Prevalence and Antibiotic Resistance of Salmonella Enteritidis and Salmonella Typhimurium in Ground Beef and Meatball Samples in Samsun,Turkey

Abstract

The aims of this study were to evaluate the prevalence of Salmonella spp., including S. Enteritidis and S. Typhimurium, their antibiotic resistance profiles, and the presence/absence of class 1 integron (intI1) in 50 raw ground beef and 50 raw, meatball samples collected in the Samsun Province, Turkey. For the detection of Salmonella, conventional culture technique and PCR assay were used. The antibiotic resistance profiles of the isolates against nine antibiotics were tested. Salmonella spp. was detected in 20 (n = 86 isolates) samples, namely 12 ground beef and 8 meatball samples. Salmonella Enteritidis (n = 12; 24 isolates) or S. Typhimurium (n = 3; 6 isolates) was detected in 15 (75.00%, n = 30 isolates) samples. At least one species-specific gene (oriC or invA) was detected in the isolates. All isolates were sensitive to two of the third-generation cephalosporins and also nalidixic acid. There was a different level of multidrug resistance (MDR) between S. Enteritidis and Typhimurium isolates. Class 1 integron was detected in four samples (n = 7 isolates); seven isolates were S. Enteritidis and four out of the seven S. Enteritidis isolates were also MDR. In conclusion, the presence of Salmonella, particularly S. Enteritidis and S. Typhimurium, in ground beef and meatballs may cause foodborne infections. The presence of antibiotic-resistant Salmonella and S. Enteritidis with the Cls1integron is important for horizontal antibiotic gene transfer.

Introduction

SAlmonellosis is one of the most important foodborne illnesses worldwide, and S. Enteritidis and S. Typhimurium are the predominant serotypes in human Salmonella infections.¹ According to the latest report published by the European Food Safety Authority (EFSA), 94,530 human Salmonella cases were confirmed in the 28 EU member states in 2016.² In the USA, from 1999 to 2016, the number of Salmonella outbreaks was 2,583. They caused 68,993 cases of illness and resulted in 7,969 (11.60%) hospitalizations and 89 (0.01%) deaths. Of these, Salmonella outbreaks originating from ground beef caused 42,931 cases of illness, 156 (16.80%) hospitalizations, and 3 (0.30%) deaths.³

Salmonella is a major pathogen of the gastrointestinal tract.⁴ During the slaughter of cattle and processing steps of carcasses, the meat can be contaminated with Salmonella. Poultry is understood to be the most important source of Salmonella in foods of animal origin, but other types of foods can also be contaminated with different serovars of Salmonella, and may pose a public health risk. The EFSA reported that Salmonella was mostly detected in poultry meat (5.60–6.50%), particularly broiler meat, with lower contamination levels in pork (1.70%) and beef (0.20%).⁵ However, some outbreaks have shown the importance of ground beef as a source of multidrug-resistant (MDR) Salmonella serotypes.⁶ Moreover, there have been reports of human Salmonella infections after the consumption of contaminated beef.^7,8

It was reported that more than 20 people became ill after consuming ground beef contaminated with S. Typhimurium in six states of USA.⁷ A recent report of the United States Department of Agriculture (USDA) stated that S. Newport in ground beef caused illness in 57 patients in USA, Colombia, and Puerto Rico.⁸ There have been many studies on the prevalence of Salmonella in ground beef in different countries, with the contamination level varying between 0.00% and 26.70%.^9–16

Antibiotics play a key role in the treatment of infectious diseases. However, antibiotic-resistant microorganisms, including Salmonella, have emerged because of the use of antibiotics in human and veterinary medicine. Therefore, drug resistance has become a serious public health concern worldwide. The EFSA reported that Salmonella isolates originating from humans were highly resistant to some antibiotics. In the EU member states, about 26.50% of the Salmonella isolates from humans were MDR.¹⁷ The same report stated that resistance levels varied across Salmonella serotypes. For example, higher or lower resistance was reported for S. Typhimurium and S. Enteritidis, respectively.¹⁸ Dolapçı et al. reported that 65 out of 66 S. Typhimurium isolates of human origin obtained from seven provinces in Turkey with resistance to five drugs were DT104.¹⁹

Antibiotic-resistant bacterial reservoirs may play an important role in the transfer of potentially resistant bacteria or their genes from animals to humans through the food chain.²⁰ During the transfer, mobile genetic elements, including transposons, plasmids, and integrons, play a significant role in the spread of MDR; mobile integrons (MIs) are responsible for the acquisition of antibiotic resistance.²¹ Although there are five classes of MIs, among them, class1 integrons are the most common and prominent in clinical terms. An integron without the integrase gene (Int1) is not capable of transferring drug resistance. This means that Int1 plays a crucial role in all classes of integrons.²² It was reported from Turkey that class 1 integrons were detected in 43 out of 65 S. Typhimurium DT104 isolates.¹⁹

The invA gene is a Salmonella-specific gene and it is therefore widely used for molecular confirmation of Salmonella isolates obtained by using conventional techniques. It is also one of the important virulence genes.^23–25 Like invA, oriC is also a highly genus-specific gene to Salmonella,^25,26 and therefore, it is widely used for the molecular detection of Salmonella.

The objectives of this study were to (1) detect the presence of Salmonella by using conventional culture techniques and PCR assay, (2) determine the distribution of S. Enteritidis and S. Typhimurium serotypes and the antibiotic resistance profiles of Salmonella isolates, and (3) investigate the presence of class 1 integron, in 100 raw, ground beef and raw, meatball samples purchased from markets in Samsun Province, Turkey.

Materials and Methods

Sample collection

In this study, a total of 100 samples consisting of 50 raw ground beef and 50 raw beef meatballs were randomly purchased from different butchers and supermarket brands (up to three times from each brand) in the Samsun Province, Turkey, between November 2012 and June 2013, and then transferred under cold conditions to the laboratory and tested immediately for Salmonella spp.

Salmonella spp. isolation

Salmonella spp. isolation was conducted in two enrichment steps with the conventional culture technique. Briefly, under aseptic conditions, 25 g of ground beef or meatball sample was weighed into a sterile polyethylene bag and 225 mL of buffered peptone water (BPW-Oxoid CM 0509) broth was added. The mixture was homogenized (Interscience-bag mixer 400) and then incubated at 37°C for 24 hr for pre-enrichment. Then, 0.1 mL of pre-enrichment broth (BPW) was suspended in 10 mL of Rappaport Vassiliadis (RV) enrichment broth (Oxoid CM 669). Following that, the tubes were incubated at 42.5°C for 24 hr in selective enrichment step.²⁷ After incubation, the enriched broth was plated onto XLT4 agar (Merck, 113919, XLT4 supl. 108981), and the plate was incubated aerobically for 24–48 hr at 37°C. Up to five susceptible colonies grown on XLT4 agar were subcultured onto Tryptone Soya Agar plates (TSA-Oxoid-CM0131-L21).

Identification procedure

Biochemical and serological tests

The presumptive Salmonella colonies were tested with the Gram staining, oxidase test (Oxoid BR 64), and the standard biochemical tests performed on Triple Sugar Iron Agar (TSIA-Oxoid CM 0277), Lysine Iron Agar (LIA-Oxoid CM 0381), and Urea Agar Base (Oxoid CM 53). For confirmation, the isolates were tested with Salmonella spp. polyvalent antiserum (Difco 2264-47-2) and the colonies showing a positive precipitation reaction were identified as Salmonella spp.²⁸ For further analysis, the isolates were kept at −80°C in cryovials containing 10% (w/v) glycerol in Brain Heart Infusion broth (BHI; CM 0225, Oxoid).

Polymerase chain reaction assay for the confirmation of Salmonella isolates

DNA extraction from the bacteria was performed by using boiling method. In addition, Salmonella spp. isolates were confirmed by the presence of the genus-specific invA and oriC genes. For that purpose, a single-target PCR technique was applied according to the methodologies of Salehi et al. for invA,²⁹ and Widjojoatmodjo et al., Fluit et al., and Erol et al. for oriC.^26,30,31 S. Enteritidis (ATCC 13076) and S. Typhimurium (ATCC 14028) were used as the reference control strains. The oligonucleotide primers and product sizes are listed in Table 1.

Table 1.

Prımers Used ın Thıs Study

Amplified	Oligonucleotide sequence (5′-3′)	Product size (bp)	Reference
invA (genus specific)	F-GTG AAA TTA TCG CCA CGT TCG GGC AA	284	²⁴
invA (genus specific)	R-TCA TCG CAG CGT CAA AGG AAC	284	²⁴
oriC (genus specific)	F-TTA TTA GGA TCG CGC CAG GC	163	^26,30,31
oriC (genus specific)	R-AAA GAA TAA CCG TTG TTC AC	163	^26,30,31
sefA-1 sefA-2	F-GCA GCG GTT ACT ATT GCA GC	330	³²
sefA-1 sefA-2	R-TGT GAC AGG GAC ATT TAG CG	330	³²
pefA-1 pefA-2	F-TTC CAT TAT TGC ACT GGG TG	497	³²
pefA-1 pefA-2	R-GGC ATC TTT CGC TGT GGC TT	497	³²
integrase (IntI1)	F-CCT CCC GCA CGA TGA TC	280	³³
integrase (IntI1)	R-TCC ACG CAC TGT CAG GC	280	³³

Single-target PCR technique for the detection of S. Enteritidis and S. Typhimurium geno-serotypes

For the detection of fimbrial sefA and pefA genes in the isolates, the method of Cortez et al. was used.³²

Class 1 integron determination using the integrase gene

A single-target assay was conducted according to the methodology of Bass et al. for the detection of intI1. The nucleotide sequences of the primers, as well as their estimated size, are shown in Table 1.³³

Antimicrobial susceptibility tests

The Salmonella isolates were tested for their susceptibility to 10 antibiotics, namely cefotaxime (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ampicillin (10 μg), erythromycin (15 μg), gentamicin (10 μg), nalidixic acid (30 μg), streptomycin (10 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (23.75/1.25 μg). For that purpose, the Kirby-Bauer disc diffusion method was used.^34,35

Statistical analysis

Chi-square test was employed to compare the resistance and susceptibility of the isolates against the antibiotics. A probability of p < 0.01 was considered statistically significant.

Results

Based on conventional culture and PCR results, Salmonella spp. were detected in 20.00% of the samples (20 of 100). Of the 20 positive samples, S. Enteritidis (n = 12; 6 ground beef and 6 meatball) or Typhimurium (n = 3; 2 ground beef and 1 meatball) was detected in 15 samples (75.00%). The Salmonella contamination rate of raw meatball samples (n = 50) was 24.00% (n = 12) and ground beef samples (n = 50) was 16.00% (n = 8), (p > 0.05) (Table 2).

Table 2.

Detection of S. Enteritidis and S. Typhimurium

	Meatball samples (n = 50) % (n)	Ground beef samples (n = 50) % (n)	Total samples (n = 100) % (n)	p-Value
Salmonella spp. (n = 86 isolates)	24 (12)	16 (8)	20 (20)	0.317
S. Enteritidis/S. Typhimurium (n = 30 isolates)	14 (7)	16 (8)	15 (15)	—
S. Enteritidis (n = 24 isolates)	12 (6)	12 (6)	12 (12)	1.000
S. Typhimurium (n = 6 isolates)	2 (1)	4 (2)	3 (3)	0.558
p-Value	0.04^*	1.141	0.001^**

p < 0.05.

p < 0.01.

In this study, isolation, identification, and molecular confirmation procedures were applied for the detection of Salmonella spp. A total of 86 suspected Salmonella spp. isolates were obtained; invA and/or oriC genes were present in all the 86 isolates, so they were all identified as Salmonella. Both of the two genes were present in 59 isolates; in the other 27 isolates, only oriC was found in 20 isolates and only invA was detected in 7 isolates, which means that the oriC and invA genes were detected in 79 and 66 isolates, respectively.

The geno-serotyping data were also evaluated according to isolate numbers. Thirty (34.88%) of the 86 Salmonella spp. isolates were S. Enteritidis (n = 24; 12 ground beef and 12 meatball) or S. Typhimurium (n = 6; five ground beef and one meatball origin) (Table 2).

The 86 Salmonella spp. isolates were also analyzed for their antibiotic resistance profiles. All of the isolates (100%) were susceptible to two of the third-generation antibiotics, namely cephalosporin (ceftriaxone and cefotaxime) and nalidixic acid (Table 3). However, some of the isolates showed resistance (intermediate or resistant) to streptomycin, tetracycline, and trimethoprim/sulfamethoxazole in the range of 31.39–68.60%.

Table 3.

Antibiotic Resistance Profiles of S. Enteritidis and S. Typhimurium Isolates

Antibiotics	Salmonella isolates (n = 86)			S. Enteritidis isolates (n = 24)			S. Typhimurium isolates (n = 6)
	S	I	R	S	I	R	S	I	R
	% (n)	% (n)	% (n)	% (n)	% (n)	% (n)	% (n)	% (n)	% (n)
Ampicillin (10 μg)	88.37 (n = 76)	3.48 (n = 3)	8.13 (n = 7)	79.16 (n = 19)	12.50 (n = 3)	8.33 (n = 2)	83.33 (n = 5)	0.00 (n = 0)	16.66 (n = 1)
Ceftriaxone (30 μg)	100 (n = 86)	0.00 (n = 0)	0.00 (n = 0)	100 (n = 24)	0.00 (n = 0)	0.00 (n = 0)	100 (n = 6)	0.00 (n = 0)	0.00 (n = 0)
Gentamicin (10 μg)	87.20 (n = 75)	9.30 (n = 8)	3.48 (n = 3)	79.16 (n = 19)	12.50 (n = 3)	8.33 (n = 2)	100 (n = 6)	0.00 (n = 0)	0.00 (n = 0)
Chloramphenicol (30 μg)	82.55 (n = 71)	16.27 (n = 14)	1.16 (n = 1)	70.83 (n = 17)	0.00 (n = 0)	29.16 (n = 7)	83.33 (n = 5)	16.66 (n = 1)	0.00 (n = 0)
Streptomycin (10 μg)	31.39 (n = 27)	30.23 (n = 26)	38.37 (n = 33)	29.16 (n = 7)	25.00 (n = 6)	45.83 (n = 11)	16.66 (n = 1)	33.33 (n = 2)	50.00 (n = 3)
Tetracycline (30 μg)	41.86 (n = 36)	4.65 (n = 4)	53.48 (n = 46)	45.83 (n = 11)	0.00 (n = 0)	54.16 (n = 13)	50.00 (n = 3)	0.00 (n = 0)	50.00 (n = 3)
Trimethoprim/Sulfamethoxazole (23.75/1.25 μg)	68.60 (n = 59)	25.58 (n = 22)	5.81 (n = 5)	66.66 (n = 16)	4.16 (n = 1)	29.16 (n = 7)	83.33 (n = 5)	0.00 (n = 0)	16.66 (n = 1)
Cefotaxime (30 μg)	100 (n = 86)	0.00 (n = 0)	0.00 (n = 0)	100 (n = 24)	0.00 (n = 0)	0.00 (n = 0)	100 (n = 6)	0.00 (n = 0)	0.00 (n = 0)
Nalidixic acid (30 μg)	100 (n = 86)	0.00 (n = 0)	0.00 (n = 0)	100 (n = 24)	0.00 (n = 0)	0.00 (n = 0)	100 (n = 6)	0.00 (n = 0)	0.00 (n = 0)

n = number of isolates; S, sensitive; I, intermediate resistance; R, resistance.

A total of 70.83%, 54.16%, 33.32%, and 29.16% of the S. Enteritidis isolates were resistant (intermediate or resistant) to streptomycin, tetracycline, trimethoprim/sulfamethoxazole, and chloramphenicol, respectively. In addition, a total of 20.83% of the S. Enteritidis isolates were found to be resistant (intermediate or resistant) to both ampicillin and gentamicin. Regarding the geno-serotypes of S. Typhimurium, there was a high level of resistance (intermediate or resistant) to streptomycin (83.33%) and tetracycline (50.00%), and a relatively low level of resistance (intermediate or resistant) to ampicillin (16.66%), trimethoprim/sulfamethoxazole (16.66%), and chloramphenicol (16.66%) (Table 3).

The detailed MDR levels of the isolates according to geno-serotype levels are shown in Table 4.

Table 4.

Multidrug Resistance of S. Enteritidis and S. Typhimurium Isolates

Antibiotics	S. Enteritidis (n = 24)			S. Typhimurium (n = 6)
Antibiotics	Number of isolates resistant	% (n)	Class 1 integron (%) (n)	Number of isolates resistant	% (n)	Class 1 integron % (n)
Ampicillin + Streptomycin + Tetracycline	3	8.33 (2)	0.00 (0)	3	0.00 (0)	0.00 (0)
Chloramphenicol + Tetracycline + Trimethoprim/Sulfamethoxazole	3	4.16 (1)	0.00 (0)	3	0.00 (0)	0.00 (0)
Gentamicin+ Chloramphenicol + Streptomycin + Trimethoprim/Sulfamethoxazole	4	8.33 (2)	8.33 (2)	4	0.00 (0)	0.00 (0)
Gentamicin + Chloramphenicol + Streptomycin + Tetracycline + Trimethoprim/Sulfamethoxazole	5	16.66 (4)	8.33 (2)	5	0.00 (0)	0.00 (0)
Ampicillin +Gentamicin+ Chloramphenicol + Streptomycin + Tetracycline + Trimethoprim/Sulfamethoxazole	6	4.16 (1)	0.00 (0)	6	0.00 (0)	0.00 (0)
Ampicillin + Chloramphenicol + Streptomycin + Tetracycline + Trimethoprim/Sulfamethoxazole	5	0.00 (0)	0.00 (0)	5	16.66 (1)	0.00 (0)

n = number of isolates; MDR (resistance to three or more antibiotics) according to Kemm et al.⁵⁰

MDR, multidrug resistant.

In this study, the class 1 integron was present in four samples (seven isolates, one isolate from ground beef sample and six isolates from three meatball samples). Regarding serotype distribution, all seven isolates were S. Enteritidis. The class 1 integron was not present in any S. Typhimurium isolate. The one ground beef origin isolate was resistant to five antibiotics. Therefore, the isolate was MDR. The remaining six isolates from meatballs were resistant to one to five different antibiotics. Two of the six isolates from meatballs were resistant to one antibiotic (ampicillin or trimethoprim/sulfamethoxazole). The other isolate of meatball origin was resistant to two antibiotics (ampicillin and streptomycin). The remaining three isolates of meatball origin were MDR [4 (n = 2)–5 (n = 1) to different antibiotics] (Table 4). Two samples (one ground beef and one meatball samples) were MDR, and they contained the class 1 integron.

Discussion

Salmonella spp. are important causative agents of food-borne infections in people across the world and can lead to morbidity, mortality, and substantial economic costs.¹³ Although there have been over 2,500 Salmonella serovars recorded, S. Enteritidis and Typhimurium are the most common serotypes causing food-borne diseases.³⁶ There have been many studies of Salmonella contamination of ground beef in different countries, with the level of contamination varying from 0.00% to 26.70%.^16,37–40 Sallam et al. isolated Salmonella from 30.00% of beef and 26.70% of ground beef samples in Egypt by using molecular techniques.¹³ In studies from Ethiopia, China, and Tunisia, Salmonella spp. were detected in 14.40%, 17.00%, and 10.70% of ground beef samples, respectively.^37,41,42 However, Salmonella was not found in 134 ground beef samples tested in Canada.⁴⁰ In Turkey, the contamination of ground beef samples by Salmonella spp. was reported to be up to 10.00%.^{9,11,14,15,43} For example, Erol isolated Salmonella from 3.30% of samples in Ankara Province⁴³ and Sırıken reported that 10.00% of samples were contaminated with Salmonella at 0.3–1,100 MPN/g in Afyon and Aydın Provinces.⁹ In this study, Salmonella spp. were found in 16.00% of ground beef samples. In contrast to these studies, Direkel et al. reported from Mersin Province¹¹ and Atasever & Atasever reported from Erzurum Province that Salmonella spp. were not found in ground beef samples.¹⁴

There have also been studies of the prevalence of Salmonella in meatball samples in Turkey. Yıldız et al. detected Salmonella spp. in 5.40% of samples in Istanbul.⁴⁴ A recent study from Amasya Province reported the presence of Salmonella spp. in 4.00% of meatball samples.¹⁵

There have been differences reported in Salmonella incidences in ground beef samples around the world and even in the same countries, including Turkey. Contamination levels may vary according to the sampling procedure, detection method, hygienic procedures in the slaughterhouses and processing plant, and seasonally. Centers for Disease Control and Prevention (CDC) reported that the occurrence of salmonellosis in the United States was two to four times higher in summer, particularly in August.⁴⁵ In addition, cross contamination may also play an important role during meat processing. In Ireland, Khen et al. reported Salmonella spp. in 0.25% of beef carcasses and that the level of contamination increased to 3.00% in ground beef samples during processing.⁴⁶ Therefore, primary and secondary contamination of ground beef are both important considerations.

In this study, the oriC gene was present in 79 isolates and the invA gene was detected in 66 isolates. In other words, the oriC and invA genes were not detected in 7 and 20 Salmonella spp. isolates, respectively. oriC-based molecular confirmation of the isolates was therefore superior to the use of the invA gene. The two isolates with only the oriC gene were identified as S. Enteritidis on the basis of geno-serotyping. On the other hand, from the seven isolates containing invA alone, two were the S. Enteritidis/Typhimurium serotypes. The 20 isolates in which invA was not detected may be S. Lichefield, S. Senftenberg, or S. Arizonae serotypes, according to Rahn et al. and Moganedi et al.^24,47 Fluit et al. reported that the oriC region of the chromosome encodes for replication and therefore includes genus and/or strain-specific sequences by which PCR detection of a particular genus can be achieved.²⁶ In another study, Gooding and Choudary compared the efficacy of the use of 5 different primer pairs on 14 Salmonella isolates from different sources. They reported that the oriC gene was superior to the remaining 4 primers because it was detected in 12 of 14 Salmonella isolates.⁴⁸ Rahn et al. reported that, except for S. Senftenberg and S. Lichtfield, the remaining Salmonella serovars contain the invA gene in the pathogenicity island 1 (SPI-1), which is highly conservative for the invA gene.²⁴ In this study, the use of the invA and oriC genes together appeared to produce better results for the molecular confirmation of Salmonella isolates. The invA gene is in the SPA-1 region, which encodes for the proteins of the Type Three Secretion System (T3SS). Therefore, the invA gene plays a significant role in the invasion of the host tissue by Salmonella.⁴⁹ Salmonella bacteria that do not contain the invA gene are not capable of invasion of host tissue or use other invasion genes in their SPI.^49,50

Epidemiological studies on food-borne salmonellosis, including serotyping and phage typing, are crucial for the identification of the underlying causes such as the source of the infection, and links between foods, animals, and human salmonellosis. Although over 2,500 serotypes of Salmonella have been identified, the predominantly identified serotypes in human salmonellosis worldwide are S. Typhimurium and S. Enteritidis.^2,51,52 In Turkey, according to the Turkey Health Report, S. Enteritidis is more common than the other Salmonella serovars. The report further stated that 171 Salmonella isolates were sent to the National Enteric Pathogens Surveys Network (NEPLSN-UEPLA) in the period of 2008 to 2011, and that 61.40% of them were identified as S. Enteritidis.⁵³ Recently, S. Enteritidis PT14b was identified as the causative agent of a food-borne infection in England.⁵¹ Terentjeva et al. reported from Lativa that a total of seven different serotypes were detected among Salmonella isolates from beef, pork, poultry, lamb, and mixed meat samples. Among the seven serotypes, S. Typhimurium (8.33%) and S. Derby (16.66%) were detected in ground meat and meat preparations.¹⁶ In this study, 34.88% of Salmonella isolates were S. Enteritidis or Typhimurium, and S. Enteritidis was more prevalent than S. Typhimurium (p < 0.01). The results of this study resemble those reported by Uzunlu et al. from Turkey.⁵⁴ They reported that S. Enteritidis was the serotype most frequently isolated from raw meatballs (çiğköfte–main ingredients are ground beef and cuscus). In another study conducted by Erol in Turkey, the serotypes S. Anatum, S. Typhimurium, and S. Telaviv were detected in ground beef samples.⁴³ There have been many studies across the world concerning the investigation of Salmonella spp. in meat products. For instance, Yang et al. concluded that the predominant serotypes in various types of meat in China were S. Typhimurium and S. Enteritidis.⁴¹ In Egypt, the predominant Salmonella serotypes in minced beef and beef burgers were S. Typhimurium and S. Enteritidis.¹³ Little et al. stated that S. Typhimurium (predominant serotype), S. Derby, and Salmonella spp., and single S. Dublin, S. Mbandaka, and S. Muenster isolates, were obtained from beef samples in England.¹⁰ However, in Indonesia, Ethiopia, and some other countries, other serotypes were most prevalent in ground beef and cattle carcasses.^{12,37,46,55–59}

Antimicrobial resistance is causing a community health care crisis worldwide. Foods can play an important role in the transmission of antibiotic-resistant bacteria and antibiotic-resistant genes to humans. In this study, all Salmonella isolates were sensitive to ceftriaxone, as well as nalidixic acid and cefotaxime. In contrast, high levels of streptomycin and tetracycline resistance and medium levels of trimethoprim/sulfamethoxazole resistance (intermediate or resistance) were observed. High levels of resistance to traditional antibiotics like ampicillin, tetracycline, and sulfonamide were also found in Salmonella isolates from foods of animal origin in Malaysia, Vietnam and Thailand.^34,59,60 The resistance level of Salmonella isolates to nalidixic acid, a first-generation quinolone, was medium to the high in Vietnam (19–29.00%), Thailand (9–36.00%), and Kampuchea (23.00%).^59–62 Tafida et al. reported that Salmonella isolates from beef in Nigeria were resistant to six different antibiotics, and had multiple resistance to lincosamide, macrolide, aminoglycoside, nitrofuran, and β-lactamase group antibiotics. However, the same study reported that the isolates were sensitive to 11 different antibiotics.⁵⁷ In another study conducted in Poland, a total of 106 Salmonella isolates, including three from ground beef, were resistant (26.40–52.80%) to nalidixic acid, tetracycline, ampicillin, streptomycin, and sulfonamide, but susceptible to one of the fourth-generation cephalosporin, three of the third-generation cephalosporins, one fluoroquinolone derivative, and two carbapenems. At the serotype level, S. Newport, S. Typhimurium, S. Hadar, S. Virchow, S. Infantis, and S. Enteritidis were resistant to at least one antibiotic. MDR properties of the isolates were also evaluated; 4, 10, 13, and 15 of the 106 isolates were resistant to 2, 3, 4, and 5 or more antibiotics, respectively.⁶³ In this research, we determined that S. Enteritidis serotypes were resistant (intermediate or resistance) to six different antibiotics (20.83–70.83%), with the highest resistance (intermediate or resistance) against streptomycin. However, the serotypes were susceptible to ceftriaxone, cefotaxime, and nalidixic acid. Salmonella Typhimurium was susceptible to four different antibiotics, namely ceftriaxone, cefotaxime, gentamicin, and nalidixic acid. In contrast, the serotypes were resistant (intermediate or resistant) to the remaining five antibiotics (16.66–83.33%). Like S. Enteritidis, the highest resistance (intermediate or resistance) was to streptomycin (83.33%). The two serotypes were also evaluated for their MDR properties, and 36.66% (n = 11) of the isolates obtained from the two serotypes were MDR. Among these, while seven S. Enteritidis isolates were resistant to four to six different antibiotics, only one S. Typhimurium isolate was resistant to five different antibiotics (Table 4).

In a study from Turkey, ≥89.28% of Salmonella isolates from poultry meat showed quite high resistance to four antibiotics, namely vancomycin, tetracycline, streptomycin, and nalidixic acid, while they showed low resistance to four other antibiotics (gentamicin, chloramphenicol, ampicillin, and ceftriaxone), and 32.14% of the isolates were found resistant to trimethoprim/sulfamethoxazole. In addition, 92.85% of the isolates showed resistant to four or more antibiotics.⁶⁴ Very different antibiotic resistance profiles of Salmonella isolates have been reported from different sources, including humans, beef, and poultry meat origin. Regarding isolates from humans in Turkey, 12.00%, 6.00%, 17.00%, and 4.00% were resistant to ampicillin, trimethoprim/sulfamethoxazole, nalidixic acid, and ciprofloxacin, respectively.⁵³ According to a report from the EFSA, between 0.80% and 27.10% of Salmonella isolates of human origin from 19 member countries were resistant to a total of 11 different antibiotics. In addition, 0.00% to 33.40% of Salmonella isolates from cattle were resistant to the same antibiotics.⁶⁵ Moreover, a recent EFSA report stated that in general, MDR in Salmonella is high throughout the EU. Nevertheless, it was noted that resistance to critically important antibiotics used in the treatment of serious Salmonella infection cases was low.¹⁷

Class 1 integron is the most common integron type found among MDR Salmonella.⁶⁶ For instance, isolates of S. Typhimurium DT104 were often resistant to multiple antibiotics, which was associated with the acquisition of the mobile genetic element SGI1 (Salmonella genomic island 1).⁶⁷ In Turkey, there have been a few studies evaluating the relationship between the presence of integron and antimicrobial resistance patterns among Salmonella serotypes, particularly S. Enteritidis and Typhimurium. Dolapçı et al. investigated whether there is a correlation between the presence of class 1 integron and antibiotic resistance acquisition by S. Typhimurium isolates¹⁹; they detected class 1 integrons 43 of 65 S. Typhimurium DT104 isolates of human origin. In this study, although class 1 integron was not detected in S. Typhimurium isolates, it was detected in seven S. Enteritidis isolates. The intI1 integron-integrase gene-positive isolates were resistant to ampicillin (n = 2), gentamicin (n = 4), chloramphenicol (n = 4), streptomycin (n = 5), and trimethoprim/sulfamethoxazole (n = 5). Among seven isolates, four (one of ground beef and three of meatball origin) isolates obtained from two samples were MDR. Therefore, the antibiotic-resistant properties of the S. Enteritidis isolates may be associated with class 1 integron.

Conclusions

In this study in Turkey, some ground beef and meatball samples were found to be contaminated with the most widespread Salmonella serotypes, S. Enteritidis, and S. Typhimurium, and may therefore have posed a public health threat. Moreover, the results of this study suggest that using both the invA and oriC genes together for the confirmation of Salmonella isolates may provide more precise results, particularly in the avoidance of false negative results. Although the isolates showed high resistance to some antibiotics used in the treatment of human salmonellosis,⁴⁸ they were sensitive to antibiotics widely used for its treatment. In addition, the presence of the class 1 integron revealed that some of the isolates can spread antibiotic resistance.

Footnotes

Acknowledgments

The author thank Gregory T. Sullivan for editing an earlier version of this article.

Disclosure Statement

All the authors declare that they have no competing interests.

Funding Information

A part of this study was a Master's thesis supported by Project Management Office (PYO), the Scientific Research Project Program (Project No: PYO. VETERINARY [VET]. 1904.12.0090), Ondokuz Mayıs University, Samsun, Turkey.

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