.0%)

S 11 (14.9) 93 (78.8)

TE 0 (0.0) 69 (58.5) 69 (35.9)

K 2 (2.7) 38 (32.2) 40 (20.8)

NA 5 (6.8) 31 (26.3) 36 (18.8)

ENR 0 (0.0) 30 (25.4) 30 (15.6)

CN 1 (1.4) 11 (9.3) 12 (6.3)

SXT 0 (0.0) 18 (15.3) 18 (9.4)

104 (54.2)

From eggs shell, albumen or yolk of egg. Resistant to one or more of the seven antimicrobial agents tested. c Sstreptomycin, TEtetracycine, Kkanamycin, NAnalidixic acid; ENRenroXoxacin; CNgentamicin, SXTsulphamethoxazole/trimethoprim.

A. Adesiyun et al. / Food Control 18 (2007) 306311 Table 3 Frequency of resistance to antimicrobial agents amongst Salmonella and E. coli isolates from egg shell and yolk/albumen (content) Type of bacteria Location of isolation from eggs Shell No. of isolates tested Salmonella spp. E. coli Total
a b

309

Albumen/Yolka No. (%) of isolates resistantb 7 (25.0) 76 (86.4) 83 (71.6) No. of isolates tested 46 30 76 No. (%) of isolates resistantb 10 (21.7) 28 (93.3) 38 (50.0)

28 88 116

A mixture of yolk and albumen. Resistant to one or more antimicrobial agents.

Table 4 Frequency of resistance to antimicrobial agents amongst Salmonella and E. coli isolates by sale outlet of table eggs Type of bacteria Sale outlet Farmsa No. of isolates tested Salmonella spp. E. coli Total
a b c d

Mallsb No. (%) of isolates resistantd 3 (16.7) 52 (86.7) 55 (70.5) No. of isolates tested 25 23 48 No. (%) of isolates resistantc 8 (32.0) 21 (91.3) 29 (60.4)

Other retailersc No. of isolates tested 31 35 66 No. (%) of isolates resistantc 6 (19.4) 31 (88.6) 37 (56.1)

18 60 78

Layer farms studied from where vendors could purchase eggs directly. Sale outlets in shopping malls across the country where all eggs are stored refrigerated. Outlets for eggs which include kiosks, roadside vendors, supermarkets (small, medium or large) selling eggs where few had refrigerated facilities. Resistant to one or more antimicrobial agents.

4. Discussion A prevalence of 22.9% for resistance to antimicrobial agents detected amongst Salmonella isolates from eggs is considerably lower than the prevalence of 50% reported for Salmonella isolates recovered from livestock in Trinidad (Adesiyun, Kaminjolo, Loregnard, & Kitson-Piggott, 1993) and elsewhere (Brown, Baggesen, Hansen, Hansen, & Bisgaard, 1994; Hangombe et al., 1999; Poppe, 1994; Neu, Cherubin, Longo, Flouton, & Winter, 1975; Wray, Beedell, & McLaren, 1991). Amongst S. Enteritidis isolates, the prevalence of multi-resistance was considerably low, 1.4%, a Wnding in agreement with report of Yang et al. (2002) but considerably lower than that found by Dias de Oliveira, Siqueira Flores, Dos Santos, and Brandelli (2005). Similarly, the prevalence of resistance, as determined by resistance to one or more antimicrobial agents, although higher than reported for isolates from table eggs by Brown et al. (1994) is signiWcantly lower than found in Brazil (Simango & Mbewe, 2000). It has been reported that inappropriate use of antimicrobial agents in livestock may result in the development of resistance amongst bacteria in these animals or their products (Washington, 1979; Waltner-Toews & McEwen, 1994). The prevalence of 88.1% for resistance to antimicrobial agents amongst E. coli isolates from table eggs is comparable to the prevalence of E. coli isolated from livestock, 89.7% (Adesiyun & Kaminjolo, 1992). The generally high prevalence of resistance to antimicrobial agents has been attributed to uncontrolled use of antimicrobial agents as

growth promoters or in the treatment of bacterial infections with the farmers having unlimited access to these agents and their use (Adesiyun & Kaminjolo, 1992). The report on the detection of antimicrobial agents (sulfonamides, macrolides and tetracyclines) in table eggs oVered for sale in Trinidad was therefore not a surprise (Adesiyun et al., 2005a). The detection that exhibition of resistance was highest to streptomycin amongst all enteric bacteria studied is similar to the Wndings for other bacteria from several sources in the country attributed to the widespread use of the antimicrobial agents in several preparations available locally (Adesiyun & Kaminjolo, 1992; Adesiyun et al., 1993). Furthermore, on the layer farms in Trinidad it has been reported that as many as 87% of the farms admitted to the use of medicated feeds that contained chlortetracycline, tylosin or nitrofurantoin (Adesiyun et al., 2005a). Unlike in other studies where Salmonella, including S. Enteritidis, have been reported to be sensitive to nalidixic acid, kanamycin and gentamicin (Dias de Oliveira et al., 2005; Hangombe et al., 1999), in the present study, together with resistance exhibited to streptomycin, the isolates were also resistant to the three antimicrobial agents albeit, at a relatively low prevalence. It was hardly a surprise that no statistically signiWcant diVerences were observed in the prevalence of resistance amongst bacteria obtained from the three sources (farms, shopping malls and other retailers). This is so because a majority of table eggs sampled from the sale outlets (malls and other retailers) actually originated from most of the 23

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layer farms studied. There are however suggestions that some of the table egg sale outlets import their eggs (Adesiyun et al., 2005b). It is of epidemiological signiWcance that all the S. Enteritidis isolates belonged to phage type 1. In an earlier report in Trinidad, S. Enteritidis phage type 4, was considered to be the most important phage type implicated in egg-borne gastroenteritis (Indar-Harrinauth et al., 2001). Enteritidis PT1 has however been recovered from cases of gastroenteritis in the country also emphasizing the clinical importance of this phage type (CAREC, 2002). Enteritidis PT1 has been implicated in egg-borne gastroenteritis in other countries (Brown et al., 1994; Kusunoki et al., 1999). It has been demonstrated that there are geographical distributions of S. Enteritidis phage types and there is also evidence of changing phage types of the pathogen even within countries and regions (Mare et al., 2000; Nygard et al., 2004). It is also known that various phage types of S. Enteritidis isolates from table eggs have been implicated in human epidemics of salmonellosis (Berghold et al., 2003; Kowalczyk-Pecka et al., 2003; Majtanova, 1997; Simango & Mbewe, 2000). The role of PT1 compared with that of PT4 in egg-borne gastroenteritis in Trinidad therefore needs further investigation to ascertain whether there is a change in pattern. It is realized that the phage typing and the antibiotic sensitivity of S. Enteritidis cannot discriminate strains of the serotype, as detected in this study where all the isolates belonged to phage type 1 but the antibiotic sensitivity patterns of some isolates were diVerent. It has been documented that a combination of phage typing, pulse-Weld gel electrophoresis (PFGE) and plasmid proWling considerably increases ability to diVerentiate S. Enteritidis isolates (Cardinale et al., 2005; Cieslik, Browm, Paciorek, Szych, & Kaluzewski, 2001; Lukinmaa, Schildt, Rinttila, & Siitonen, 1999). An earlier study of S. Enteritidis from the Caribbean region employed PFGE to diVerentiate isolates from various countries in the region (Adesiyun, Carson, McAdoo, & Bailey, 2001). In that study, the PFGE patterns of S. Enteritidis strains from Trinidad and Tobago and Barbados were distinctly diVerent. Kowalczyk-Pecka et al. (2003) have however stated that the use of phage typing and antibiotic sensitivity could be used to initially assess the relatedness of S. Enteritidis isolates with a correlation in 18.3% of the isolates while to the contrary, Dias de Oliveira et al. (2005) reported that there was no predominant resistance pattern related to phage types. It was concluded that the relatively high prevalence of resistance to antimicrobial agents amongst isolates of Salmonella and E. coli may pose therapeutic implications, particularly in egg-borne salmonellosis. Acknowledgements We are grateful the staV of the Poultry Surveillance unit of the Ministry of Agriculture, Land and Marine Resources for their assistance in sample collection. The University of

the West Indies, St. Augustine Campus Research Fund Committee kindly funded the project. The technical assistance of Shakti Dookeran and Gerard Ramirez is appreciated. References
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