Epidemiological, molecular characterization and antibiotic resistance of Salmonella enterica serovars isolated from chicken farms in Egypt

Background Salmonella is one of major causes of foodborne outbreaks globally. This study was conducted to estimate the prevalence, typing and antibiotic susceptibilities of Salmonella enterica serovars isolated from 41 broiler chicken farms located in Kafr El-Sheikh Province in Northern Egypt during 2014–2015. The clinical signs and mortalities were observed. Results In total 615 clinical samples were collected from broiler flocks from different organs (liver, intestinal content and gall bladder). Salmonella infection was identified in 17 (41%) broiler chicken flocks and 67 Salmonella isolates were collected. Recovered isolates were serotyped as 58 (86.6%) S. enterica serovar Typhimurium, 6 (9%) S. enterica serovar Enteritidis and 3 (4.5%) were non-typable. The significant high mortality rate was observed only in 1-week-old chicks. sopE gene was detected in 92.5% of the isolates which indicating their ability to infect humans. All S. enterica serovar Enteritidis isolates were susceptible to all tested antimicrobials. The phenotypically resistant S. enterica serovar Typhimurium isolates against ampicillin, tetracycline, sulphamethoxazole and chloramphenicol were harbouring BlaTEM, (tetA and tetC), (sul1 and sul3) and (cat1 and floR), respectively. The sensitivity rate of S. enterica serovar Typhimurium to gentamycin, trimethoprim/sulphamethoxazole and streptomycin were 100, 94.8, 89.7%, respectively. The silent streptomycin antimicrobial cassettes were detected in all Salmonella serovars. A class one integron (dfrA12, orfF and aadA2) was identified in three of S. enterica serovar Typhimurium strains. Conclusions To the best of our knowledge, this study considered first report discussing the prevalence, genotyping, antibiotic susceptibility and public health significance of S. enterica serovars in broilers farms of different ages in Delta Egypt. Further studies are mandatory to verify the location of some resistance genes that are within or associated with the class one integron.


Background
In spite of significant improvement in technology and hygienic practices at all stages of poultry production accompanied with advanced improvement in public sanitation, salmonellosis and Salmonella infections remains a persistent threat to human and animal health. In many countries high incidence of salmonellosis in man appears to be caused by infection derived from contaminated eggs, poultry meat and meat-products. The contaminated products cause disease as a result of inadequate cooking or cross contamination of working surfaces in kitchen environment [1][2][3].
The genus Salmonella of the family Enterobacteriaceae includes more than 3000 distinct serovars that have many host species and cause different diseases; most of which show little specificity for their host species [4][5][6][7]. The genus Salmonella can roughly be classified into three categories or groups: Group 1, highly host-adapted and invasive serovars: this group includes species restricted and invasive Salmonella such as S. Pullorum, S. Gallinarum in poultry and S. Typhi in humans. Group 2, non-host-adapted and invasive serovars: this group consists of approximately 10-20 serovars that are able to cause an invasive infection in poultry and may be capable of infecting humans. Currently, the most important serovars are S. Enteritidis, S. Typhimurium, S. Hadar, S. Heidelberg, S. Saintpaul and S. Infantis. Group 3, nonhost-adapted and non-invasive serovars: most serovars of the genus Salmonella belong to this group and may cause disease in humans and other animals [8][9][10][11][12][13][14].
Although the acute enteritis caused by Salmonella species in humans is usually self-limiting, salmonellosis may be complicated especially in younger and older ages by severe systemic sequelae depending on serotype and on host-specific factors [15][16][17].
Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis have been identified as the predominant serotypes present in Egyptian poultry farms [18].
Salmonella enterica serovar Enteritidis has been associated with disease in broiler breeding stock and can be transmitted vertically to their progeny [19]. Infection of adult chickens with S. enterica serovar Typhimurium is usually without clinical manifestation [20]. S. enterica serovar Enteritidis can inhabit the intestinal tract of several bird species such as chickens, turkeys and game birds and has the ability to survive outside of the host for over 1 year. S. enterica serovar Enteritidis infection in adult poultry is usually asymptomatic and infected bird will become a chronic carrier [21,22]. In chickens up to 6 weeks of age S. enterica serovar Enteritidis may produce clinical symptoms including depression, disinclination to move, and diarrhoea, with high mortality especially in chicks less than 1 week of age [23], while older chicks may show uneven growth and stunting. Laying hens sometimes produce S. enterica serovar Enteritidis contaminated eggs leading to public health concerns [19]. The diseased birds may show lesions of pericarditis, perihepatitis and septicaemia. The mortality and morbidity vary and has been found to depend upon the dosage and phage type of the S. enterica serovar Enteritidis infection [24,25].
Antimicrobial resistance is increasingly becoming an issue with salmonellosis infections in both animals and humans [26]. Understanding the key mechanisms involved in the evolution of antibiotics resistance in bacteria may aid scientific innovations aimed at controlling antimicrobial resistance [27,28]. Bacteria can acquire resistance genes through mobile elements such as plasmids, which provide flexibility to host bacteria and help in the spread and distribution of these genes across diverse bacterial populations [29].
The inappropriate use of antibiotics in chicken farms in developing countries, including Egypt, is thought to be one of the main reasons for the increase in multidrug resistant bacteria [30]. These multidrug resistant bacteria including both S. enterica serovar Typhimurium, and S. enterica serovar Enteritidis that have the potential to infect humans and with a consequent failure of treatment can lead to systemic infection and death [31].
In this study, the incidence and antimicrobial resistance of S. enterica serovars Typhimurium and Enteritidis isolated from broiler chicken farms in Kafr El-Sheikh Province, Northern Egypt was reported. Determination of genes associated with antimicrobial resistance was investigated by examining the distribution of mobile integrons that carry the multidrug resistance cassettes within the genome of the isolated strains.

Sampling strategy and Salmonella isolation
This study was conducted in 41 broiler flocks located in Kafr El-Sheikh Province in Delta Egypt. Twenty flocks of 1-week-old birds and 21 flocks of 5-week-old birds were investigated. The observed clinical symptoms were observed and recorded (Table 1). Five living morbid birds from each flock were randomly selected and humanly sacrificed. At necropsy, sections of liver and intestinal wall plus contents were collected aseptically and processed for Salmonella isolation. From the same bird bile was aspirated from the gall bladder. Wetted cotton swabs in bacteriological transport media were used to collect samples from each specimen. Collected swabs and tissue samples were immediately frozen on ice and stored at −20 °C for further investigation within 5 h. Each tissue sample and swabs were inoculated in 10 ml selenite F broth (Oxoid, UK) and incubated at 37 °C overnight. A loopful of inoculated broth was streaked on selective Salmonella Shigella (SS) agar (Oxoid, UK) and incubated at 37 °C overnight. The suspected colony was sub-cultured on Xylose lysine deoxycholate (XLD) agar (Oxoid, UK) and on brilliant green (BG) agar (Oxoid, UK) and incubated at 37 °C for 16-18 h. The suspected colonies were collected for further biochemical identification using API 20E (BioMérieux, Marcy-l'Étoile, France).

Genomic DNA extraction and purification
The identified bacterial cultures were cultivated on SS agar and inoculated on Luria-Bertani (LB) broth (Oxoid, UK) and incubated at 37 °C overnight. The DNA was extracted from bacterial cultures on broth using Qiagen DNA extraction kit (Qiagen, UK) according to the manufacturer's instructions.

Molecular biological identification and differentiation of Salmonella serovars
In order to make a rapid and definite diagnosis of Salmonella, PCR was conducted using primers to detect the gene marker for S. enterica invA [32], sdfI primers specific for detection of S. enterica serovars Enteritidis [33], and Typh, Sal and fliC specific primers for serovar S. Typhimurium [34,35] (Table 2). invA positive strains were tested for the presence of the sefA gene, which encodes for SEF14 fimbriae that can be detected in S. enterica serovar Enteritidis strains and will also be present in the poultry-associated serotype S. Gallinarum.
In order to detect the zoonotic potential of our isolated strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium we screened for the presence of the sopE gene [36].
The PCR reaction was geared to a previously described protocol for Salmonella [32][33][34][35][36]. Conserved forward and reverse primers (Eurofins, Japan) were used to generate the target amplicon ( Table 1). The PCR cycling conditions were carried out as the following: initial denaturation at 94 °C for 5 min. Thirty cycles of amplification were run for 5 s, at 94 °C, 10 s at 68 °C and 20 s at 72 °C, with the final extension continuing at 72 °C for 7 min. Different annealing temperatures were used as described in Table 1. Five microliter aliquots of reaction mixture were electrophoresed through 1.5% agarose gels (Nippongene, Japan).

Determination and sequencing of class 1 integrons
The class one integrons PCR fragments were purified from the agarose gel using Nucleospin Gel Extraction Kit (Macherey-Nagel, Germany) and sequenced (Genome centre-Gifu University, Japan). The sequencing results were analysed using BLAST webpage (http://blast.ncbi. nlm.nih.gov/Blast.cgi).

Antimicrobial susceptibility testing
The antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion test [37] at the Clinical Veterinary Microbiology Laboratory of the Royal Dick School of Veterinary Study, University of Edinburgh. Briefly, one colony from the SS agar plate of each strain was picked up and streaked onto Mueller-Hinton blood agar (Oxoid, UK) and incubated at 37 °C overnight. Bacterial colonies were suspended in 0.9% NaCl to obtain a McFarland turbidity of 0.5 (Dr. Lange, photometer CADAS 30, Berlin, Germany) that containing about 1-2 × 10 8 colony forming units (CFU)/ml of Escherichia coli strain American Type Culture Collection (ATCC) 25922. Approximately, 300 μl of the saline suspension was spread onto the surface of a Mueller-Hinton agar plate (Oxoid, UK) using a sterile swab.

Typh-R CCCTGACAGCCGTTAGATATT
The antimicrobial discs (Oxoid, UK) of six clinically used antibiotics that are used in the Egyptian poultry production (tetracycline 30 μg, ampicillin 10 μg, sulfamethoxazole/trimethoprim 25 μg, gentamicin 10 μg, streptomycin 25 μg and chloramphenicol 30 μg) were distributed onto the surface of the Mueller-Hinton agar plates using a Multi-disc dispenser (Oxoid, UK). The plates were incubated at 37 °C overnight. The diameters of the inhibited zones were measured using sliding callipers and interpreted using standard break points according to the method described by The European Committee on Antimicrobial Susceptibility Testing [38] ( Table 3). The gene associated with antibiotic resistance was tested in isolated Salmonella strains. Isolates were screened for the presence of 18 genes known to be associated with resistance to the seven tested antibiotics (Table 4).

Statistical analysis
The mortality rate associated with Salmonella infection and the rate of S. enterica serovar Typhimurium isolation from internal organs were analysed by the student t test [39].

Clinical signs, mortality and incidence of Salmonella isolation from broiler flocks
Clinical symptoms of Salmonella infection observed in the 1-week-old broiler chicks included pasty diarrhea, inappetence, dehydration, growth retardation, blindness and lameness. The main gross lesions were hepatomegaly with necrotic foci, splenomegaly, pericarditis, panophthalmitis, and arthritis (Table 1).
The mean mortality rate (5.23% ± 2.85) of the 1-weekold flocks was significantly higher (P < 0.01) than the mean mortality rate (2.68% ± 0.52) in the 5-week-old flocks. When grouped by Salmonella infection status, the mortality rate observed in the 1-week-old birds was significantly higher (P < 0.001) in the Salmonella positive flocks (7.8% ± 2.07) compared to negative flocks (3.1% ± 0.45) (Table 1). While, there was no significant difference in mortality rate between the infected and non-infected 5-week-old flocks (P = 0.15, Table 1).

Molecular biological identification of Salmonella serovars and public health significance
Both S. Enteritidis and S. Typhimurium serovars were isolated and identified from both the 1-and 5-week old sacrificed chicks ( Table 5). Three of the collected 67 isolates were Salmonella positive but un-typable serovars.
The isolation rate of Salmonella serovars from different organs were demonstrated in Table 5. Briefly, there was highly significant difference (P < 0.001) of S. enterica serovars Typhimurium isolated from the gallbladder (14.63%) and liver (9.76%) (P < 0.05) compared to those isolated from the intestine (3.9%) ( Table 5). There was no significance difference (P = 0.28) between the isolation rate of S. enterica serovars Enteritidis form liver, intestine and gallbladder. The three un-typable serovars were found only at one farm and were isolated only from the intestinal samples (Table 5).    The sopE gene was amplified in 62 (92.5%) Salmonella isolates, indicating zoonotic and public health significance of isolated strains (Fig. 1b).

Phenotypic and genotypic antimicrobial resistance
All Salmonella serovars isolated in this study were sensitive to gentamicin. Fifty-two (89.7%) S. enterica serovars Typhimurium isolates were susceptible to streptomycin, while six isolates (10.3%) were intermediate. Fifty-five (94.8%) S. enterica serovars Typhimurium isolates were sensitive to trimethoprim/sulphamethoxazole, while 3 (5.2%) isolates were resistant. All non-typable Salmonella strains were sensitive to trimethoprim/sulphamethoxazole and streptomycin. All S. enterica serovars Typhimurium and non-typable Salmonella strains isolated were resistant to ampicillin, chloramphenicol, and tetracycline. However, all S. enterica serovars Enteritidis isolates were sensitive to all tested antimicrobial agents ( Table 3).
The amplicons of intI integrons were identified with size of 2 kbp in three S. enteric serovar Typhimurium strains ( Table 6). The sequencing data indicated that these integrons contain dfrA12-orfF-aadA2.

Discussion
Salmonella enterica serovars Typhimurium is known to be able to cause high rates of mortality in early ages of broiler chickens [20]. The InvA protein is a putative inner membrane component of the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion system (TTSS) [40]. It has been reported that invA is present only in Salmonella species and therefore is used as a golden marker in genetic diagnosis of Salmonella species [35]. In this study 17 broiler flocks were positive and 67 Salmonella strains were isolated. The overall rate of incidence of Salmonella was (41%) in the screened broiler chicken flocks which was considerably higher than the infection rates that reported in the UK (10.7%), Lithuania (29%), Italy (20%), Netherlands (11%) and Germany (27.5% in chickens and 33.3% in turkeys) [41][42][43][44][45]. The higher infection rate found in this study compared to that of Abd El-Ghany et al. [18] shows the increased sensitivity of the use of the invA gene marker for diagnosis compared to isolation through culture on specific agar. Although the S. enterica serovars Enteritidis is closely related to other pathogenic S. enteric serovars, this serovar has some characteristics that appear to discriminate it from others serovars. As S. enterica is known to contain the Salmonella difference fragments (sdf), a group of chromosomally encoded genes, which to date are of unknown function. sdfI was reported by Agron et al. [33] to be found only in S. enterica serovars Enteritidis strains and considered to be a strong marker for this Salmonella serovar. sdfI was used as a target for phylotying of the serotype-specific S. enterica serovars Enteritidis. In this study, sdfI was present in 6 of the 67 invA positive isolates. These sdfI positive strains were isolated from three of 41 screened farms. Our findings indicated that the sdfIII gene marker was associated with the sdfI positive strains. Interestingly, sdfII was detected in all 67 strains isolated in this study of different serovars. This indicates that there is some degree of diversity within serovars that can be detected by the primers which in agreement with previous observation [33,35].
Bacteria use the fimbriae in the adherence to one another and to the host cells and in some instance to inanimate objects. Sef14 fimbriae have been shown to consist of a repeating major subunit of the 14.3 kDa protein SefA, encoded for by the sefA gene and are required for macrophage uptake and survival in intraperitoneal infections [46]. The sefA gene is known to be specific to the poultry-associated Salmonella serotypes Gallinarum and Enteritidis. It is also detected in serotype Dublin, although this serotype is more commonly associated with cattle [47]. In the present study, sefA was detected in all isolates of S. enterica serovar Enteritidis. In the current study same six S. enterica serovar Enteritidis isolates positive for sdfI and sdfIII markers were also positive for the sefA gene; these six strains came from three Salmonella infected chicken farms that were isolated from 41 screened farms.
In this study the S. enteric serovar Typhimurium serotype specific virulent flagella genes Typh and fliC were used for phenotyping as recommended previously [34,35]. Flagella are multi-functional organelles that play different roles in the biology of bacteria. The motility functions of flagella help bacteria to acquire nutrients, move away from toxic materials, and move to specific colonization sites within hosts and to disperse in the environment during the course of transmission between hosts [48]. The flagellum also primes the host immune system through activation of TLR5 receptors [49].
sopE is a translocated effector protein that plays an important part in the systemic phase of salmonellosis infection; sopE has been shown to be involved in actin cytoskeletal rearrangements and membrane ruffling [36]. As a virulence factor that is frequently transferred by bacteriophages, the sopE gene is encoded in the SPI-1, and has been identified in isolates involved in major  epidemics; sopE has therefore been identified as playing a key role in the emergence of epidemic strains [50]. In study conducted by Rahman et al. [51] indicated that sopE gene appeared to be distributed and conserved among only a few serovars of Salmonella (Enteritidis, Gallinarum and Virchow) irrespective of their source of isolation and the presence of sopE gene in Salmonella provides an important pathogenic means to invade epithelial cells [51]. Moreover Prager et al. [52] identified sopE in all isolates of S. enterica serovar Enteritidis and carrying of sopE in S. Enteritidis may contribute to their epidemiological success [52]. In another study, all Salmonella Enteritidis isolated from human, chicken, and egg houses tested positive for sopE which may indicate its importance in pathogenesis [53].
In this study 92.5% of the Salmonella stains were harboured sopE gene that suggested that these strains could have zoonotic potential as previously reported [50][51][52][53].
There was a significant difference in mortality rate between Salmonella infected and non-infected flocks at the 1st week of life, however, there was no difference in mortality between Salmonella infected and non-infected flocks at the 5th week of age; a similar finding was previously reported [20,23]. According to previous study, the results suggest that the age at infection plays an important role in the persistence of S. enteritidis infection in chickens and may cause severe infections and high mortality in young chickens [54]. Unfortunately, in this study we did not investigate other possible causes of mortality which may act as co-factors.
There was a higher rate of Salmonella isolation from the sampled internal organs, in the gall bladder and liver samples compared to the intestine samples indicating the ability of Salmonella to cause systemic infection which in agreement with previous study [55].
In this study All S. enterica serovars Typhimurium and non-typable Salmonella strains isolated in this study were resistance to ampicillin, chloramphenicol, and tetracycline.
Most of the phenotypically antibiotic resistance isolates were positive for some of the antibiotic resistance marker genes for each of the screened antibiotics.
The blaTEM gene was detected only in 65.5% of ampicillin resistant S. enterica serovar Typhimurium isolates. All of the isolated strains of S. enterica serovar Enteritidis were susceptible to ampicillin and were negative for blaTEM. The three non-typable Salmonella strains showed phenotypical resistance to ampicillin without harbouring the blaTEM gene, indicating that these strains possess another ampicillin resistance mechanism.
In this study, tetracycline resistance in the S. enterica serovar Typhimurium isolates correlated with the presence of tetC (96.6%), and tetA (84.5%). All tested strains were negative for tetB codon. tetA codon was also found in all of the non-typable Salmonella strains. All S. enterica serovar Enteritidis were sensitive to tetracycline. However, two of the strains were harboured both tetC and tetA determinants and one strain was harbouring tetA determinant. These cassettes were silent in this serotype strain in vitro, however, they may turn on in vivo.
All of the S. enterica serovar Enteritidis and non-typable Salmonella strains were sensitive to trimethoprimsulphamethoxazole and all these strains were negative for the dfr1 codon and did not possess integron that contains dfrA12 trimethoprim resistance cassette. Although one strain of S. enterica serovar Enteritidis carried sul1 gene but not possessed any trimethoprim genes. All S. enterica serovar Typhimurium isolates were sensitive to trimethoprim-sulphamethoxazole despite 98% of isolates being positive for sul3 and 57% being positive for sul1, both of which confer sulphamethoxazole resistance. Interestingly, the three S. Typhimurium strains that were resistant to trimethoprim-sulphamethoxazole were found to harbour the 2 kp integron that contains the dfrA12 trimethoprim resistant marker.
All of the Salmonella isolates were sensitive to the streptomycin despite the presence of streptomycin modifying enzyme gene cassettes (aadA1, aadA2 and strA). This suggests that some of the antimicrobial resistance genes are silent in bacteria in vitro; however, these silent genes can spread to other bacteria or turn on in vivo, especially under antimicrobial pressure which in agreement with previous reports [31,57].
The cat1 gene, encoding chloramphenicol acetyltransferase, was identified in all resistant strains. In S. enterica serovar Typhimurium, the cat2 gene was not found in any of the tested strains. The floR gene which also confers chloramphenicol resistance was detected in 80% of S. enterica serovar Typhimurium strains. One of the non-typable Salmonella strains carried the cat1 gene but the other two isolates did not possess cat1, cat2 or floR gene indicating that these two strains harbour another chloramphenicol resistance mechanism. Of six S. enterica serovar Enteritidis strains, one strain possessed both, cat1 and, floR, and one strain harboured only the cat1 gene, however, phenotypically they were all sensitive to chloramphenicol indicating that this resistant cassette is silent in vitro in this Salmonella serovar.
Multiple drug resistance genes have been found to be clustered on individual mobile elements, which mean that multi-resistance can be readily transferred and increase the multi-drug resistant bacterial population as reported previously [58].
Gene cassettes are a major source of the resistance genes found in clinical, commensal, and environmental isolates of bacteria that are resistant to antibiotics [59,60]. Most commonly, they are found in association with class 1 or class 2 integrons [61].
In this study, a class one integron in three S. enterica serovar Typhimurium strains with size of 2 kb was identified. The sequencing data indicated that these integrons contained dfrA12-orfF-aadA2. The presence of the dfrA12-orfF-aadA2 open reading frames revealed the basis for the streptomycin and trimethoprim/sulphamethoxazole resistance seen in these strains. It also provides an indication of the mapping distribution of antibiotic resistance alleles in this region of the Salmonella genome/chromosome.
In this study the higher infection rate in the investigated flocks may regarding to low biosecurity and hygienic measures inside these farms and easily to spread the infection through different reservoirs and the workers in the farms.
The screening of antimicrobial resistance in the Salmonella strains isolated in this study provides evidence for confirming the mechanisms employed by S. enterica serovars to resist cluster antibiotics used for treatment of broiler chicken in Egypt. Future work, in this regard, should address if allele distribution in chicken and human Salmonella isolates from the same region share the same resistance mechanisms in order to highlight potential horizontal gene transfer by this zoonotic organism and the origin of antimicrobial resistance in human isolates. Finally, we believe that this is the first report of the presence of a class one integron in the S. enterica serovar Typhimurium serotype together with the verification of the location of some resistance genes that are within or associated with the class one integron.