Antimicrobial susceptibility of Salmonella strains isolated from retail meat products in Poland between 2008 and 2012

Food Control 36 (2014) 199e204

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Antimicrobial susceptibility of Salmonella strains isolated from retail meat products in Poland between 2008 and 2012  zy _ _ n  ska a, Kamila Paw1owska a, qukasz Ma˛ ka a, *, Elzbieta Ma ckiw a, Halina Scie Magdalena Popowska b a Laboratory of Food Microbiology, Department of Food Safety, National Institute of Public Health e National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland b Department of Applied Microbiology, Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 March 2013 Received in revised form 13 August 2013 Accepted 20 August 2013

A total of 106 Salmonella strains were isolated in the years 2008e2012 from retail meat products sampled in Poland. Strains from poultry meat (n ¼ 81), pork (n ¼ 7), beef (n ¼ 3) and mixed meat (n ¼ 15) were serotyped and subjected to antimicrobial susceptibility testing by the disk diffusion method (19 antibiotics). Twenty-one Salmonella serotypes were identified, with the three most common being Salmonella Enteritidis (34.9%), Salmonella Infantis (14.2%) and Salmonella Typhimurium (10.4%). The majority of the Salmonella strains (68.9%; n ¼ 73) were resistant to one or more antimicrobial compound. Among the resistant isolates, 31 were resistant to one antibiotic, 4 to two, 10 to three, 13 to four, and 15 to five or more antibiotics. Of the Salmonella Enteritidis isolates, 54% were resistant to at least one antibiotic, while much higher frequencies of resistance were found in Salmonella Newport (100%), Salmonella Typhimurium (91%), Salmonella Hadar (85.7%), Salmonella Virchow (80%) and Salmonella Infantis (80%). The most common resistance observed among the Salmonella isolates was to nalidixic acid (52.8%). The isolates were also frequently resistant to tetracycline (32.1%), ampicillin (28.3%), streptomycin (28.3%) and sulphonamides (26.4%). All of the tested strains were susceptible to cefepime, cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, ertapenem and imipenem. Salmonella strains isolated from poultry meat showed the widest spectrum of resistance (to 12 of the 19 tested antimicrobials) compared with isolates from the other meat sources. The level of resistance among Salmonella strains isolated between 2008 and 2012 was consistently high: 59.1% in 2010, 84.6% in 2011 and 64.7% in 2012. In addition, there was an increase in the number of multiresistant strains over this period, from 23.1% in 2010 to 81.8% in 2012. The demonstration that meat products are a source of antibiotic resistant Salmonella strains is a serious concern for public health and food safety. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Salmonella Retail food Meat products Prevalence Antimicrobial susceptibility

1. Introduction Members of the bacterial genus Salmonella, classified within the family Enterobacteriaceae, are among the most common foodborne pathogens. Salmonellae are widespread in nature, being present in domestic and wild animals as pathogens or commensal microorganisms. These robust bacteria can survive in the environment outside their hosts for long periods. More than

* Corresponding author. Tel.: þ48 225421384; fax: þ48 225421225. E-mail address: [email protected] (q. Ma˛ ka). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.08.025

2500 serovars comprise the Salmonella genus (Grimont & Weill, 2007) and new serovars are regularly described (Guibourdenche et al., 2010). S. Enteritidis and S. Typhimurium are the most commonly reported serovars in the European Union (EU), being associated with 52.3% and 23.3% of all confirmed human infections (salmonellosis), respectively. Since 2006, S. Infantis has been the third most common serovar in the EU (EFSA and ECDC, 2011). Over the same time period, S. Enteritidis and S. Typhimurium represented over 80% of the Salmonella isolated from human cases in Poland. The percentage of cases associated with S. Enteritidis decreased from 77.6% in 2006 to 68.5% in 2010. Moreover, in 2010, the second most prevalent serotype was

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S. Mbandaka (8.79%), replacing S. Typhimurium (8.02%) (Czarkowski, Cieleba˛ k, Kondej, & Staszewska, 2009; 2011). Based on the Reports on cases of infectious diseases and poisonings in Poland (http://www. pzh.gov.pl/oldpage/epimeld/index_p.html#01) can be concluded, that Salmonella spp. are a major bacterial agent of food poisoning in Poland. In the USA, the annual costs resulting from salmonellosis amount to several billions of dollars (Voetsch et al., 2004). The European Food Safety Authority (EFSA) has estimated that the overall economic burden due to human cases in the EU could be as high as 3 billion euros per year (EFSA, 2011a). The majority of Salmonella infections are associated with contaminated food: chicken, pork, dairy products, eggs, fruits, vegetables and others (Oliveira, Flores, Santos, & Brandelli, 2005; Yan et al., 2010; Zhao et al., 2008). Antimicrobial agents are widely use in human and veterinary medicine, and to improve the growth of animals and plants. Although the use of antibiotic growth promoters in animal nutrition is now prohibited by EU legislation (Regulation (EC) No. 1831/ 2003), some practices in human and animal healthcare, often resulting from commercial pressure to prescribe and sell antibiotics, may lead to inappropriate and overuse of these agents (Council of the European Union conclusions, 2012). The use of antibiotics in animals, not only to treat disease, but also as growthpromoting substances and to prevent from diseases, promotes the development and spread of antimicrobial-resistant bacteria potentially worldwide (Aminov, 2010). Antibiotics released into the environment can provoke the formation of resistance, and even cross- and multiple resistance in bacteria (Allen et al., 2010; Aminov, 2011; Popowska et al., 2012; Popowska, Miernik, Rzeczycka, & qopaciuk, 2010). Pathogens as well as commensal bacteria are affected, the latter constituting a potential reservoir of resistance genes for pathogenic bacteria. The transfer of such pathogens through the food chain is possible and consequently lowers the success of pharmacotherapies for curing humans (Doyle et al., 2006; EFSA, 2008; Singer et al., 2003). Resistance genes active against beta-lactams (3rd and 4th generation) and fluoroquinolones, which are extremely important antimicrobial agents in human medicine, have been identified on many livestock farms, and particularly in the poultry industry where fluoroquinolones are used, so that meat products may be contaminated with bacterial strains resistant to this antimicrobial agent. Genes encoding enzymes of the AmpC and Extended Spectrum Beta-lactamase (ESBL) families are often found in Salmonella and E. coli isolates (EFSA, 2011b; Zhao et al., 2008). Genetic analyses of the bacterial strains and resistance genes isolated from farm animals, foods and human subjects have identified strong similarities (Leverstein-Van Hall et al., 2011). Such studies indicate that resistant strains, ESBL genes and mobile genetic elements might be transmitted to humans through the food chain (Gastmeier, 2010). The molecular mechanisms of resistance to antibiotics have been studied extensively and a great deal is now known about the genetics and biochemistry of many different facets of bacterial cell function (Alekshun & Levy, 2007; Andersson & Hughes, 2010; Roe & Pillai, 2003; Walsh & Amyes, 2004). However, it is also very important to identify any trends in antimicrobial resistance by conducting regular screening. Most surveys of resistance in Salmonella have focused on clinical and veterinary strains. To fully appreciate the scale of antimicrobial resistance in order to counter this growing problem, it is also necessary to investigate consumer exposure to resistant strains present in food. In this study, we have examined the antimicrobial susceptibility of Salmonella strains isolated from retail meat products in Poland over the last five years.

2. Materials and methods 2.1. Strain collection Between 2008 and 2012, a total of 106 Salmonella strains were collected by 26 Sanitary and Epidemiological Stations across Poland in the course of their Official Control and Monitoring Program (18 strains in 2008, 23 in 2009, 22 in 2010, 26 in 2011 and 17 in 2012). The strains were isolated from retail meat products sampled according to PN-EN ISO 6579:2003/A1:2007 “Horizontal method for the detection of Salmonella spp.”, from poultry meat (n ¼ 81), pork (n ¼ 7), beef (n ¼ 3) and mixed meat (n ¼ 15) (pork-beef meat). 2.2. Antimicrobial susceptibility testing The antimicrobial susceptibility of Salmonella isolates was assessed by tests performed in 2011 and 2012. The antibiotic resistance profile of each strain was determined using the disk diffusion method on Mueller-Hinton agar (OXOID, PO5007A), according to the methodology of the European Committee on Antimicrobial Susceptibility Testing e EUCAST (EUCAST, 2012). Discs containing the following antibiotics (OXOID) were used: Aztreonam (30 mg), Amoxycillin/clavulanic acid (20/10 mg), Ampicillin (10 mg), Cefepime (30 mg), Cefotaxime (5 mg), Cefoxitin (30 mg), Ceftazidime (10 mg), Ceftriaxone (30 mg), Chloramphenicol (30 mg), Ciprofloxacin (5 mg), Ertapenem (10 mg), Gentamicin (10 mg), Imipenem (10 mg), Nalidixic acid (30 mg), Sulphonamides compound (300 mg), Streptomycin (10 mg), Tetracycline (30 mg), Trimethoprim (5 mg), Trimethoprim/sulphametoxazole (1.25/ 23.75 mg). Individual colonies were suspended in saline to a density of 0.5 on the McFarland turbidity standard, measured using a densitometer (Biomerieux). Each cell suspension was spread over the entire surface of a plate by swabbing in three directions and the antibiotic discs were applied. The plates were then incubated at 35  C for 16e20 h. The diameters of the zones of inhibition were measured and compared with EUCAST (EUCAST, 2012) or Clinical and Laboratory Standards Institute (CLSI) standards (CLSI, 2012). In cases where EUCAST breakpoints were absent, the results were interpreted according to the CLSI breakpoints. Quality control tests were performed using E. coli ATCC 25922. 2.3. Salmonella serotyping Isolates were serotyped by Sanitary and Epidemiological Stations and sent to the Laboratory of Food Microbiology, National Institute of Public Health e National Institute of Hygiene (NIPHe NIH), Warsaw, Poland. Where the serotype of a Salmonella isolate was undefined, serotyping was carried out within the Department of Bacteriology at the NIPHeNIH. Strains were serotyped by slide agglutination test with specific O and H antisera (Immunolab, Biomed) and classified according to the WhiteeKauffmann-Le Minor scheme (Grimont & Weill, 2007). 3. Results and discussion 3.1. Prevalence of Salmonella serotypes in retail meat products The Salmonella serotypes isolated from retail meat products of different animal origin in Poland are listed in Table 1. Most of the 106 Salmonella strains were isolated from poultry meat (n ¼ 81), while isolates from pork (n ¼ 7), beef (n ¼ 3) and mixed meat (n ¼ 15) samples were less frequent. A total of twenty-one different Salmonella serotypes were identified.

Ł. Ma˛ ka et al. / Food Control 36 (2014) 199e204 Table 1 Incidence of Salmonella serotypes isolated from different retail meats. Source Poultry

Pork

Beef Mixed

Total

No. of isolates

Serotype

81

Salmonella Enteritidis Salmonella Infantis Salmonella Hadar Salmonella Newport Salmonella Typhimurium Salmonella Virchow Salmonella Saintpaul Salmonella Chester Salmonella Duisburg Salmonella Sandiego Salmonella Agona Salmonella Anatum Salmonella Derby Salmonella Glostrup Salmonella Mbandaka Salmonella Enteritidis Salmonella Typhimurium Salmonella Brandenburg Salmonella Agona Salmonella Wippra Salmonella Enteritidis Salmonella Indiana Salmonella Enteritidis Salmonella Typhimurium Salmonella Chester Salmonella Agona Salmonella Eko Salmonella Derby Salmonella Heidelberg Salmonella Kottbus

7

3 15

%

201

Table 2 Incidence of Salmonella serotypes in retail meat products. n

34.6 18.5 8.6 7.4 7.4

28 15 7 6 6

6.1 3.7 2.5 2.5 2.5 1.2 1.2 1.2 1.2 1.2 42.9 14.3

5 3 2 2 2 1 1 1 1 1 3 1

14.3 14.3 14.3 66.7 33.3 26.7 26.7 13.3 6.7 6.7 6.7 6.7 6.7

1 1 1 2 1 4 4 2 1 1 1 1 1 106

Salmonella Enteritidis was the prevalent serotype in all studied types of retail meat product and accounted for 34.9% of all strains. The second most common serotype was Salmonella Infantis, which comprised 14.2% of all strains. Salmonella Typhimurium was the third most common serotype. The three Salmonella serotypes most frequently isolated from retail meat products (S. Enteritidis, S. Infantis, S. Typhimurium) accounted for 59.4% of all isolates (Table 2). Salmonella Enteritidis and Salmonella Typhimurium, which comprised 45.3% (n ¼ 48) of all isolates in the present study, are also the most frequently isolated Salmonella serotypes in many countries of Europe, and both are clinically important (EFSA and ECDC, 2011). In the UK, S. Typhimurium was the predominant serotype (54.2%) in fresh raw red meat samples from retail outlets or food services (Little, Richardson, Owen, De Pinna, & Threlfall, 2008). According to Zhao et al. (2008), Salmonella Heidelberg was the serotype isolated most frequently from retail meats in North America. However, Aslam et al. (2012) recently reported that the most prevalent serotype found in retail chicken meat in Alberta, Canada, was S. Hadar, followed by S. Heidelberg and S. Kentucky. In turkey samples, S. Heidelberg was the most common, followed by S. Hadar. This study also isolated Salmonella from 2% of pork samples, but no strains were detected in ground beef. Bosilevac, Guerini, Kalchayanand, and Koohmaraie (2009) reported that the overall incidence of Salmonella isolated from ground beef in the USA was 4.2%. A study of Salmonella strains in retail meat samples from Tehran, Iran, found that S. Thompson was the most prevalent serotype followed by S. Hadar (Dallal et al. 2010). In Vietnam, S. Anatum was the most common serotype found in retail chicken and pork samples, followed by S. Infantis and S. Emek (Thai, Hirai, Lan, &

Salmonella serotype

%

n

Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Total

34.9 14.2 10.4 6.6 5.7 4.7 3.8 2.8 2.8 1.9 1.9 1.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9

37 15 11 7 6 5 4 3 3 2 2 2 1 1 1 1 1 1 1 1 1 106

Enteritidis Infantis Typhimurium Hadar Newport Virchow Chester Agona Saintpaul Derby Duisburg Sandiego Anatum Brandenburg Eko Glostrup Heidelberg Indiana Kottbus Mbandaka Wippra

Yamaguchi, 2012). The findings summarized above indicate that different Salmonella serotypes are prevalent in separate regions of the world and that the trade in food has had an impact on worldwide serotype distribution. 3.2. Antimicrobial susceptibility of Salmonella The results of antimicrobial susceptibility tests are shown in Table 3, Fig. 1 and Fig. 2. Salmonella strains isolated from poultry meat displayed the widest spectrum of antibiotic resistance (to 12 of 19 tested compounds). Isolates from mixed meat samples showed the same resistance spectrum as poultry meat products, excluding aztreonam, cefoxitin and gentamicin. It is likely that most Salmonella isolates from mixed meat originated from poultry. The seven isolates from pork showed resistance to three antimicrobials (ampicillin, nalidixic acid and tetracycline), while the three isolates from beef were resistant to two (nalidixic acid and gentamicin). However, due to the small number of isolates from pork and beef it is hard to draw firm conclusions from this finding. All of the tested strains were susceptible to cefepime, cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, ertapenem and imipenem. The most common antibiotic resistance among all Salmonella isolates (52.8% of strains) was to nalidixic acid. Resistance to this quinolone was observed in strains isolated from all meat sources. This prevalence of resistance to nalidixic acid is in agreement with the findings of previous studies. All tested Salmonella strains isolated from poultry meat in Spain were resistant to nalidixic acid (Álvarez-Fernández, Alonso-Calleja, García-Fernández, & Capita, 2012). In Mexico, higher levels of quinolone resistance were found in isolates from poultry meat compared with those from other foods (Miranda, Mondragon, Martinez, Guarddon, & Rodriguez, 2009). Nalidixic acid resistance was most frequent in isolates from chicken meat, but it was also found in the most common serotypes (except for S. Derby) isolated from fresh pork sausages (Mürmann, Dos Santos, & Cardoso, 2009; Yan et al., 2010). Besides nalidixic acid, the most frequent antibiotic resistance occurring in the Salmonella isolates tested in the present study was also resistance to tetracycline (32.1%), ampicillin (28.3%), streptomycin (28.3%) and sulphonamides (26.4%). The incidence of resistance to the aforementioned antimicrobials is similar to that found in strains isolated from raw, chilled, retail chickens in the

ATM e Aztreonam; AMC e Amoxycillin/clavulanic ac.; AMP e Ampicillin; FEP e Cefepime; CTX e Cefotaxime; FOX e Cefoxitin; CAZ e Ceftazidime; CRO e Ceftriaxone; C e Chloramphenicol; CIP e Ciprofloxacin; ETP e Ertapenem; CN e Gentamicin; IPM e Imipenem; NA e Nalidixic Ac.; SUL e Sulphonamides comp.; STR e Streptomycin; TE e Tetracycline; W e Trimethoprim; SXT e Trimethoprim/sulphametoxazol.

2.7(1) 60(9) 0 28.6(2) 81.8(9) 50(3) 0 33.3(1) 0 0 100(1) 0 50(2) 100(2) 0 0 100(1) 0 100(1) 100(1) 100(1) 2.7(1) 66.7(10) 0 0 54.5(6) 33.3(2) 0 66.7(2) 0 0 0 0 50(2) 100(2) 50(1) 0 0 0 100(1) 0 100(1) Enteritidis Infantis Virchow Hadar Typhimurium Newport Agona Saintpaul Anatum Mbandaka Glostrup Duisburg Chester Sandiego Derby Brandenburg Wippra Indiana Eko Heidelberg Kottbus

37 15 5 7 11 6 3 3 1 1 1 2 4 2 2 1 1 1 1 1 1

0 0 0 0 0 16.7(1) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 6.7(1) 20(1) 57(4) 36.4(4) 16.7(1) 0 100(3) 0 0 0 0 0 100(2) 0 0 0 0 0 100(1) 0

2.7(1) 6.7(1) 20(1) 71.4(5) 72.7(8) 83.3(5) 0 100(3) 0 0 0 0 0 100(2) 50(1) 0 100(1) 0 100(1) 0 100(1)

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 6.7(1) 20 (1) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 6.7(1) 0 0 45.4(5) 0 0 33.3(1) 0 0 0 0 0 0 0 0 0 0 0 100(1) 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 18.2(20) 0 0 66.7(2) 0 0 0 0 0 100(2) 0 0 0 100(1) 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

54(20) 73.3(11) 60(3) 71.4(5) 36.4(4) 50(3) 0 100(3) 0 0 100(1) 100(2) 0 100(2) 0 0 0 0 100(1) 0 100(1)

2.7(1) 60(9) 0 71.4(5) 54.5(6) 0 0 66.7(2) 0 0 100(1) 0 50(2) 100(2) 50(1) 0 0 0 100(1) 0 0

2.7(1) 0 0 0 9.1(1) 0 0 0 0 0 0 0 0 0 50(1) 0 0 0 100(1) 0 0

SXT W TE STR SUL NA IPM CN ETP CIP C CRO CAZ FOX CTX FEP AMP AMC ATM

Antimicrobial

No. of isolates Serotype

Table 3 Antimicrobial resistance among different serotypes of Salmonella % (n).

2.7(1) 0 0 0 9.1(1) 0 0 0 0 0 0 0 0 0 50(1) 0 0 0 100(1) 0 0

Ł. Ma˛ ka et al. / Food Control 36 (2014) 199e204

202

Fig. 1. Antimicrobial susceptibility of Salmonella isolated from various meat sources to tested antibiotics. ATM e Aztreonam; AMC e Amoxycillin/clavulanic ac.; AMP e Ampicillin; FEP e Cefepime; CTX e Cefotaxime; FOX e Cefoxitin; CAZ e Ceftazidime; CRO e Ceftriaxone; C e Chloramphenicol; CIP e Ciprofloxacin; ETP e Ertapenem; CN e Gentamicin; IPM e Imipenem; NA e Nalidixic Ac.; SUL e Sulphonamides comp.; STR e Streptomycin; TE e Tetracycline; W e Trimethoprim; SXT e Trimethoprim/ sulphametoxazol.

UK: sulfonamides (52%), streptomycin (26%), tetracycline (22%) and ampicillin (17%) (Wilson, 2004). Among Salmonella strains isolated from poultry and beef products in Iran by Dallal et al. (2010), resistance to nalidixic acid (82%), tetracycline (69%), trimethoprim (63%) and streptomycin (52%) was extremely widespread. The percentage of antibiotic-resistant strains among Iranian isolates was far higher than found in the present study, most notably, the incidence of resistance to trimethoprim (63% vs. 3.8%). As in Poland, all Salmonella isolates from Iran were sensitive to ciprofloxacin, imipenem, ceftazidime (Dallal et al., 2010). Resistance to sulphonamides, streptomycin, tetracycline and ampicillin has been frequently reported among Salmonella isolates from retail meats sampled in East Asia, but unlike the present study, the tested strains were often also resistant to chloramphenicol (37.3e42.1%), ciprofloxacin (42.1%) and trimethoprim (34.0%) (Thai et al., 2012; Yan et al., 2010). None of the Salmonella strains isolated in North Vietnam by Thai et al. (2012) were resistant to ceftazidime. In a study examining Salmonella in raw red meats in the UK, the most frequent antimicrobial resistance found in isolates was to tetracyclines (67.5%), sulphonamides (56.6%), streptomycin (50.6%), ampicillin (43.4%) and chloramphenicol (33.7%) (Little et al., 2008). In Austria, the highest resistance rate among Salmonella isolated from meat samples was seen for nalidixic acid (42%), followed by

Fig. 2. Percentage of Salmonella isolates resistant to the tested antibiotics. ATM e Aztreonam; AMC e Amoxycillin/clavulanic ac.; AMP e Ampicillin; FEP e Cefepime; CTX e Cefotaxime; FOX e Cefoxitin; CAZ e Ceftazidime; CRO e Ceftriaxone; C e Chloramphenicol; CIP e Ciprofloxacin; ETP e Ertapenem; CN e Gentamicin; IPM e Imipenem; NA e Nalidixic Ac.; SUL e Sulphonamides comp.; STR e Streptomycin; TE e Tetracycline; W e Trimethoprim; SXT e Trimethoprim/sulphametoxazol.

Ł. Ma˛ ka et al. / Food Control 36 (2014) 199e204

tetracycline (almost 33%), streptomycin (27%), ampicillin and chloramphenicol (both 17%), and ciprofloxacin (9.6%) (Mayrhofer, Paulsen, Smulders, & Friederike, 2004). The results of the present study support the notion that the frequency of antimicrobial resistance is higher among Salmonella strains isolated from poultry meat than in those from other meat sources, e.g. pork (Thai et al., 2012). Examination of the resistance profiles of individual Salmonella serotypes (Table 3) showed that among Salmonella Enteritidis isolates, 54% (n ¼ 20) were resistant to nalidixic acid and one was multiresistant (AMP, NA, SUL, STR, TE, W, SXT). Of the Salmonella Infantis strains, 73.3% (n ¼ 11), were resistant to nalidixic acid, 66.7% (n ¼ 10) were resistant to sulphonamides, while 60% (n ¼ 9) showed resistance to both streptomycin and tetracycline. Salmonella Typhimurium displayed resistance to the widest spectrum of antimicrobials: 10 of the 19 tested compounds. Among these strains, resistance to tetracycline was most prevalent (81.8%, n ¼ 9) and many strains were resistant to ampicillin (72.7%, n ¼ 8), and sulphonamides and streptomycin (54.5%, n ¼ 6). Of all the isolates, only one strain of Salmonella Newport was resistant to aztreonam. 3.3. Multiresistance Of the 106 Salmonella strains isolated from meat samples in the present study, 68.9% (n ¼ 73) displayed antibiotic resistance. The frequency of resistance among isolates was high and varied in particular years between 59.1% in 2010 and 84.6% in 2011 (Fig. 3). Overall, 42.5% (n ¼ 31) of the resistant isolates showed resistance to one antibiotic, 5.5% (n ¼ 4) to two, 13.7% (n ¼ 10) to three, 17.8% (n ¼ 13) to four, and 20.5% (n ¼ 15) were resistant to five or more antibiotics (Table 4). Among the 37 Salmonella Enteritidis isolates, 54% (n ¼ 20) displayed antibiotic resistance. A higher percentage of resistant isolates was found in some of the less frequently identified serotypes: Salmonella Newport (100%, i.e. 6 resistant strains/6 isolates), Salmonella Typhimurium (91%, 10/11), Salmonella Hadar (85.7%, 6/7), Salmonella Virchow (80%, 4/5) and Salmonella Infantis (80% 12/15). Of the 20 resistant S. Enteritidis isolates, 19 strains were resistant to one antibiotic (nalidixic acid) and one was resistant to 7 antibiotics. Multiresistance (resistance to 3 or more antimicrobials) was seen in a higher proportion of some less frequently identified serotypes: Salmonella Newport (3 multiresistant strains among 6 resistant isolates), Salmonella Typhimurium (7/10), Salmonella Hadar (5/6), Salmonella Infantis (10/12). The percentage of multiresistant strains among antibiotic resistant isolates varied between 23.1% in 2010 and 81.8% in 2012. The greatest differences was observed between the year and the frequency of multiresistant isolates for the scope of years 2010e2012 (Fig. 3).

Fig. 3. Percentage of resistant and multiresistant Salmonella isolates by year.

203

Table 4 Multiresistance in various Salmonella serotypes. Serotype

Number of antimicrobials 1

Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Total:

Enteritidis Infantis Typhimurium Hadar Virchow Newport Saintpaul Eko Derby Heidelberg Glostrup Wippra Indiana Duisburg Kottbus Chester Sandiego

2

19 1 2 1 3 2

3

4

5

6

7

8

1 1 1

1

1 1 1 1 2

8 1 3

1 1 1

2

1 1

2

1 1

1 1 1 1 1 2 1 2 31

4

10

13

6

2 6

3

Our findings are similar to those of previous studies with respect to the antibiotic resistance rate in particular Salmonella serotypes: resistance has been observed more frequently in S. Typhimurium, S. Infantis and S. Virchow than in S. Enteritidis (Ma˛ ka et al., 2010; Thai et al., 2012; Threlfall, Ward, Frost, & Willshaw, 2000). In Turkey, 62% of Salmonella strains isolated from meat were resistant to three or more antimicrobial agents (Arslan & Eyi, 2010). In Spain, the average number of antibiotic resistance determinants per strain was lowest in isolates of Salmonella Enteritidis (4.64), while higher values were seen in S. Infantis (5.5), S. Newport (6.5) and S. Typhimurium (6.0) (Álvarez-Fernández et al., 2012). Among strains isolated from meat samples in Austria, 36% of S. Enteritidis isolates were resistant to at least one of the antimicrobials tested, all four S. Virchow isolates showed resistance, one of the three isolates of S. Typhimurium was sensitive to all tested antimicrobials, and the single S. Hadar isolate was multiresistant (Mayrhofer et al., 2004). Multiresistance has become a significant public health problem. Antibiotic resistance is a factor that can influence the severity of salmonellosis. Moreover, infections caused by multiresistant bacteria are often much more difficult to treat because the panel of effective antimicrobials is reduced and antimicrobial therapy can be delayed or inadequate (Magiorakos et al., 2012; Roberts et al., 2009). 4. Conclusions This study examined Salmonella strains isolated in Poland between 2008 and 2012 from retail meat products; mostly from poultry, but also from beef, pork and mixed meat. The serotype found most often was Salmonella Enteritidis. Salmonella strains isolated from poultry products were resistant to a wider spectrum of antimicrobials that those of other origins. Among the isolates, resistance to nalidixic acid was prevalent, but tetracycline, ampicillin, streptomycin and sulphonamides resistance was also common. Multiresistance was more frequently observed among isolates of S. Newport, S. Typhimurium, S. Hadar, S. Virchow and S. Infantis, than in the more abundant S. Enteritidis strains. Although the number of Salmonella strains isolated from retail meats in Poland decreased over the 5-year study period, other potential problems remain. In particular, levels of antibiotic resistance and multiresistance are high among the isolated strains. This represents an additional risk for consumers because therapy becomes more complicated in the case of illness. Even where the consumption of contaminated food does not cause illness, the

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introduction of antibiotic resistant bacterial strains can lead to the transfer of resistance genes to the host bacteria via HGT. In view of the observed increase in the resistance level of Salmonella strains, regular surveillance is essential for the early detection of any shift in the antimicrobial resistance profile of isolates from food and the environment. Our findings indicate that food is a potential source of resistant bacteria that are a serious concern for public health and food safety. Acknowledgments This work was supported by a grant from the National Center of Science DEC-2011/01/N/NZ9/00197 and partly supported by a grant from the State Committee for Scientific Research, Poland (741/NCOST/2010/0). The research was conducted as part of European Cooperation in the field of Scientific and Technical (COST) Research Action TD0803 “Detecting evolutionary hot spots of antibiotic resistances in Europe (DARE)” (2009e2013). The authors would like to thank Dr. Jolanta Szych, Monika Wasiak and the Department of Bacteriology of the National Institute of Public Health e National Institute of Hygiene (Warsaw, Poland) for serotyping the Salmonella isolates. References Alekshun, M. N., & Levy, S. B. (2007). Molecular mechanisms of antibacterial multidrug resistance. Cell, 128, 1037e1050. Allen, H. K., Donato, J., Wang, H. H., Cloud-Hansen, K. A., Davies, J., & Handelsman, J. (2010). Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology, 8, 251e259. Álvarez-Fernández, E., Alonso-Calleja, C., García-Fernández, C., & Capita, R. (2012). Prevalence and antimicrobial resistance of Salmonella serotypes isolated from poultry in Spain: comparison between 1993 and 2006. International Journal of Food Microbiology, 153(3), 281e287. Aminov, R. I. (2010). A brief history of the antibiotic era: lessons learned and challenges for the future. Frontiers in Microbiology, 1, 134. Aminov, R. I. (2011). Horizontal gene exchange in environmental microbiota. Frontiers in Microbiology, 2, 158. Andersson, D. I., & Hughes, D. (2010). Antibiotic resistance and its cost: is it possible to reverse resistance? Nature Reviews Microbiology, 8(4), 260e271. Arslan, S., & Eyi, A. (2010). Occurrence and antimicrobial resistance profiles of Salmonella species in retail meat products. Journal of Food Protection, 73, 1613e 1617. Aslam, M., Checkley, S., Avery, B., Chalmers, G., Bohaychuk, V., Gensler, G., et al. (2012). Phenotypic and genetic characterization of antimicrobial resistance in Salmonella serovars isolated from retail meats in Alberta, Canada. Food Microbiology, 32(1), 110e117. Bosilevac, J. M., Guerini, M. N., Kalchayanand, N., & Koohmaraie, M. (2009). Prevalence and characterization of Salmonellae in commercial ground beef in the United States. Applied and Environmental Microbiology, 75(7), 1892e1900. CLSI. Clinical and Laboratory Standards Institute. (2012). Supplement M100-S22. Performance standards for antimicrobial susceptibility testing; twenty-second information (Vol. 32) (3). Wayne, PA. Council conclusions of 22 June 2012 on the impact of antimicrobial resistance in the human health sector and in the veterinary sector e a “One Health” perspective. Official Journal of the European Union C211, 55, 2e5. Czarkowski, M. P., Cieleba˛ k, E., Kondej, B., & Staszewska, E. (2009). Infectious diseases and poisonings in Poland in 2008. Warsaw, Poland. Czarkowski, M. P., Cieleba˛ k, E., Kondej, B., & Staszewska, E. (2011). Infectious diseases and poisonings in Poland in 2010. Warsaw, Poland. Dallal, M. M. S., Doyle, M. P., Rezadehbashi, M., Dabiri, H., Sanaei, M., Modarresi, S., et al. (2010). Prevalence and antimicrobial resistance profiles of Salmonella serotypes, Campylobacter and Yersinia spp. isolated from retail chicken and beef, Tehran, Iran. Food Control, 21(4), 388e392. Doyle, M. P., Busta, F., Cords, B. R., Davidson, P. M., Hawke, J., Hurd, H. S., et al. (2006). Antimicrobial resistance: implications for the food system e an expert report, funded by the IFT Foundation. Comprehensive Reviews in Food Science and Food Safety, 5, 71e137. EFSA. (2008). Scientific opinion of the Panel on Biological Hazards on a request from the European Food Safety Authority on foodborne antimicrobial resistance as a biological hazard. The EFSA Journal, 765, 1e87. EFSA (2011a). http://www.efsa.europa.eu/en/corporate/doc/factsheetsalmonella.pdf. EFSA, & EFSA Panel on Biological Hazards (BIOHAZ).. (2011b). Scientific opinion on the public health risks of bacterial strains producing extended-spectrum blactamases and/or AmpC b-lactamases in food and food-producing animals. The EFSA Journal, 9(8), 1e95, 2322.

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