Prevalence and genotypic characterization of Salmonella spp. from chicken meats marketed in the province of Skikda, Algeria

Here, we aim to determine the prevalence of Salmonella contamination of poultry meat from butcheries of the province of Skikda and to investigate antibiotic resistance. Salmonella spp. isolates were screened from 70 samples, including chicken breasts (n = 40 samples) and chicken thighs (n = 30 samples) collected from 14 butcheries. All suspected Salmonella colonies from selective media were confirmed by MALDI-TOF MS and serotyped. The susceptibility profile to 16 antibiotics was studied. According to the antibiotic susceptibility results, resistance genes were investigated by standard PCR targeting various genes such as blaSHV, blaTEM, aac3, aac6-Ibcr, aad, qnrA and qnrB. Of the 14 butcheries studied, samples from eight butcheries were contaminated with Salmonella (57.14%). 19 Salmonella strains were isolated, including five serotypes with a predominance of Kentucky serotype (n = 9), Enteridis (n = 3), followed by Heidelberg (n = 3), Virchow (n = 3), and Manhattan (n= 1). All isolates were resistant to Rifampicin (100%; n = 19), and to other antibiotics such as Ciprofloxacin (47.36%), Amoxicillin-clavulanic acid (47.36%; n = 9), Amoxicillin, (47.36%; n = 9), Ticarcillin-clavulanic acid (47.36%; n = 9), and Gentamycin (47.36%; n = 9). All isolates showing multidrug resistance (47.36%; n = 9) were positive by PCR to the blaTEM-1 β-lactamase gene, from which 8 strains carried the aminoglycoside resistance aad7 gene. However, none was positive for the tested blaSHV, Aac3, Aac6-Ibcr, qnrA, qnrB, ArmA and ArmB genes. Our findings show a worrying rate of Salmonella contamination of poultry meats.


Introduction
Salmonella is one of the most important causes of foodborne diseases worldwide. It is frequently associated with consumption of contaminated products such as poultry, eggs, meat, milk and seafood [1]. S. enterica infection leads to severe public health consequences and significant economic losses [2]. Algeria has seen a significant development of the poultry industry over the last decade and chicken meat is the most popular because of its relatively low price and easy digestibility [3,4]. According to the statistics of the Ministry of Agriculture, Algeria produces about 460,000 tons of white meat and 6 billion eggs annually [5]. In Algeria, the poultry meat contamination occurs during the transport of live birds, their housing, slaughter and marketing without compliance with basic hygiene criteria. This meat is generally implicated in human salmonellosis outbreaks causing acute gastroenteritis, especially in young and immunodeficient patients [6]. Furthermore, poultry has been reported as a source of non-typhoidal Salmonella resistant to clinically relevant antibiotics with a higher incidence in middle-income countries [7]. The emergence and spread of antimicrobial-resistant Salmonella strains, particularly multi-drug resistant (MDR), is a major public health concern [7]. Genes conferring resistance to these antibiotics have been found on different plasmid types. The latter carry multiple antibiotic resistance genes that are transferable to other Salmonella strains and other bacterial species [7]. In this scope, the present study was undertaken to study the prevalence of Salmonella contamination in marketed poultry meat in Skikda province and to characterize the antibiotic resistance mechanisms of the Salmonella isolates.

Study locations
The present study was carried out from 14 butcheries, located in the province of Skikda (northeastern Algeria), over a period from December 2014 to February 2016. We have tried to cover the most accessible municipalities of the province. For technical reasons, including purchase of poultry meats, a total of 70 samples were collected. Samples consisted of three breasts and two thighs. All samples were transported to the laboratory into ice packs within a period not exceeding two hours to be treated on the same day or kept in the refrigerator overnight.

Data collection and analysis
Bacteriological analyses were performed according to the EN/ISO 6579-2002/Amd1:2007 protocol for Salmonella detection in food and animal feedstuffs [8]. Samples (25g) of meat and skin of breast and thigh were individually pre-enriched with 225 mL of buffered peptone water broth (PWB) (Fluka, Sigma Aldrich, St. Quentin Fallavier, France). All samples were incubated at 37°C for 18-20 hours. From each pre-enrichment solution, 1 mL and 0.1 mL were respectively transferred into 10 mL of enrichment Muller-Kauffmann tetrathionate / novobiocin broth (AES Chemunex Combourg, Bretagne, France) and 10 mL of Rappaport Vassiliadis broth (Merck Darmstadt, Land Hessen, Germany) and incubated at 37 °C and 42 °C for 24 hours, respectively. Both enriched samples were then streaked on XLD (Fluka analytical Steinheim, Buchs, Switzerland) and Hektoen agars (Pasteur Institute of Algeria) and incubated at 37 °C for 24 hours. Suspected colonies were first identified with the API 20E System (bioMérieux, Crappone, France), then confirmed with MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time OF light Mass Spectrometry) (Bruker Daltonics GmbH, Germany) [9]. The protein mass profiles were obtained using the Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Brême, Germany), with Flex Control software (Bruker Daltonics, Brême, Germany). The spectrum profiles obtained were visualized with Flex analysis v.3.3 software and exported to MALDI-Biotyper v.3.0 (Bruker Daltonics, Germany) for data processing (smoothing, baseline subtraction and spectra selection). The phyloproteomic analysis of Salmonella strains was assessed through construction and comparison of their reference spectra (main spectra) with the MALDI-Biotyper v.3.0 software (Bruker Daltonics, Germany). Cluster analysis was performed based on a pairwise comparison of specific main spectra (MSP: mean spectra projection dendrogram) of the different strains to generate a dendrogram of similarities among spectra profiles using the software default correlation function.

Prevalence of poultry meat contamination by Salmonella
Of the 14 butcher shops studied, eight had poultry meat contaminated with Salmonella, resulting in a prevalence rate of 57.14%. The number of contaminated samples with Salmonella varied according to the nature of the sample: 10 breasts (n = 40), and 9 thighs (n = 30).

Distribution and antimicrobial resistance of Salmonella serotypes
Nineteen Salmonella strains were isolated from poultry meat samples and confirmed by MALDI-TOF MS and gave very good scores ranging from 2.00 to 3.00. The phyloproteomic analysis of the multidrug resistant Salmonella strains (n = 9) from poultry meats of the present study and isolates from human and poultry of our previous study [20] was assessed through the construction and comparison of their characteristic reference spectra (main spectra). As shown on Figure 1, we noted that all Salmonella strains from poultry meats and five poultry farms avian strains clustered together (A distance level of 150). Interestingly, we observe that Salmonella strains of the present study (in red) clustered together with strains of our previous study, suggesting the presence of a Salmonella clone which contaminate poultry meats and farm environment.

Discussion
The results of the present study highlight the prevalence of contamination of poultry meat sold in some butcheries in the province of Skikda. The recorded prevalence rates (57.14%) are less with those reported in other similar studies 35, 5% in Mexico [21; 33% in Niger [22]; 22.6% in Egypt [4] and 5.92% in Saudi Arabia [23]. As previously reported, white meat had significantly more bacteria than other types of meat [24][25][26][27]. Poultry and cutting meats are often contaminated with the gastrointestinal flora, which could possibly be the cause of food borne pathogens [28].
Contamination from one commodity to another, usually through the hands of operators, utensils and work tops or cutting boards [29]. Studies in this area clearly show that without adequate precautions, the bacteria present on the surface of chicken carcasses can be disseminated in the kitchen after raw meat cutting operations [30,31]. We have noted a higher contamination of the breast meat (23.80%). This finding is in accordance with the results reported by Khalafalla et al. with Salmonella contamination of 20% for breast meat and 33.3% for thighs [4].
The results of the serotyping showed a very heterogeneous distribution of the serotypes. This distribution suggests that most Salmonella serotypes are primarily transmitted during breeding and slaughter.Two serotypes were identified in most of the butcheries studied namely, Kentucky (n = 9) and Enteritidis (n = 3). Khalafalla et al. also recorded the predominance of serotype Enteritidis in their study on butcheries in Egypt [4]. This serotype is also very common in animal production, especially in poultry farming [32]. The white meat isolates were predominantly resistant to rifampicin. These results agreed with Abd El-Tawab et al. who reported 100% of resistance to cephalexin and rifampicin in Salmonella isolated from chicken meats in Dubai [33]. We have noted resistance to certain antibiotics, such as ciprofloxacin; amoxicillin-clavulanic acid, amoxicillin; ticarcillin-clavulanic acid and gentamycin. Penicillin and tetracycline are commonly used in poultry feed as antimicrobial agents. The resistance to these antimicrobials has been previously demonstrated and related to poultry production units [34,35]. By comparing our results with those of a study carried out in the same region on farms and slaughterhouses [20], it appears that antibiotic resistance in poultry meat is less expressed, which reinforces the hypothesis that amplifying factors such as breeding conditions (drinking water, nature of the soil on the farm and in the slaughterhouse) are involved in Salmonella contamination. Shea suggested that prolonged antibiotic therapy is the major cause of antimicrobial resistance. Fluoroquinolones are among the most widely used antibiotics for treating human and animal salmonellosis because of their broad antimicrobial activity spectrum [36]. The most prevalent serovar carrying resistance genes was Salmonella Kentucky. It has become the most commonly detected serovar in chickens, while S. Typhimurium remains the most common cause of human infections. The prevalence of multidrug resistance (MDR) S. Kentucky isolates from poultry is significant [37]. The present study demonstrates the presence of TEM genes. This finding is partly consistent with the results of previous studies, which confirmed the presence of β-lactamase encoding the bla TEM gene conferring resistance to penicillins and first-generation cephalosporins [38,39]. ESBLs are mostly located on mobile genetic elements (plasmids or integrons) that can facilitate their mobility from a bacterial species to another by horizontal gene transfer [38]. We have reported the presence of aad genes that confer resistance to streptomycin, gentamicin and tobramycin. Aminoglycoside resistance in Salmonella is generally associated with the expression of aminoglycosidemodifying enzymes [40]. Our results are in accordance with those of Djeghout et al., who reported the presence of aadA7, aadA2 and aadA3 genes on most of streptomycin-resistant strains of Salmonella isolated from human and poultry in four Algerian cities [41]. Moawad et al. [38] and Sheng et al. [42] reported the presence of the aadA2 gene in isolates from retail meats in Egypt and Japan respectively [38,42]. Moreover, in Algeria, several studies have reported various contaminations of avian products by Salmonella spp. These contaminations may take place through the food chain, occupational exposure or direct contact with live animals and their environment in the broiler chicken industry [41,43,44,45].

Conclusions
The results of the present study demonstrate the existence of a worrying rate of Salmonella contamination in poultry meat that is sold in butcheries of the Skikda province. The potential implications of contaminated surfaces (slaughterhouses, kitchens and butcher shops) in the direct transmission of highly pathogenic micro-organisms such as Salmonella spp. to poultry meat are very frequent. The emergence of antimicrobial resistance of S. enterica isolates is a serious public concern in Algeria. Significantly, high rates of resistance have been detected to penicillin, cephalosporins, fluoroquinolones and aminoglycosides. Presence of genes encoding for antibiotic resistance was confirmed. In perspective, it would be interesting to carry out similar or more extensive studies on much larger samples in order to compare the results and evaluate these circulating clones with those of the study we previously performed on farms and slaughterhouses in the same region [20].