Subtyping of Salmonella Food Isolates Suggests the Geographic Clustering of Serotype Telaviv

Abstract

Salmonella is commonly found in a variety of food products and is a major cause of bacterial foodborne illness throughout the world. In this study, we investigated the prevalence and diversity of Salmonella in eight different food types: sheep ground meat, cow ground meat, chicken meat, cow offal, traditional Sanliurfa cheese, unripened feta cheese, pistachios, and isot (a spice blend of dried red peppers specific to Sanliurfa), traditionally and commonly consumed in Turkey. Among 192 food samples, Salmonella was detected in 59 samples, with the highest prevalence in raw poultry parts (58%) and offal (58%) samples, while Salmonella was not detected in pistachios and dried red pepper. Resultant Salmonella isolates were characterized by serotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Ten different serotypes represented 10 MLST sequence types (STs) with 1 novel ST and 17 PFGE types. Antimicrobial resistance profiling revealed that 30.5% of the isolates were resistant to two or more antimicrobials. Salmonella enterica subsp. enterica serotype Telaviv, which is rare throughout the world, was the second most common serotype isolated from food samples in this study, suggesting that this serotype might be one of the subtypes that is endemic to Turkey.

Introduction

S almonella enterica subsp. enterica is considered to be one of the leading causes of food-related illnesses (CDC, 2014a), due to its great survival ability on different foods, such as raw meat and poultry, fresh produce, as well as dried nuts, pastes, and spices (Zweifel and Stephan, 2012; CDC, 2014b). It is estimated that that 93.8 million cases of gastroenteritis and 155,000 deaths caused by nontyphoidal Salmonella occur annually worldwide (Majowicz et al., 2010). Antimicrobials, such as ampicillin, trimethoprim–sulfamethoxazole, or ciprofloxacin, might be required to treat severe Salmonella infections in humans. Excessive and unnecessary use of antimicrobials such as growth promoters in livestock might facilitate Salmonella isolates to become resistant to antimicrobials (Kumar et al., 2005).

Among many methods, serotyping represents the cornerstone for Salmonella classification and nomenclature (Wiedmann, 2002; Grimont and Weil, 2007; Wattiau et al., 2011), but it lacks the ability to reveal the phylogenic relation between different subtypes. DNA-based methods are commonly used to further subtyping of strains (Harbottle et al., 2006; Soyer et al., 2010). Among molecular subtyping tools, multilocus sequence typing (MLST), based on comparison of nucleotide sequences of the housekeeping genes, has the advantage of providing portable subtyping data, which are easier to interpret, exchange, and compare between different laboratories through the open-access internet-based databases (Wiedmann, 2002; Urwin and Maiden, 2003). Thus, the analysis displays true evolutionary relationship of the isolates (Alcaine et al., 2006; Achtman et al., 2012). However, MLST may not reach the discriminatory power of pulsed-field gel electrophoresis (PFGE), a valuable tool in epidemiological investigation and surveillance (Refsum et al., 2002; Wonderling et al., 2003).

Retrospective knowledge about the relationship between subtype and food source or geographical location of isolation can facilitate the outbreak investigation process. Hence, we aim to determine the spatial and temporal groups of different Salmonella subtypes from various food samples in a pilot region (i.e., Sanliurfa) in Turkey by using phenotypic (serotyping and antimicrobial susceptibility test by disk diffusion) and genotypic (MLST and PFGE) methods.

Materials and Methods

Food samples

Eight different types of samples (raw ground sheep meat, raw ground cow meat, chicken meat parts, cow offal, traditional Sanliurfa cheese, unripened feta cheese, pistachios, and isot [a spice blend of dried red peppers specific to Sanliurfa]) were collected from retail and street vendors in different districts in the south part of Turkey, the city Sanliurfa. Food samples were collected over four different sampling periods, representing the seasons of a year (i.e., spring, summer, fall, and winter), between April 2012 and December 2012. During each sample collection, two different districts in Sanliurfa city were designated as the sampling districts. For each food type, three food samples were collected from each district, resulting in the collection of six food samples for each food type in a sampling period. In total, 192 food samples were collected. Food samples were transferred overnight in cold-chain conditions to the Food Engineering Department at Middle East Technical University, Ankara, where all analysis was conducted.

Isolation of Salmonella

Food samples were microbiologically analyzed to detect and isolate Salmonella in compliance with the standard of ISO 6579:2002 (ISO, 2002). For Salmonella confirmation of presumptive positive colonies, polymerase chain reaction (PCR) was performed by the amplification of genus-specific 678-bp region of Salmonella, invA gene (Kim et al., 2007). Amplified DNA fragments were observed under ultraviolet light after gel electrophoresis procedure for 30 min at 110 V. Confirmed Salmonella isolates were stored at −80°C in a solution in 15% glycerol.

Serotyping

Serotyping of isolates was conducted according to the White-Kauffmann–Le Minor scheme (Grimont and Weil, 2007) at the laboratory of the Public Health Agency of Turkey in Ankara, where a second Salmonella confirmation was also performed by using biochemical tests.

Antimicrobial susceptibility test (AST)

The disk diffusion method was performed to characterize the antimicrobial resistance profile of the isolates. Eighteen different antimicrobials (Oxoid Ltd., Basingstoke, UK) were analyzed: amikacin 30 μg (AK), gentamicin 10 μg (GN), kanamycin 30 μg (K), streptomycin 10 μg (S), ciprofloxacin 5 μg (CIP), nalidixic acid 30 μg (N), ampicillin 10 μg (AMP), amoxicillin–clavulanic acid 20/10 μg (AMC), tetracycline 30 μg (TE), cefoxitin 30 μg (FOX), cephalothin 30 μg (KF), ertapenem 10 μg (ETP), ceftriaxone 30 μg (CRO), ceftiofur 30 μg (EFT), sulfisoxazole 300 μg (SF), sulfamethoxazole–trimethoprim 23.75/1.25 μg (SXT), chloramphenicol 30 μg (C), and imipenem 10 μg (IPM) (CLSI, 2002). The quality control strain was Escherichia coli ATCC 25922 (Favier et al., 2013; Rothrock et al., 2015). The susceptibility limits of antimicrobial agents except ceftiofur were determined by the Clinical Laboratory Standards Institute's (CLSI) latest report (CLSI, 2013), and the limits for ceftiofur were set according to the CLSI report in 2002 (CLSI, 2002). Isolates showing resistance to two or more antimicrobial agents were considered to be multidrug resistant (MDR).

MLST

PCR amplification and the subsequent DNA sequencing of seven housekeeping loci were performed according to S. enterica MLST at University of Warwick (UoW) (available on http://mlst.warwick.ac.uk/mlst/). Seven housekeeping fragments—aroC (639 nt), dnaN (833 nt), hemD (666 nt), hisD (894 nt), purE (510 nt), sucA (643 nt), and thrA (852 nt)—were amplified. Genomic DNA was purified using Nanobiz Bacterial Genomic DNA Isolation Kit (Nanobiz, Ankara, Turkey). DNA sequencing was conducted by Macrogen Inc. (Geumchon-gu, Seoul, Korea). All sequences were trimmed, proofread, and assembled by using SeqMan and SeqBuilder software (DNAStar, Madison, WI). The trimmed sequences were aligned by Clustal W algorithm using MegAlign software (DNAStar). Assignment of gene alleles was implemented in compliance with the allelic numbers specified in the UoW MLST Database. Unique combinations of seven allelic types were then used to assign sequence types (STs) to each Salmonella isolate.

PFGE

PFGE was performed using the PulseNet standardized protocol with additional 50 μM thiourea to the running buffer (Murase et al., 2004; Ribot et al., 2006). PFGE plugs were digested with XbaI (Roche Applied Science, Mannheim, Germany) and Salmonella Braenderup H9812 was used as a molecular size standard in all PFGE gels run on the CHEF-DR III system (Bio-Rad Laboratories, Hercules, CA). The gel pictures were analyzed using BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). Similarity analysis was conducted using Dice coefficient and clustering was performed using the unweighted-pair group method by arithmetic mean. PFGE types were assigned unique numerical identifiers (i.e., PT 1).

Phylogenetic and statistical analyses

Phylogenetic analysis was performed, and phylogenetic trees were constructed using MegAlign software (DNAStar). Via the concatenation of seven genes for each isolate, an overall phylogenetic tree was formed. Neighbor-joining format was used for the display of the trees.

Due to the low values of many integers, 2 × 2 two-tailed Fischer exact test was used in order to examine the relationship between food types with STs and serotypes. P values of <0.05 were evaluated as statistically significant. Simpson's Index of Diversity (SID) was calculated for the comparison of the discriminatory power of serotyping, MLST, and PFGE.

All information for isolates, including isolate source, serotypes, STs, antimicrobial resistance profiles, and PFGE types, is publicly available at the Pathogen Detector website (pathogendetector-metu.rhcloud.com/).

Results

The high prevalence of Salmonella among food samples

A total of 59 among 192 food samples were as positive for Salmonella (30.1%). The highest prevalence of Salmonella (39.6%) was observed in the food samples collected in the fall, which was followed by the prevalence of summer (N = 15, 31.3%), spring (N = 13, 27%), and winter (N = 12, 25%) periods, respectively. Among eight food categories, the highest prevalence of Salmonella was found in chicken meat and offal; 58.3% of samples were Salmonella-positive in chicken meat and offal groups. Meanwhile, prevalence in cattle and sheep ground meat was obtained in 13 samples (54.2%) and 12 samples (50%), respectively. Lower prevalence values were observed in cheese groups (i.e., four unripened feta cheese, 16.7%, and two Sanliurfa cheese, 8.3%). In dried food samples (i.e., pistachios and isot), Salmonella was not detected.

Serotyping and MLST

For three isolates, multiple serotyping attempts resulted in different serotypes that did not match with the genotyping analysis (i.e., MLST and PFGE). Therefore, three isolates were excluded from the isolate set that is reported from this point. A total of 56 S. enterica subsp. enterica isolates represented 10. Salmonella Infantis (n = 15), Salmonella Telaviv (n = 13), and Salmonella Anatum (n = 11) were the 3 dominant serotypes accounting for approximately 67.8% of 56 isolates, which were followed by Salmonella Montevideo (n = 7), Salmonella Reading (n = 3), and Salmonella Kentucky (n = 2), Salmonella Newport (n = 2). Furthermore, one isolate was classified as Salmonella Typhimurium, Salmonella Hadar, and Salmonella Othmarschen serotypes (Table 1). As a result, the SID calculated for serotyping of 56 isolates was 0.83.

Table 1.

Distribution of 10 Sequence Types (STs) and 10 Serotypes of 56 Salmonella Isolates over Food Type

ST	Correspondent serotype	Total no.	Cattle ground meat	Sheep ground meat	Chicken meat	Offal	Unripened cheese	Urfa cheese
32	Infantis	15	0	0	14	1	0	0
1068	Telaviv	13	2	3	0	3	4	1
64	Anatum	11	6	5	0	0	0	0
195^a	Montevideo	7	1	1	0	5	0	0
93	Reading	3	1	1	0	1	0	0
166	Newport	2	1	0	0	1	0	0
314	Kentucky	2	1	0	0	1	0	0
33	Hadar	1	0	0	0	0	0	1
1832	Othmarschen	1	0	1	0	0	0	0
19	Typhimurium	1	0	0	0	1	0	0
Total		56	13	12	14	14	4	2

One cattle ground meat isolate, one sheep ground meat isolate, and one offal isolate, all with ST 195 resulted in inaccurate serotyping.

Similarly, 10 STs were detected among the 56 Salmonella isolates characterized. Four of the sequence types represented 82.14% of 56 Salmonella isolates, including ST 32 (n = 15), ST 1068 (n = 13), ST 64 (n = 11), and ST 195 (n = 7). Other sequence types detected were ST 93 (n = 3), ST 166 (n = 2), ST 314 (n = 2), ST 33 (n = 1), ST 81 (n = 1), and ST 19 (n = 1). Therefore, for MLST, SID was calculated as 0.83, which is the same with the result concerning serotyping (0.83). Results of serotyping and MLST were concordant, where each ST corresponds to one serotype and the SID for MLST and serotyping was 0.83 (Table 1). Among 10 STs, only 1 novel sequence (ST 1832, Salmonella Othmarschen) was detected. It is noteworthy that novel ST 1832 was closely related to ST 195 (Montevideo), sharing the same allelic types for four of the seven alleles (Fig. 1).

FIG. 1.

Phylogenetic tree of detected 10 subtypes according to their concatenated sequences of seven housekeeping genes in Salmonella enterica multilocus sequence typing scheme. Sequences of seven genes from one representative of each serovar-sequence typing subtype group were concatenated, and a 3336-base-pair sequence was formed for each subtype. Concatenation was conducted with following order: aroc (501 nt), dnan (501 nt), hemd (432 nt), hisd (501 nt), pure (399 nt), suca (501 nt), thra (501 nt). In order to avoid negative branch lengths in the figure, a cladogramic view of neighbor-joining tree was selected. Phylogenetic tree of the detected sequence types (STs) were rooted by ST 1 of serovar Typhi as the outgroup.

AST

Almost half of Salmonella isolates (45.8%) showed resistance to at least one antimicrobial agent. Antimicrobial-resistant Salmonella isolates showed resistance to aminoglycosides (66.7%), tetracyclines (63.0%), quinolones (59.3%), and sulfonamides (85.2%). Among aminoglycosides, streptomycin and kanamycin were dominant (Table 2). The MDR isolates were the 30.5% of Salmonella isolates and made up 66.7% of resistant isolates. Antimicrobial susceptibility test by disc diffusion method resulted in 11 different antimicrobial resistance profiles among 56 Salmonella isolates.

Table 2.

Antimicrobial Resistance Profiles of 56 Salmonella Isolates with a Comparison of Food Type

Source	Serotype/ST/PT ^a	Antimicrobial resistance profile ^b	Number of isolates
Cheese (unripened) (n = 4)	Telaviv/ST 1068/PT 33, PT 34	—	4
Cheese (Urfa) (n = 2)	Hadar/ST 33/PT 41	S, N, AMP, T, KF	1
	Telaviv/ST 1068/PT 36	—	1
Chicken meat (n = 14)	Infantis/ST 32/PT 7, PT 8, PT 9	K, S, N, T, SF	6
		S, N, T, SF	5
		K, S, N, AMP, T, SF	1
		K, S, N, AMP, T, KF, SF, SXT, C	1
		S, N, T	1
Cow ground meat (n = 13)	Anatum/ST 64/PT 42	—	5
	Anatum/ST 64/PT 42	S, SF	1
	Kentucky/ST 314/PT 10	SF	1
	Montevideo/ST 195/PT 28	—	1
	Newport/ST 166/PT 40	—	1
	Reading/ST 93/PT 32	—	1
	Telaviv/ST 1068/PT 33, PT 34	—	2
Offal (n = 14)	Infantis/ST 32/PT 8	S, N, T, SF	1
	Kentucky/ST 314/PT 10	—	1
	Montevideo/ST195/PT 21, PT 25	—	4
	Montevideo/ST195/PT 21	SF	1
	Typhimurium/ST 19/PT 13	AMP, T	1
	Newport/ST 166/PT 40	—	1
	Reading/ST 93/PT 32	—	1
	Telaviv/ST 1068/PT 33	—	2
	Telaviv/ST 1068/PT 33	S	1
Sheep ground meat (n = 12)	Anatum/ST 64/PT 42	SF	3
	Anatum/ST 64/PT 42	—	2
	Montevideo/ST 195/PT 21	—	1
	Othmarschen/ST 1832/PT 27	—	1
	Reading/ST 93/PT 17	—	1
	Telaviv/ST 1068/PT 33	—	2
	Telaviv/ST 1068/PT 33	SF	1

ST, sequence type; PT, pulsed-field electrophoresis type.

K, kanamycin; S, streptomycin; N, nalidixic acid; AMP, ampicillin; T, tetracycline; KF, cephalothin; SF, sulfisoxazole; SXT, sulfamethoxazole–trimethoprim; C, chloramphenicol.

Of note, all Salmonella Infantis isolates were resistant to at least to two antimicrobial agents and thus were classified as MDR (Table 3). All Salmonella Infantis isolates were resistant to nalidixic acid and tetracycline, and nearly all of them were resistant to streptomycin and sulfisoxazole. It was observed that 51.9% and 83.3% of the resistant and MDR isolates, respectively, belonged to Salmonella Infantis. The other MDR profiles were observed in serotypes Salmonella Hadar (5.6%), Salmonella Anatum (5.6%), and Salmonella Newport (5.6%) isolates. In contrast, 85.7% of Salmonella Montevideo isolates, and 84.6% of Salmonella Telaviv serotypes were susceptible to all antimicrobial agents (Table 3). None of the isolates showed resistance to amikacin, gentamicin, ciprofloxacin, amoxicillin–clavulanic acid, cefoxitin, ceftriaxone, ceftiofur, imipenem, and ertapenem.

Table 3.

Comparison of Subtyping Results and Combined Subtype Profiles

Serotype/ST	PFGE type	Antimicrobial resistance profile ^a	Combined subtype profile ID	Total no.
Infantis/ST 32	8	K, S, N, AMP, T, SF	1	1
	8	K, S, N, AMP, T, KF, SF, SXT, C	2	1
	8	K, S, N, T, SF	3	4
	8	S, N, T, SF	4	5
	9	S, N, T, SF	5	1
	7	K, S, N, T, SF	6	2
	8	S, N, T	7	1
Montevideo/ST 195^b	25	—	15	2
	21	—	18	2
	21	SF	19	1
	28	—	20	1
	25	—	21	1
Telaviv/ST 1068	33	S	9	1
	33	—	10	7
	34	—	11	3
	36	—	12	1
	33	SF	14	1
Anatum/ST 64	42	SF	22	4
	42	—	23	6
	42	S, SF	24	1
Reading	32	—	27	2
	17	—	28	1
Kentucky	10	SF	25	1
	10	—	26	1
Newport	40	—	30	2
Hadar	41	S, N, AMP, T, KF	17	1
Typhimurium	13	AMP, T	8	1
Othmarschen	27	—	16	1

K, kanamycin; S, streptomycin; N, nalidixic acid; AMP, ampicillin; T, tetracycline; KF, cephalothin; SF, sulfisoxazole; SXT, sulfamethoxazole–trimethoprim; C, chloramphenicol.

Three isolates belonging to ST 195 were excluded due to their inaccurate serotyping results.

ST, sequence type; PT, pulsed–field electrophoresis type.

PFGE

The 56 Salmonella isolates characterized in the current study represented unique 17 PFGE types (PTs) (Fig. 2). All the serotypes and STs were further diversified by PFGE typing as each serotype and ST included more than one PFGE pattern. The SID value calculated for PFGE (0.89) was higher than serotype and MLST (0.83). PFGE provided discrimination beyond the serotype and ST level for isolates belonging to Salmonella Infantis (ST 32, n = 15), Telaviv (ST 1068, n = 13) (Fig. 3), and Salmonella Montevideo (ST 195). The most frequently detected PFGE pattern was PT 8, which was observed in 12 of 15 Salmonella Infantis (ST 32) isolates. Since 93% of subtype Infantis (ST 32) isolates were recovered from chicken meat, PFGE typing revealed a potentially common and highly clonal Salmonella Infantis strain present in retail poultry samples in Turkey. PT 8 was further differentiated into five different MDR profiles. Similarly, PT64, represented by 11 Salmonella Anatum isolates (ST 64), was categorized into three groups of antimicrobial resistance profiles including susceptible (n = 6), resistant to sulfisoxazole (n = 4), and resistant to sulfisoxazole and streptomycin (n = 1). PFGE type 33 representing nine Salmonella Telaviv (ST 1068) isolates (Fig. 3), was the third most common PT and PT 33 isolates were susceptible to all antimicrobial agents, except one isolate, which was resistant to sulfisoxazole (Table 3).

FIG. 2.

Dendrogram for 56 isolates were determined by the restriction fragments created by XbaI enzyme. Bionumerics software was used to build the dendrogram. Similarities were determined by Dice coefficient and the patterns were compared by unweighted-pair group method with arithmetic mean. 2.5% band position tolerance and 1.5% optimization was used. PFGE, pulsed-field gel electrophoresis.

FIG. 3.

Dendrogram for serovar Telaviv showed three clusters of approximately 92.1% similarity. Having the most number of isolates, PT 33 was found in nine isolates. The isolates showed clonal dissemination through various food isolates by presenting only a three-band difference among 13 isolates. PFGE, pulsed-field gel electrophoresis; Sa. enterica, Salmonella enterica; G. Meat, ground meat.

Discussion

Distribution of serotyping and MLST results in different food types indicates a possible affiliation between some subtypes and food types

Salmonella Infantis isolates (ST 32), the most common serotype in our study, were all from chicken meat samples (p < 0.05). In addition, 6 of 11 Salmonella Anatum isolates were acquired from raw ground beef (p < 0.01), while the remaining 5 isolates were from ground mutton (p < 0.05). In addition, five out of seven Salmonella Montevideo (ST 195) isolates were isolated from cow offal samples (p < 0.01). However, serotype Salmonella Telaviv (ST 1068) was collected in a wider range food samples (i.e., cattle ground meat, sheep ground meat, offal, unripened cheese, and Urfa cheese) and was not over-represented within a given food type. It is also notable that all of the four isolates isolated from unripened feta cheese represented Salmonella Telaviv (p < 0.01) (Table 1). These analyses support that specific Salmonella serotypes may be over-represented among certain types of food samples collected in Turkey.

Despite the fact that there is a lack of data to assess the source and geographical clustering of Salmonella subtypes in Turkey, Salmonella Infantis was previously isolated from chicken products in different areas of Turkey (Sarımehmetoğlu et al., 1996; Cetinkaya et al., 2008). Reports by the European Food Safety Authority (EFSA) also indicated that the prevalence of serotype Salmonella Infantis in European Union (EU) countries at broiler facilities has increased in recent years. Along with Salmonella Enteritidis and Salmonella Typhimurium, Salmonella Infantis was the most commonly reported serotype isolated from chicken, broiler meat, and eggs in the EU (EFSA, 2013). Another study by EFSA also showed that Salmonella Infantis, isolated in 15 different EU member states, was the most widely distributed serotype across the EU as well as the most dominant serotype in several countries, accounting for 97.8%, 57.1%, and 40% of Salmonella isolates in Hungary, Slovenia, and Switzerland, respectively (EFSA, 2011). Apart from the EU, Salmonella Infantis persistence in broiler flocks had been described as an emerging problem worldwide, including countries such as Japan, Saudi Arabia, Algeria, and the United States on different continents (Miller et al., 2010).

Consistent with previous studies, the Salmonella Infantis in our study showed MDR. The most prevalent MDR profiles were K-S-N-T-SF (6/27) and S-N-T-SF (5/27), and they were characteristic of nearly all Salmonella Infantis isolates in the current study (Table 3). Researchers from Germany showed that Salmonella isolates from food were commonly resistant to streptomycin (93.7%), sulfamethoxazole (92.5%), and tetracycline (80.9%) (Miko et al., 2005). In another study, tetracycline (80.0%), streptomycin (73.0%), and sulfamethoxazole (60.0%) resistance was demonstrated by Salmonella isolated from retail meat samples such as chicken, beef, pork, and turkey in the United States (White et al., 2001). These three antimicrobials were also shown to inhibit growth of food-origin Salmonella isolated from Turkey in our study.

Comparison of international references with our findings and other studies in Turkey indicating that subtype Telaviv representing ST 1068 and PTs 33, 34, and 36 might be a characteristic serotype to Sanliurfa, as well as Turkey

In the current study, Salmonella Telaviv was the second most common serotype, where Salmonella Telaviv was isolated from 13 food samples, including 5 different food types. It was also noteworthy that all of the isolates collected from unripened cheese samples (n = 4) were classified as Salmonella Telaviv (ST 1068), as well as one of the two isolates collected from Urfa cheese samples. The isolate from Urfa cheese (MET S1-391) was the only isolate representing PT 36. MET S1-391 had a single band difference (approx. 120 kb) when compared to PT 33, which might be attributed to an insertion or a deletion mutation or plasmid acquisition (Tenover et al., 1995). PT 33 was represented by Salmonella Telaviv isolates collected from cow offal (MET S1-63, MET S1-74, MET S1-528) and ground sheep samples (MET S1-430, MET S1-440, MET S1-557), while PT 34 was represented by Salmonella isolates collected from one ground beef sample (MET S1-485) and two unripened cheese (MET S1-119, MET S1-530) samples (Fig. 3). All Salmonella Telaviv isolates were susceptible to antimicrobial agents. Although Salmonella Telaviv seems to be a rare serotype in the United States (CDC, 2009) and other countries, a Turkish study, mentioned above (Erol, 1999) reported one Salmonella Telaviv isolate isolated from a ground beef sample from Ankara. In addition, there were three notifications in 2003 and one notification in 2011 regarding detection of Salmonella Telaviv in EU by the Rapid Alert System for the Food and Feed (RASFF) annual reports. Products exported to EU that were reported to be contaminated with Salmonella Telaviv included pet food in 2011 and meal of lamb tripe, cumin, and peppermint in 2003; all products originated from Turkey (RASFF, 2011). To our knowledge, a sole previous surveillance study described the presence of Salmonella Telaviv among 300 fecal samples from adult cattle in Europe (Vella and Cuschieri, 1995). In another study, our research team observed that Salmonella Telaviv was also significantly present in cattle feces and sheep feces collected from slaughterhouses and pastures, but had not obtained clinical human cases in the last 5 years in the Sanliurfa region in Turkey (Acar et al., 2015, unpublished data). In the UoW S. enterica MLST Database, there was a single record of Salmonella serotype Telaviv (out of 6174 records). This single Salmonella Telaviv isolate was originated from a chicken in Israel in 1940 and shared the identical sequence type with Telaviv isolates in the current study, ST 1068.

Conclusions

As one of the important exporters of fresh fruits, fresh vegetables, and also processed foodstuff to EU and many other countries, Turkey lacks the retrospective literature data focusing on relations of subtype-source and subtype-geographical region through the use of conventional and molecular characterization methods. This study suggested a possible geographic clustering of serotype Telaviv in Turkey. To confirm this result, studies with a larger sample size from different regions of Turkey should be conducted in future.

Footnotes

Acknowledgments

The authors thank Dr. Huseyin Avni Kirmaci in Department of Food Engineering at Harran University for sending food samples, Dr. Martin Wiedmann for providing access to BioNumerics in Food Science Department at Cornell University, and Dr. Kendra K. Nightingale for reviewing the manuscript. The authors are very grateful for the financial research support and technical support provided by The Scientific and Technological Research Council of Turkey (TUBITAK) under project number of TUBITAK 111O192, and Middle East Technical University BAP.

Disclosure Statement

No competing financial interests exist.

References

Achtman

, Wain

, Weill

, et al. Multilocus sequence typing as a replacement for serotyping in Salmonella enterica . PLoS Pathogens, 2012; 8:e1002776.

Alcaine

, Soyer

, Warnick

, et al. Multilocus sequence typing supports the hypothesis that cow- and human-associated Salmonella isolates represent distinct and overlapping populations. Appl Environ Microbiol, 2006; 72:7575–7585.

[CDC] Centers for Disease Control and Prevention.. National Salmonella Surveillance Annual Summary 2009. Atlanta, GA: CDC, 2009.

CDC. General Information on Salmonella . Volume 2014. Atlanta, GA: CDC, 2014a.

CDC. Salmonella Montevideo and Salmonella Mbandaka Infections Linked to Tahini Sesame Paste. Volume 2014. Atlanta, GA: CDC, 2014b.

Cetinkaya

, Cibik

, Soyutemiz

, Ozakin

, Kayali

, Levent

. Shigella and Salmonella contamination in various foodstuffs in Turkey. Food Control, 2008; 19:1059–1063.

[CLSI] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard, Volume 22. Wayne, PA: CLSI, 2002.

CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement, Volume 33. Wayne, PA: CLSI, 2013.

[EFSA] European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses, Part B: Analysis of factors associated with Salmonella contamination of broiler carcasses. EFSA J, 2011; 9:2090.

10.

EFSA. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA J, 2013; 11:3129.

11.

Erol İ. Ankara'da tüketime sunulan kıymalarda Salmonellaların varlığı ve serotip dağılımı [Salmonella prevalence and serotype distribution on ground meats collected from retails in Ankara]. Turk J Vet Anim Sci, 1999; 23:321–325. (in Turkish.)

12.

Favier

, Estrada

CSL

, Otero

, Escudero

. Prevalence, antimicrobial susceptibility, and molecular characterization by PCR and pulsed field gel electrophoresis (PFGE) of Salmonella spp. isolated from foods of animal origin in San Luis, Argentina. Food Control, 2013; 29:49–54.

13.

Grimont

, Weil

. Antigenic Formulae of the Salmonella Servovars, 9th ed. Paris: World Health Organization Centre for Reference and Research on Salmonella: Pasteur Institute, 2007.

14.

Harbottle

, White

, McDermott

, Walker

, Zhao

. Comparison of multilocus sequence typing, pulsed-field gel electrophoresis, and antimicrobial susceptibility typing for characterization of Salmonella enterica serotype Newport isolates. J Clin Microbiol, 2006; 44:2449–2457.

15.

[ISQ] International Organization for Standardization. 6579, 2002: Microbiology of Food and Animal Feeding Stuffs—Horizontal Method for the Detection of Salmonella spp. London: British Standard Institute, 2002.

16.

Kim

, Lee

, Park

, et al. A novel multiplex PCR assay for rapid and simultaneous detection of five pathogenic bacteria: Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus . J Food Prot, 2007; 70:1656–1662.

17.

Kumar

, Gupta

, Chander

, Singh

. Antibiotic use in agriculture and its impact on the terrestrial environment. Adv Agron, 2005; 87:1–54.

18.

Majowicz

, Musto

, Scallan

, et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis, 2010; 50:882–889.

19.

Miko

, Pries

, Schroeter

, Helmuth

. Molecular mechanisms of resistance in multidrug-resistant serovars of Salmonella enterica isolated from foods in Germany. J Antimicrob Chemother, 2005; 56:1025–1033.

20.

Miller

, Prager

, Rabsch

, Fehlhaber

, Voss

. Epidemiological relationship between Salmonella Infantis isolates of human and broiler origin. Lohmann Inform, 2010; 45:27–31.

21.

Murase

, Nagato

, Shirota

, Katoh

, Otsuki

. Pulsed-field gel electrophoresis-based subtyping of DNA degradation-sensitive Salmonella enterica subsp enterica serovar Livingstone and serovar Cerro isolates obtained from a chicken layer farm. Vet Microbiol, 2004; 99:139–143.

22.

RASFF.. RASFF Portal. Rapid Alert System for Food and Feed. European Union Commission, 2011.

23.

Refsum

, Heir

, Kapperud

, Vardund

, Holstad

. Molecular epidemiology of Salmonella enterica serovar Typhimurium isolates determined by pulsed-field gel electrophoresis: Comparison of isolates from avian wildlife, domestic animals, and the environment in Norway. Appl Environ Microbiol, 2002; 68:5600–5606.

24.

Ribot

, Fair

, Gautom

, et al. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis, 2006; 3:59–67.

25.

Rothrock

, Ingram

, Gamble

, et al. The characterization of Salmonella enterica serotypes isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multidrug-resistant Heidelberg strain. Poultry Sci, 2015; 94:467–472.

26.

Sarımehmetoğlu

, Küplülü

, Erol

, Özdemir

. Tavuk kesimhanelerinde Salmonella kontaminasyonu ve serotip dağılımı [Contamination and serotype distribution of Salmonella in broilers]. Ankara Üniv Vet Fak Derg, 1996; 43:85–90. (in Turkish.)

27.

Soyer

, Alcaine

, Schoonmaker-Bopp

, et al. Pulsed-field gel electrophoresis diversity of human and bovine clinical Salmonella isolates. Foodborne Pathog Dis, 2010; 7:707–717.

28.

Tenover

, Arbeit

, Goering

, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol, 1995; 33:2233–2239.

29.

Urwin

, Maiden

MCJ

. Multi-locus sequence typing: A tool for global epidemiology. Trends Microbiol, 2003; 11:479–487.

30.

Vella

, Cuschieri

. Salmonella excretion in adult cattle on the Maltese island of Gozo. Rev Sci Tech Oie, 1995; 14:777–787.

31.

Wattiau

, Boland

, Bertrand

. Methodologies for Salmonella enterica subsp enterica subtyping: Gold standards and alternatives. Appl Environ Microbiol, 2011; 77:7877–7885.

32.

White

, Zhao

, Sudler

, et al. The isolation of antibiotic-resistant Salmonella from retail ground meats. New Engl J Med, 2001; 345:1147–1154.

33.

Wiedmann

. Subtyping of bacterial foodborne pathogens. Nutr Rev, 2002; 60:201–208.

34.

Wonderling

, Pearce

, Wallace

, et al. Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of Salmonella isolates obtained from the carcasses and feces of swine at slaughter. Appl Environ Microbiol, 2003; 69:4177–4182.

35.

Zweifel

, Stephan

. Spices and herbs as source of Salmonella-related foodborne diseases. Food Res Int, 2012; 45:765–769.