First study of Salmonella in meat in Romania

Introduction: The increasing antimicrobial resistance of Salmonella isolates is of major public health concern, but information regarding these aspects is still lacking in Romania. This study focused on a detailed and accurate investigation concerning prevalence, serotypes, and antimicrobial resistance patterns of Salmonella strains, isolated from pork and chicken meat, collected from all regions of Romania in 2011. Methodology: The research was conducted on 650 samples of chicken and pork meat collected from production units and retail markets located in various regions of Romania. A total of 149 Salmonella isolates were recovered (22.92%), serotyped, confirmed by PCR, and further tested for antimicrobial susceptibility. Results: Thirteen Salmonella serovars were identified; predominant serovars included Infantis, Typhimurium, Derby and Colindale. Multiple resistance was found in 83.22% (n = 124) of the isolates. The isolates were frequently resistant to tetracycline (80.53%), streptomycin (81.21%), sulfamethoxazole (87.25%), nalidixic acid (65.10%), and ciprofloxacin (42.95%). Additionally, a markedly lower resistance rate was observed for ampicillin (20.81%), chloramphenicol (16.78%), and ceftazidime (11.41%). Among 137 resistant Salmonella isolates, 35 different resistance patterns were found. Conclusion: A high prevalence of Salmonella spp. and a relatively high resistance rate to multiple antimicrobials was found. This data indicates that chicken and pork meat could constitute a source of human exposure to multidrug-resistant Salmonella and therefore could be considered a potential vehicle of resistant Salmonella foodborne diseases. Further actions are needed to succesfully implement a national surveillance program for better monitoring of these resistant pathogens.


Introduction
Numerous epidemiological studies have implicated foods of animal origin as major vehicles associated with illnesses caused by foodborne pathogens, which lead to the development of antimicrobial-resistant pathogens [1].Salmonella species are considered to be among the most important foodborne pathogens in the world and salmonellosis is still one of the most widespread foodborne bacterial illnesses in humans, with clinical manifestations ranging from asymptomatic state to severe disease [2].The majority of infections are associated with the ingestion of contaminated foods such as poultry, beef, pork, eggs, milk, cheese, seafood, fruits, juices, and vegetables [3,4], although most infections caused by multidrug-resistant Salmonella are acquired through contaminated foods of animal origin.
Strains of Salmonella that are resistant to antimicrobial agents have become a worldwide public health concern, since resistance in Salmonella limits the therapeutic options available to physicians in the treatment of salmonellosis in humans.
Surveillance of antimicrobial resistance in zoonotic bacteria such as Salmonella is essential for providing information on the magnitude and trends of resistance in foodborne pathogens in each country, because the resistance prevalence varies widely between and within countries and over time [5].The use of antimicrobials in one country affects the spread of resistance in others.
Information about the prevalence of multidrugresistant (MDR) Salmonella isolated from chicken and pork meat and about Salmonella resistance trends is lacking in Romania.Moreover, little is known about the potential role of meat in the dissemination of MDR Salmonella, because very limited research work concerning these factors had been done in our country.
The present study was undertaken to provide baseline data on antimicrobial resistance in Salmonella strains isolated from chicken and pork meat in Romania.We reported prevalence, serotypes, and antibiotic resistance patterns of Salmonella strains isolated from pork and chicken meat, collected from all regions of the country, in 2011.

Methodology
The study was conducted on 650 food samples, including pork (n = 208) and chicken (n = 442), randomly collected from production units and retail markets in Romania, during the period of January to December 2011.

Salmonella spp. isolation
The isolation protocol followed the steps recommended by the International Organization for Standardization (ISO) 6579 and previously described by Molla and Mesfin (2003) [6].Briefly, the meat samples (25 g) were previously homogenized in buffered peptone water (225 mL) with a laboratory blender (Stomacher 400, Seward Ltd., Worthing, England) for approximately two minutes.After incubation for 18 to 24 hours at 37°C, 0.1 mL was inoculated in 10 mL Rappaport-Vassiliadis (RV) green broth (LabM Limited, Heywood, England) and incubated for 18 to 24 hours at 42°C.Another 1 mL from the culture obtained was inoculated into 10 mL of selenite cysteine (SC) broth (LabM Limited, Heywood, England) and incubated at 37°C for 18 to 24 hours.From both enrichment broths obtained, 1 mL was streaked onto brilliant green-phenol red-lactosesucrose (BPLS) agar (Merck, Darmstadt, Germany) and xylose lysine deoxycholate (XLD) agar (Oxoid, Basingstoke, England).Following the incubation at 37°C for 24 hours, presumptive Salmonella colonies were characterized by their biochemical properties through slide agglutination using standard protocols.The positive colonies where then identified as Salmonella using the Sensititre Automated Microbiology System Aris 2X (Thermo Scientific, Waltham, USA) following the protocol stated by the producer.

Serotyping
The serovar was established with the antisera commercially available, Salmonella antisera test group (Denka Seiken Co., Tokyo, Japan), which provides specific agglutinins for each Salmonella antigen.The steps were followed in accordance with the protocol mentioned in the test performed.

PCR confirmation
All strains of Salmonella spp.were confirmed by PCR, targeting the common sequence ompC, using the set of primers previously described by Modaressi and Thong (2010) (F: 5´-ATCGCTGACTTATGCAATCG-3´, R: 5´ CGGGTTGCGTTATAGGTCTG-3´) [7].The bacterial DNA extraction followed the basic steps previously described by Yang et al. (2008) with a few particularities [8].Briefly, 150 µL of CHELEX (10%) reactive (BioRad, Berkeley, USA) was added in Eppendorff tubes (1.5 mL) (Ratiolab, Dreieich, Germany).The tubes were subjected to UV sterilization in a microbiological laminal flow, class II to remove any possible contaminants from the manipulation performed earlier.One to two colonies were harvested with a sterile microbiological loop and immersed in the CHELEX reactive.The following extraction temperatures were used: 57ºC -30'; 94ºC -5'.The last step included a high-speed centrifugation (14.000 rotations per minute) for one minute.
The amplification for ompC was carried out in a final volume of 25 μL containing 25 pmol of each primer, 12.5 µL of MasterMix (Bioline, London, UK), 4 µL of DNA template, and 6.5 µL PCR water grade (Sigma, Saint Louis, USA).For each experiment, a negative control containing the same reactive except for the DNA template, was used.For the positive control, Salmonella Typhimurium ATCC 14028 was used.
The PCR conditions were adjusted for the primers and Taq-polymerase used, consisting of an initial denaturation at 95°C for 4 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and 1 minute at 72°C; the final elongation was performed at 72°C for 5 minutes.
Given the fact that Salmonella serovars Typhimurium and Enteritidis are the most common cause of human salmonellosis worldwide, the accurate confirmation of these strains was made by PCR, amplifying specific sequences for S. Enteritidis (ENT) and S. Thyphimurium (STM).This multiplex PCR was selected to insure the accuracy of the results.

The statistical analysis
The statistical package SPSS Statistics version 19 was used.The differences were considered significant at a p value less than 0.05.

Prevalence of Salmonella in chicken and pork meat
A total of 149 Salmonella isolates were recovered, representing 22.92% of the samples tested.Out of the 149 Salmonella isolates, 48 were from pork samples (32.21%) while 101 (67.78%) were from chicken samples.

Antimicrobial susceptibility
The results of the antimicrobial resistance tests of 149 Salmonella isolates are shown in Table 2.
Isolates showing resistance to three or more antimicrobials were classsified as multidrug resistant (MDR).
The resistance to multiple antimicrobial agents was predominantely seen in serovars Infantis, Colindale, Derby and Typhymurium.Among the 19 S. Typhimurium isolates, 11 were MDR, all of them showing resistance to more than five antimicrobials (Table 3).
MDR was seen in 64.58% of the pork isolates and in 92.07% of the chicken isolates.Among 137 resistant Salmonella isolates, 35 different resistance patterns were found, and most of them were represented by five strains.More than 37% of the isolates showed resistance to five antimicrobials, while 22.8% were resistant to six antimicrobials.Two isolates (one isolate of S. Colindale from pork and one chicken isolate of S. Typhimurium) were resistant to eight antimicrobials, the highest number of resistant traits shown by the isolates.Antimicrobial resistance patterns exhibited by Salmonella isolates are presented in Table 3.The most prominent pattern was sulfamethoxazole, streptomycin, ciprofloxacin, nalidixic acid, and tetracycline (20.8% of the isolates).

Discussion
The increasing antimicrobial resistance in the foodborne zoonotic bacteria Salmonella is a major public health concern.Although in 2010, the number of reported human Salmonella cases in 14 countriesall members of the European Union -showed a significant decreasing level, Malta and Romania presented an increasing trend [10].
The present study, the first report about substantial MDR in Salmonella serovars isolated from Romania, revealed a high antimicrobial resistance in Salmonella enterica subsp.enterica isolated from chicken and pork meat There are few other reports about resistance to antimicrobial agents, but these reports focused on particular products in Romania [11].
Our study demonstrated that pork and chicken meat were contaminated by Salmonella; a total of 149 Salmonella isolates were recovered, representing 22.92% of the samples tested.Different prevalence rates of Salmonella spp. in food, especially poultry and poultry products, have been reported by different authors [12][13][14][15][16][17].Salmonella contamination rates observed in our country are relatively high (22.92%)and confirm the widespread contamination of pork and chicken meat with Salmonella spp.The data reported by Romania in 2010 in the European Surveillence Program showed that samples tested at retail were less contaminated than samples tested earlier in the food chain [10], which shows that even higher levels are expected at farms or slaughterhouses.Moreover, the high contamination rates obtained in our study suggest that both poultry and pork meat could be a potential vehicle of transmission of Salmonella spp.from animals to humans.Given our results, a microbiological risk assessment (MRA) must be taken into consideration in this country, in order to improve food control systems and to produce safer food, reduce the number of foodborne illnesses, and facilitate the European trade.
Among the 149 Salmonella isolates, 83.22% were MDR.Multidrug resistance was seen in 64.58% of the pork isolates and in 92.07% of the chicken isolates.
The level was much higher than earlier figures reported by Thong and Modaressi (2011) in Malaysia (67%) [12], Yan et al. (2010) in China (20.9%) [15], Bouchrif et al. (2009) in Morocco (44%) [20], Hao Van et al. (2007) in Vietnam (34%) [21], and lower than figures reported by Yildirim et al. (2011) (97% of isolates from raw chicken carcasses exhibited multidrug resistance) [13].When comparing the two types of meat sampled (pork and chicken), a significant difference (p = 0.001) was found in the multidrug resistance pattern; it was much higher in chicken (92.07%) than in pork (64.58%).The high rates of resistance revealed by this study can be explained by the extensive use of antimicrobial agents given in large units of animal growth, especially chickens, as prophylaxis, growth promoters, or treatment.The use of antimicrobials is under strict surveillance in Romania and growth promoters have been banned, but as it is well observed, farmers still use antimicrobials to achieve their production goals with minimal losses, regardless of the health costs [22].
The high resistance to sulfamethoxazole was not surprising, since sulfamethoxazole (in combination with trimethoprim) is widely used in Romania, both in human and veterinary medicine.
The widespread occurence of antimicrobial resistance to sulfonamides and tetracycline was also demonstrated in Salmonella strains isolated from fresh pork sausages by Murmann et al. in 2009 [15].
Resistance to tetracycline (80.53%) and streptomycin (81.20%) tended to occur at relatively high frequencies, higher than reported by Thong and Modaressi in 2011 (73.8% and 57.9%) [11], Yildirim et al. in 2011 (67.6% and 61.7%) [13], and Zhao et al. in 2009, who observed resistance of S. Typhimurium to streptomycin (63%) and tetracycline (61%) [23].A higher resistance rate to tetracycline was reported by Mahmud et al. in 2011 (93%) [14].S. Typhimurium and S. Infantis isolates had higher resistance rates to these antibiotics.One of the serovars commonly involved in foodborne diseases, S. Typhimurium from chicken, was resistant to eight antimicrobials, the greatest number of resistant phenotypes.Lower rates (62%-65%) were mentioned in a number of studies focused on these specific strains [24,25] while higher rates of 84%-97% were mentioned less frequently [26].Other studies concluded that this resistance prevalence for tetracycline and streptomycin is due to their frequent administration in veterinary medicine [27].
Nalidixic acid resistance was especially prevalent in chicken isolates (84.15%).This finding is in accordance with studies from other countries [12,15].The prevalence of Salmonella isolates resistant to nalidixic acid was lower than the one reported by Shrestha et al. (2010) in Nepal [28].The emergence of quinolone-resistant Salmonella isolates from food animals in Europe has increased substantially following the licensing of fluoroquinolones such as enrofloxacin for veterinary use [29].Since enrofloxacin is the second most used antibiotic in veterinary practice in Romania after tetracycline, an even higher resistance was expected.Resistance to nalidixic acid is a matter of concern since nalidixic acid resistance has been associated with a decreased susceptibility to fluoroquinolones, which are used to treat salmonellosis in humans [30].Treatment of serious human enteric infections with an effective fluoroquinolone can reduce the duration of illness, and most likely prevent complications and adverse outcomes, including hospitalization [31].Therefore, resistance to fluoroquinolones can be considered an important public health concern, since these antibiotics are being widely used in veterinary medicine as well as in poultry production, which causes the resistence genes to be transmitted to humans through the food chain [32].
Resistance to ciprofloxacin (42.95%) was found to be high.Ciprofloxacin is widely used in Romania, especially in the treatment of human urinary tract infections, respiratory tract infections, and infections of the gastrointestinal tract.Simple observations of current medical practice reveal that many fluoroquinolones are used even in minor infections where no proper diagnosis has been made; the criteria set for a prudent use of antimicrobials, therefore, is not being observed.
In our study, few strains of Salmonella (11.40%) were resistant to third-generation cephalosporins (ceftazidime).However, the resistance rate is higher than the one reported by Yan et al. (2010) [15], Chao et al. (2007) [33], and Thong and Modarressi (2011) [12].Concerning food-borne Salmonella, the resistances of most concern are those against quinolones and cephalosporins, both of which are mentioned in the WHO list of critically important antibiotics for human medicine [5].
In this study, the resistance to ampicillin was found to be only 20.80%, notably lower than the resistance reported in other countries by Mahmud et al. (2011) in Bangladesh (100%) [14] and Thai et al. (2012) in Vietnam [34].This finding is not surprising, since ampicillin is not frequently used in animal therapy, although it is still prefered in the classical therapeutical protocol of salmonellosis in humans.
Among 137 resistant Salmonella isolates, 35 different resistance patterns were found; most of them were represented by five strains of antimicrobials.One of the most prominent patterns was sulfamethoxazole, streptomycin, ciprofloxacin, nalidixic acid, and tetracycline (SMX, NA, CIP, S, TET).

Conclusion
Overall, the frequencies and resistance patterns tend to vary remarkably from one country to another.Given the fact that, in Romania, this is the first report concerning the prevalence of MDR Salmonella isolates, we could not establish a correlation with the year, area, and environmental factors, but the general trends have been established regarding Salmonella spp.sensitivity to individual antimicrobial agents.The relatively high MDR Salmonella spp.isolates from chicken and pork meat observed in our study could be considered as one of the potential sources of human salmonellosis in Romania.
In conclusion, chicken and pork meat obtained in Romania pose a risk in the dissemination of MDR Salmonella in the European market.The results obtained provide baseline data for further antimicrobial resistance studies and also for the epidemiological inquiries that lack information about our country and that could be important, not only for epidemiologists monitoring the spread of MDR Salmonella in Romania, but also beyond our borders.From this study, it is evident that MDR Salmonella is more prevalent in poultry than in pork meat, which demonstrates the necessity for stricter surveillance of antimicrobial use in poultry production in Romania.This single investigation might not be enough for a complete risk assessment concerning the possible Salmonella threat in food products, but it can draw a warning signal for further actions.This study recommends a closer cooperation between the parties involved in the prevention and control of diseases transmitted from food to humans.

Table 1 .
Serotype of Salmonella isolates

Table 2 .
Antimicrobial resistance of Salmonella isolates from chicken and pork meat