International Journal of Food Microbiology 191 (2014) 125–128

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Short communication

Multilocus sequence typing reveals genetic diversity of foodborne Arcobacter butzleri isolates in the North of Spain Rodrigo Alonso, Cecilia Girbau, Irati Martinez-Malaxetxebarria, Aurora Fernández-Astorga ⁎ Department of Immunology, Microbiology and Parasitology, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, Vitoria-Gasteiz 01006, Spain

a r t i c l e

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Article history: Received 3 April 2014 Received in revised form 7 September 2014 Accepted 14 September 2014 Available online 18 September 2014 Keywords: Arcobacter butzleri Food MLST Gene transfer

a b s t r a c t The emerging pathogen Arcobacter butzleri is being increasingly isolated from different animal food products but the routes of its transmission to human are not well established yet. Typing methods would be useful in gaining such knowledge. Here we report the great genetic diversity observed among A. butzleri isolates from different food products. Forty-five isolates were analyzed by Multilocus Sequence Typing (MLST). A total of 157 alleles were identified across all seven loci, ranging from 16 alleles at glnA to 31 at glyA. MLST differentiated the isolates into 34 sequence types (STs), with the majority of isolates containing a unique sequence type. Seventy-four new alleles were identified, which resulted in the assignment of 33 new STs. No association of alleles or STs with food source was observed. For the first time, lateral gene transfer from Arcobacter skirrowii to A. butzleri at the glyA locus is also reported. © 2014 Published by Elsevier B.V.

1. Introduction Arcobacter butzleri, an emerging food and waterborne pathogen (ICMS, 2002) belonging to the Campylobacteraceae family, is associated with human gastroenteric illness similar to campylobacteriosis (Vandamme et al., 1992; Vandenberg et al., 2004). Water environments and animal hosts seem to be the main reservoirs for this organism (Collado and Figueras, 2011; Jacob et al., 1993; Moreno et al., 2003; Rice et al., 1999). In addition A. butzleri is frequently isolated from food products of animal origin (Collado and Figueras, 2011; Ho et al., 2006; Morita et al., 2004; Nieva-Echevarria et al., 2013; Prouzet-Mauleon et al., 2006), which suggest not only the consumption of faecally contaminated water (Collado et al., 2008) but also that of raw or poorly cooked animal food products to be important sources of A. butzleri infections in humans. However, the routes of transmission of A. butzleri are not well established yet. Typing methods are useful in studying transmission routes and source tracking during outbreaks and extended epidemiological investigations. Multilocus Sequence Typing (MLST) is a method that facilitates the discrimination of microbial isolates by comparing the sequences of housekeeping gene fragments (Jolley et al., 2004). The first multilocus sequence typing method for A. butzleri was described by Miller et al. (2009). The Arcobacter MLST gene set is identical to that of Campylobacter jejuni comprising aspA, atpA(uncA), glnA, gltA, glyA, pgm and tkt genes and it has proven for strain characterization, lineage identification and epidemiology purposes. Besides the primary use of MLST data for strain

⁎ Corresponding author. Tel.: +34 945 013909; fax: +34 945 013014. E-mail address: [email protected] (A. Fernández-Astorga).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.012 0168-1605/© 2014 Published by Elsevier B.V.

typing, these are a valuable resource for studies on lateral gene transfer and evolution (Kinana et al., 2007; Miller et al., 2009, 2012). Few works have used this technique to evaluate the diversity of Arcobacter isolates. Studies held by Merga et al. showed great diversity among Arcobacter spp. isolates from cattle faecal samples (2011) and among A. butzleri isolates from the same origin (2013). Rasmussen et al. (2013) used MLST to evaluate the heterogeneity of A. butzleri isolates obtained from a Danish slaughterhouse on two consecutive production days and after the sanitizing in between. The results showed high strain variability; however they suggested that the repeated detection of two sequence types (STs) could be related to crosscontamination. In view of the diversity reported for Arcobacter populations, the aim of this study was to determine the level of heterogeneity among 45 A. butzleri isolates obtained from different products of animal origin and to study potential lateral gene transfer between the isolates.

2. Materials and methods 2.1. Bacterial strains A total of 45 A. butzleri isolates recovered from food products were analyzed in this study. These strains were isolated during a surveillance study conducted in the North of Spain (Nieva-Echevarria et al., 2013). The isolates from food products included 12 isolated from poultry, 13 from raw milk, 4 from mussels, 10 from clams, 5 from pork meats, and one from minced beef. Bacteria were routinely grown in Arcobacter broth (Oxoid) and on Columbia agar blood plates (Oxoid) at 30 °C under aerobic conditions for 24 h.

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2.2. DNA extraction Arcobacter DNA was isolated from broth cultures using the PrepMan™ Ultra reagent (Applied Biosystems) according to the manufacturer's specifications, diluted to 20 ng/μl and stored at −20 °C. 2.3. Multilocus sequence typing MLST was carried out according to the method of Miller et al. (2009). Allele numbers and sequence types (STs) were assigned using the PubMLST database (http://pubmlst.org/arcobacter/). Any new alleles were submitted to the database curator to be assigned new allele numbers. The relationships between sequence types were investigated by concatenating the allele sequences comprising each unique ST. Dendograms were constructed using the neighbor-joining method with the Kimura 2-parameter distance estimation method. Cluster analyses were performed using MEGA v5.2 (Tamura et al., 2011). At the same time, a phylogenetic tree was constructed for each locus using MEGA v5.2. Polymorphic sites, dN/dS ratios and index of association were determined using START2 program (Jolley et al., 2001). 2.4. Detection of recombination MLST sequences (downloaded from the Arcobacter PubMLST database on May 2014) were concatenated in order to produce the multiple alignments used for the recombination analysis. Recombination events were detected in concatenated sequences using the RDP program, which is implemented in RDP3 software package (Martin et al., 2010). The RDP analysis was carried out with the window size set to 30 nucleotides, no reference sequence was selected, the percentage of identity between recombinant sequences was set from 0 to 100, and the user-defined cutoff value was set to 0.05. 3. Results and discussion The A. butzleri population was diverse as revealed by the MLST data. A large number of alleles and sequence types were identified in this study (Table 1). A total of 157 alleles were identified across all seven loci, ranging from 16 alleles at glnA to 31 at glyA. Overall, 74 out of the 157 (47%) alleles were previously unreported, ranging their frequency at a locus from 35% (atpA) to 68% (glyA). These results suggest A. butzleri isolates of this study to constitute a more diverse population than those previously observed (Merga et al., 2011, 2013; Miller et al., 2009; Rasmussen et al., 2013). The high number of new alleles could reflect the heterogeneity of food isolates, but also the relatively limited number of food isolates previously available in the MLST database. Allelic density (number of alleles/number of strains) ranged from approximately 35% at the glnA locus to 69% at the glyA locus (Table 2). Although a low number of strains were analyzed, the allelic density of A. butzleri isolates in this study is higher than that observed by other authors (Merga et al., 2011; Miller et al., 2009), probably as a result of the diversity of food isolates. Further MLST studies on the diversity of Arcobacter spp. in foods will elucidate whether allelic density is consistently higher in Arcobacter isolates from foods. The most variable locus was glyA with 13.6% variable sites and the least variable locus was atpA with only 5.1% variable sites. The dN/dS ratios varied across the seven loci, ranging from 0.000 for gltA to 0.094 for glyA. These ratios are in agreement with those previously reported by Miller et al. (2009). A total of 34 STs were identified among the 45 A. butzleri isolates analyzed (Table 1). Overall, 33 (97%) STs were previously unreported and resulted from new allele's sequences. Of note, ST-193, found in one pork meat isolate (Ab-C2), was the only known ST observed. Most of the new STs (26/33, 78%) were represented by a single isolate, and eight STs by more than one. Despite the reported association between MLST alleles and certain food animal products in Campylobacter coli (Miller et al.,

Table 1 Origin and MLST typing data of A. butzleri strains analyzed in this study. Strain

Source

aspA

atpA

glnA

gltA

glyA

pgm

tkt

ST

Ab-A1 Ab-A2 Ab-A3 Ab-A4 Ab-A5 Ab-A6 Ab-A7 Ab-A8 Ab-A9 Ab-A11 Ab-M1 Ab-M2 Ab-M3 Ab-M4 Ab-C2 Ab-C3 Ab-C4 Ab-C5 Ab-C6 Ab-L1 Ab-L2 Ab-L3 Ab-L4 Ab-L5 Ab-L6 Ab-L7 Ab-L8 Ab-L10 Ab-L11 Ab-L12 Ab-L13 Ab-L14 Ab-P1 Ab-P2 Ab-P3 Ab-P4 Ab-P6 Ab-P7 Ab-P8 Ab-P9 Ab-P13 Ab-P14 Ab-P17 Ab-P18 Ab-V1

Clam Clam Clam Clam Clam Clam Clam Clam Clam Clam Mussel Mussel Mussel Mussel Pork Pork Pork Pork Pork Milk Milk Milk Milk Milk Milk Milk Milk Milk Milk Milk Milk Milk Poultry Poultry Poultry Poultry Poultry Poultry Poultry Poultry Poultry Poultry Poultry Poultry Beef

14 212 212 15 15 203 4 216 214 214 212 20 211 213 71 153 20 44 5 20 20 20 20 20 20 20 182 10 5 5 5 10 208 209 209 48 73 210 20 20 23 203 30 5 5

151 149 149 10 10 140 4 62 62 62 149 12 147 140 25 4 39 150 5 39 12 39 39 39 39 39 58 145 5 5 5 145 2 146 146 25 12 65 34 34 7 24 5 5 5

26 26 26 5 5 26 4 26 128 128 26 45 129 26 7 7 34 11 5 34 11 34 34 34 34 34 4 124 5 5 5 124 26 26 26 41 1 125 9 9 26 126 9 5 127

55 144 144 15 15 140 4 144 148 148 144 15 147 140 2 19 19 23 19 19 11 19 19 19 19 19 146 19 15 15 15 19 142 143 143 19 9 144 5 5 15 145 30 15 24

466 469 469 36 36 385 468 474 465 465 462 5 34 120 102 467 470 471 472 460 458 460 460 460 460 441 185 454 451 451 451 454 455 453 453 440 120 457 120 120 459 452 456 461 463

241 38 38 243 243 38 4 246 244 244 38 102 242 242 106 102 102 245 11 87 87 234 234 234 234 26 236 102 11 11 11 102 39 237 237 102 10 240 239 239 102 235 35 238 107

56 183 183 2 2 86 185 188 186 186 183 58 184 86 2 158 51 187 65 51 178 51 51 51 51 51 160 11 10 10 10 11 19 182 182 2 179 180 2 2 158 181 4 2 63

396 404 404 397 397 358 393 407 406 406 403 398 402 405 193 401 399 400 394 386 387 388 388 388 388 360 389 390 391 391 391 390 377 378 378 359 379 380 381 381 382 383 384 385 395

Boldface entries represent novel alleles or STs.

2006), no host-associated MLST alleles or STs could be identified in this study. This result is in agreement with that previously reported by Miller et al. (2009). However, ST-388 was observed in four out of the 13 (30.8%) raw milk isolates. These four strains were isolated from samples recovered in the same farm. Genetic variability among raw milk Arcobacter isolates has been previously observed although the technique used was not MLST. Giacometti et al. (2013) have suggested that the environmental conditions in a dairy plant constitute a good ecological niche for the colonization of A. butzleri and, in some cases, the genetic identity of strains isolated from the same facilities on

Table 2 Allelic diversity among 45 A. butzleri isolates. Locus No. of alleles

No. (%) of new alleles

Allelic density

No. of variable sites

dN/dS

aspA atpA glnA gltA glyA pgm tkt

9 (39) 7 (35) 6 (37.5) 8 (42) 21 (68) 13 (54) 10 (41.5)

51.1 44.4 35.6 42.2 68.9 53.3 53.3

32 25 25 15 69 43 41

0.006 0.092 0.030 0.000 0.094 0.023 0.043

23 20 16 19 31 24 24

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different sampling days showed that these strains could be persistent in this environment. On the other hand, genotyping of A. butzleri by pulsed-field gel electrophoresis by Revez et al. (2013) revealed a heterogeneous population with no dominant clone observed in the raw milk samples investigated. These authors have also suggested that the bulk tanks may be contaminated with multiple strains. Besides their primary use for strain typing, MLST data are a valuable resource for studies on lateral gene transfer and evolution (Kinana et al., 2007; Miller et al., 2009, 2012). For these purposes, a radial neighborjoining tree of the concatenated sequences from the unique STs (n = 34) was generated by using MEGA. This phylogenetic analysis resulted in a tree that was not very robust (few bootstrap values above 50) and showed no clustering of isolates. However, the tree revealed one divergent sequence type (ST-393) which clustered away from the main group of A. butzleri isolates. Of note, this ST was reported for the first time in this study. In order to rule out any possible misidentification, the A. butzleri identity of this isolate (Ab-A7) was confirmed by PCR-RFLP (Figueras et al., 2012). To determine whether one of the MLST genes influenced the tree topology, the seven trees constructed independently from each gene were analyzed. A divergent glyA-468 allele was identified within ST-393. This allele had a similarity percentage at the nucleotide level of 94.08% to glyA-429 and glyA-310 alleles and 93.84% to glyA-222, all of them first identified in Arcobacter skirrowii (Miller, W., personal communication). Alleles at the other six loci for the Ab-A7 isolate clustered with other A. butzleri alleles, indicating that Ab-A7 could be a strain in which a putative lateral transfer event has occurred at the glyA locus. In order to identify possible recombination unique events located within each concatenate sequence (n = 439), the RDP3 package was used. Although 43 potential recombination events were detected in this way, further analysis was focused on ST-393. The RDP algorithm indicated a single recombination event occurred at the region between nucleotide positions 1935 and 2227 of the alignment, involving the sequence of glyA allele. Besides, the results indicated that ST-296 (major parent; glyA-347, A. butzleri) and ST-245 (minor parent; glyA-222, A. skirrowii) are the potential parents, with the support of five inferred methods: RDP (3.246 × 10− 25), Geneconv (1.477 × 10− 23), MaxChi (2.329 × 10−5), Chimaera (7.657 × 10−5), and 3Seq (1.525 × 10−22). Arcobacter strains contain two unlinked glyA genes in their genomes, the lysS-linked glyA1 (included in the MLST method) and the ada-linked glyA2 genes. In order to determine if the glyA2 gene was also divergent, it was amplified and sequenced. Sequence analysis revealed that Ab-A7 strain contained the glyA-139 allele at the glyA2 locus, previously described in A. butzleri. So, it was assumed that the recombination event at this locus could be excluded. Also, it has been shown that a large number of STs contain different glyA1 and glyA2 alleles (Miller et al., 2009). Lateral transfer events identified by MLST have been reported previously by Miller et al. (2009) in Arcobacter spp. Alelles related phylogenetically to those identified in A. butzleri have been identified within A. cibarius and A. skirrowii; similarly, A.skirrowii alleles have been identified within A. cryaerophilus and A. thereius, and an A. thereius allele has been identified in A. cryaerophilus. However, lateral transfer events with A. butzleri as recipient have never been reported. So, to our knowledge, this is the first time that this event is observed in an A. butzleri isolate. The exchange of DNA in this study was seen as recombination of two alleles, from two different species, resulting in an allele with mosaic structure of nucleotides from both species. The index of association determines the clonality of the population. An absolute value of zero indicates that the population is freely recombinating and is not clonal, whereas a value significantly different from 0 indicates the high genetic diversity of the population in which recombination has been rare or absent (Jolley et al., 2001). The standarized index of association observed for the 45 strains was 0.389. Additionally, analysis of standarized index of association with the STs as unit was performed and a value of 0.178 was obtained, which

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indicates that the A. butzleri population of this study is recombining to some degree (Smith et al., 1993). In conclusion, the MLST genotyping of foodborne A. butzleri isolates showed a large amount of diversity among them. There were no links between STs and food sources but lateral gene transfer from A. skirrowii to A. butzleri was observed, which may play a role in the evolution of the genus. As our results showed that recombination is possible in A. butzleri strains, further analysis should be carried out to determine the role of mutation and recombination in the great genetic diversity of this species. In view of the relatively limited number of food isolates available in the MLST database, typing more A. butzleri strains from other food sources and geographical locations could be of interest for further epidemiological and genetic studies of A. butzleri. Acknowledgments This word was supported by a grant from the Basque Country Government Ref. IT-528-10. 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