Occurrence of Salmonella spp. in broiler chicken carcasses and their susceptibility to antimicrobial agents.

The present study was carried out to evaluate the occurrence of Salmonellae in broiler chicken carcasses and to determine the antimicrobial resistance profile of the isolated strains. Twenty-five out of the 260 broiler chicken carcasses samples (9.6%) were positive for Salmonella. S. Enteritidis was the most frequent serovar. Nineteen Salmonella isolates were tested for antimicrobial resistance, and the results indicated that 94.7% were resistant to at least one antimicrobial agent. Resistance to streptomycin (73.7%), nitrofurantoin (52.3%), tetracycline (31.6%), and nalidixic acid (21%) were the prevalent amongst Salmonella strains tested.

The consumption of poultry meat has been increasing annually, bringing with it the concern about the cases of foodborne illness events caused by Salmonella (30), since poultry have been identified as a significant reservoir of Salmonella in the environment (29).

Regarding poultry health, there are three differents salmonellosis syndromes, two of them caused by no motile serovars and host-adapted to poultry, which are Pullorum Disease, caused by Salmonella Pullorum, and Fowl Typhoid, caused by Salmonella Gallinarum; the other syndrome is Paratyphoid Infections, caused by motile Salmonella serovars (29), which are also responsible for outbreaks of foodborne illness in humans.

The presence of Salmonella in the intestinal tract, on the skin and among the feathers of chickens, may cause carcasses contamination during slaughtering and processing (16) and is responsible by the introduction of Salmonella in the slaughterhouses, where will progress along the processing line, affecting the final microbiological quality and jeopardizing the consumers health(6).

The occurrence of Salmonella in chicken carcasses, in studies undertaken in different countries, can vary for instance from 13% in Poland (21) to 66% in Thailand (17). In Brazilian studies, they vary from 5.9% (20) to 42% (14).

In veterinary medicine, antimicrobial agents are used therapeutically, metaphylatically, prophylactically, and as growth promoters (28). However, their indiscriminate use is causing increasing resistance amongst Salmonella strains and other bacteria (7).

The present study was carried out to evaluate the occurrence of Salmonella spp. in broiler chicken carcasses and to estimate the resistance profiles of the isolates.

Two hundred and sixty broiler chicken carcasses were purchased in five processing plant located in Northeastern Brazil from April to December 2004. They were collected in sterilized plastic bags and transported, under refrigeration, to the Laboratory of Food Microbiology of the “Laboratório Nacional Agropecuário – Ministério da Agricultura, Pecuária e Abastecimento”, Recife, Pernambuco, Brazil.

Salmonella were isolated using the microbiological method recommended by the Brazilian Agriculture Ministry (“Ministério da Agricultura, Pecuária e Abastecimento”) (5). Complete antigenic characterization and serovar identification were performed by the Enteric Pathogens Laboratory from Oswaldo Cruz Institute Foundation, Rio de Janeiro (FIOCRUZ-RJ).

Nineteen Salmonella strains were tested for antimicrobial resistance by the disk diffusion method on Mueller-Hinton agar, according to the Clinical and Laboratory Standards Institute (CLSI, formely NCCLS)(11). The antimicrobials agents were: ampicillin 10 µg, amoxicillin/clavulonic acid, cefotaxime, ciprofloxacin 5 µg, chloranphenicol 30 µg, enrofloxacin 5 µg, gentamicin 10 µg, kanamycin 30 µg, nalidixic acid 30 µg, nitrofurantoin 300 µg, norfloxacin 10 µg, streptomycin 10 µg, polymyxin B 300 I.U., tetracycline 30 µg and trimethoprim/sulfamethoxazole. Escherichia coli ATCC 25922 was used as a reference strain. An isolate was classified as multiple resistant when demonstrated resistance to two or more agents (10).

Twenty-five out of the 260 broiler chicken carcasses samples (9.6%) were positive for Salmonellae. Others researches investigating the occurrence of Salmonella in different Brazilian States reported different levels in broiler chicken carcasses, 13% in Santa Catarina (19), 5.9% (20%), 32% (27), and 42% (14) in São Paulo, and 11.8% (23) in Ceará.

Surveys performed in different countries, using broiler chicken carcasses showed 13% in Poland ( 21), 29.3% in Belgian (31), 29.7% in UK (24), 35.8% in Spain (12), 62.5% in Senegal (3) and 66% in Thailand (17).

Twenty strains of Salmonella isolated in this study were identified at serovar level, from where it was identified 11 different Salmonella serovars. S. Enteritidis was the most frequent serovar (25%). The results obtained agree with the fact that S. Enteritidis was the predominant serovar associated with outbreaks in humans, as showed by the WHO Salmonella surveillance date for 1990-1995 (15), and with the fact that S. Enteritidis is the predominant in poultry carcasses and their products as showed in Belgium (31), in the United Kingdom (24), Spain (12) and Portugal (1).

In Brazil, S. Enteritidis has been the most prevalent serovar detected in human infections (13), in broiler chicken carcasses (27), in broiler chicken parts (26), and in poultry flocks (18).

The other isolates were S. Typhimurium (3 isolates), S. Saintpaul (3), S. Mbandaka (2), S. Corvallis (1), S. Heidelberg (1), S. Infantis (1), S. Munchen (1), S. Newport (1), S. Panama (1) and Salmonella. enterica subsp. enterica (O9,12) (1).

Antimicrobial resistance is a widely studied issue in all bacteria genera, mainly amongst zoonotic bacteria, like Salmonellae is.

Nineteen Salmonella isolates were tested for antimicrobial resistance, and the results (Table 1) indicated that 94.7% (n: 18) were resistant to at least one antimicrobial agent. This data is higher than the 58.1% observed in Greece (2), 67.3% in the United States (8), 75% in Portugal (1), but lower than the 100% obtained in Salmonellae isolated from broiler chicken carcasses by Cardoso et al. (9) in the State of Rio Grande do Sul, Brazil.

Table 1

Antimicrobial resistence in Salmonella strain isolated from broiler chicken carcasses

Samples tested	Number of strains tested	Number of the resistance to antimicrobial agent
		Amp.	C	Kan	Enr	Nor	Tet	Nal	CTX	Nit	Est	Gen	Cip	SXT	Pol B	AmC
S. Enteritidis	5	-	-	1	-	-	1	-	-	5	3	-	-	-	-	-
S. Typhimurium	3	-	-	-	-	-	-	-	-	-	3	-	-	-	-	-
S. Saintpaul	2	-	-	-	-	-	-	1	-	1	1	-	-	-	-	-
S. Mbandaka	2	1	1	1	-	-	1	-	-	-	2	-	-	1	-	1
S. Infantis	1	-	-	-	-	1	1	1	-	1	1	-	-	-	-	-
S. Muenchen	1	-	-	-	-	-	1	-	-	-	-	-	-	-	-	-
S. Newport	1	-	-	1	1	1	-	1	-	-	1	-	1	-	-	-
S. Heidelberg	1	1	-	1	-	-	1	-	-	1	1	-	-	-	-	1
S. Panama	1	-	-	-	-	-	-	1	-	-	1	-	-	-	-	-
S. Corvallis Salmonella	1	-	-	-	-	-	-	-	-	1	1	-	-	-	-	-
enterica subsp. enterica (O9,12)	1	-	-	-	-	-	1	-	-	1	1	-	-	-	-	-
Total	19	2	1	4	1	2	6	4	0	10	14	0	1	1	0	2

Resistance to streptomycin (73.7%), were higher than the 39% obtained by Antunes et al., (1) in Portugal, however lower than the 100% obtained by Ribeiro et al., (25) in S. Hadar isolated from broiler chicken carcasses in in the State of Rio Grande do Sul, Brazil.

The results regarding the resistance to nitrofurantoin (52.6%) and to tetracycline (31.6%) are supported by Oliveira et al. (22), who showed that 52.8% of S. Enteritidis strains isolated from bird carcasses, food, humans and poultry-related samples were resistant to nitrofurantoin. As well, it is supported by Antunes et al., (1), who found 36% resistance to tetracycline in Salmonella isolated from parts and broiler chicken carcasses in Portugal.

Resistance to the nalidixic acid (21%), norfloxacin (5.2%), enrofloxacin (5.2%) and ciprofloxacin (5.2%) were showed by some the Salmonella strains.

It was also observed resistence to Kanamycin (15.8%), ampicillin (10.5%), amoxicillin/clavulonic acid (10.5%), chloramphenicol (5.2%), trimethoprim/sulfamethoxazole (5.2%). None of Salmonella isolated showed resistance to cefotaxime, gentamicin and polimyxin B.

Fourteen different patterns of resistance were observed (table 3), two of them should be a concern because they showed resistance to eight different antimicrobial agents: CIP, ENR, KAN, NAL, NIT, NOR STR, TET, found in S. Newport and AMP, AMC, CHL, KAN, NIT, STR,SXT, TET, found in one S. Mbandaka.

Multiple resistance was not observed in 7 out of 19 tested strains, and 6 were resistant only to one antimicrobial agent, nalidixic acid (n: 1), nitrofurantoin (n: 2) and streptomycin (n: 3). One isolate identified as S. Muenchen, was susceptible to all antimicrobial agents.

Different factors may contribute to the Salmonella entry to the processing plants and to reach processed carcasses. Therefore any successful Salmonella control plan will need to implement actions to control contamination and dissemination of the bacteria in poultry raising sites (layers, hatcheries and chicken farms) (4), and in the slaughterhouses with a systematic laborers training, good manufacture practices and HACCP. Regarding the resistance to antimicrobial agents, the data obtained emphasize the need of a judiciously use of these agents in animal production, based on susceptibility tests.

Note: The opinions in this article are those of the authors and do not necessarily reflect views and policies of the organization to which they are affiliated.

Table 2

Distribution of antimicrobial resistence patterns in Salmonella strains

Patterns	Salmonella
Susceptible	1
Est.	3
Nal	1
Nit	2
Est, Nal	1
Est, Nit	3
Est, Tet	1
Nit, Tet	1
Est, Kan, Nit	1
Est, Nit, Tet	1
Amp, AmC, Est, Kan	1
Est, Nal, Nor, Tet	1
Amp, AmC, C, Est, Kan, Nit, SXT, Tet	1
Cip, Enr, Est, Kan, Nal, Nor, Nit, Tet	1

AMP: ampicilIN; AmC: amoxixilna + ácido clavulônico, CHL: chloramphenicol; CIP: ciprofloxacin;

ENR: enrofloxacin, STR: streptomycin; KAN: Kanamycin; NAL: nalidixic acid, NIT: nitrofurantoin;

NOR: norfloxacin, SXT: sulfametoxazol + trimetoprim, TET: tetracycline