# Genetic Diversity and Incidence of Virulence-Associated Genes ofArcobacter butzleri and Arcobacter cryaerophilus Isolates from Pork, Beef, and Chicken Meat in Poland

Authors: Iwona Zacharow; Jarosław Bystroń; Ewa Wałecka-Zacharska; Magdalena Podkowik; Jacek Bania
Journal: BioMed Research International
 (2015)

---

## Abstract

Incidence of 9 virulence-associated genes and genetic diversity was determined in 79A. butzleri and 6A. cryaerophilus isolates from pork, beef, and chicken meat. AllA. butzleri isolates harboured thetlyA gene, and most of them carriedciaB,mviN,pldA,cadF, andcj1349 genes.ciaB was found to occur with higher frequency in poultry if compared with pork (p
=
0.0007), whileirgA was more frequent in poultry than in beef (p
=
0.007). All 6A. cryaerophilus isolates harboured theciaB gene, whilemviN andtlyA were detected in 3 out of these isolates. Only one isolate carried thecadF gene. All beef-derivedA. cryaerophilus isolates carriedciaB,mviN, andtlyA genes.A. cryaerophilus isolates from chicken meat harbouredciaB gene only. The pork-derived isolate harbouredciaB andcadF genes. Seventy-four genotypes were distinguished within 79A. butzleri isolates. Nineteen from 21 isolates derived from beef and pork were found to be closely related toA. butzleri from chicken meat. Each of the 6A. cryaerophilus isolates was found to have unique genotype. We demonstrated that closely related genotypes can spread within pork, beef, and chicken meat populations ofA. butzleri but notA. cryaerophilus.


---

## Body

## 1. Introduction

GenusArcobacter, formerly known as “aerotolerantCampylobacter,” was included into the family Campylobacteraceae in 1991 [1] and currently consists of twenty species [2, 3]. ThreeArcobacter species, that is,A. butzleri,A. cryaerophilus, andA. skirrowii, have been mainly associated with enteritis in humans and abortion in pigs [4–6].A. butzleri has been classified as a serious hazard to human health by the International Commission on Microbiological Specifications for Foods in 2002 [7]. The bacteria have been isolated from food, mainly from products of animal origin, with the highest prevalence in poultry, followed by pork and beef [8–10]. Consumption ofArcobacter-contaminated food is regarded as the most likely source of human infection [11].Arcobacter spp. were also isolated from drinking water reservoirs and sewage [12]. Recent evidence suggests thatArcobacter spp., especiallyA. butzleri andA. cryaerophilus, may be involved in human enteric diseases [4, 13, 14]. Infections involvingArcobacter spp. may result in abdominal pain with acute diarrhoea or prolonged watery diarrhoea [15].Analysis of complete genome ofA. butzleri ATCC (American Type Culture Collection) 49616 revealed some homologues to virulence genes identified inCampylobacter and other bacteria, includingE. coli andVibrio spp. Already identified virulence-associated genes ofA. butzleri includecadF, cj1349, ciaB, mviN, pldA, tlyA,irgA,hecA, andhecB [16].cadF andcj1349 encode fibronectin-binding proteins;ciaB encodesCampylobacter invasive antigen (CiaB), which may contribute to host cell invasion;hecA encodes HecA protein, a member of the filamentous hemagglutinin;hecB encodes a hemolysin activation protein;tlyA encodes hemolysin;mviN encodes essential protein MViN required for peptidoglycan biosynthesis;pldA encodes outer membrane phospholipase PldA associated with lysis of erythrocytes; andirgA encodes iron-regulated outer membrane protein IrgA. Although some authors reported on abundance of virulence factors, pathogenicity, and genetic diversity of food-derivedArcobacter isolates throughout the world, characteristics ofArcobacter spp. in Poland are still unknown [17–21].The aim of this work was to determine the incidence of virulence-associated genes and genetic diversity ofA. butzleri andA. cryaerophilus isolates from pork, beef, and chicken meat in Poland.


## 2. Materials and Methods

### 2.1. Bacterial Strains

Seventy-nineA. butzleri and 6A. cryaerophilus cultures were isolated from meat samples, purchased from retail market in Lower Silesia region (Poland) as described previously [22]. Among 79A. butzleri isolates, 58, 11, and 10 were obtained from chicken, beef, and pork samples, respectively. WithinA. cryaerophilus cultures, 3 isolates were from beef, 2 were from chicken, and one was from pork meat.Arcobacter spp. isolates were cultured according to Houf et al. [23] with modifications, inArcobacter broth (Oxoid) with selective supplement containing cefoperazone, amphotericin B, and teicoplanin (CAT, Oxoid). Additionally, novobiocin (32 mg/L), 5-fluorouracil (100 mg/L), and trimethoprim (64 mg/L) (Sigma) were added to the broth. After 48-hour incubation in aerobic atmosphere, at 30°C, the bacteria were subcultured onArcobacter agar plates (supplemented with chemotherapeutics mentioned above) and in parallel on agar plates with defibrinated sheep blood (Oxoid). Phenotypically suspected colonies were transferred to blood agar plates and incubated in aerobic conditions for 48 h at 30°C. OneArcobacter spp. isolate per sample was taken for further characterization. The isolates were preserved by freezing in Cryobank (Mast Diagnostics) at −80°C.


### 2.2. Preparation of Bacterial DNA

Total DNA was isolated as described by Agersborg et al. [24]. Briefly, the bacteria from 1 mL overnight culture were pelleted by centrifugation and suspended in 200 μL of distilled water containing 1% Triton X-100. The mixture was boiled for 10 min, and then the tubes were centrifuged for 5 min at 13 000 ×g. The supernatant containing DNA was used in PCR.


### 2.3. Species Identification of Arcobacter Isolates

The isolates were identified at species level using multiplex PCR according to Houf et al. [25]. Amplification products were resolved in 1.5% agarose containing 0.5 μg/mL ethidium bromide and documented using GelDocXR System (BioRad, Hercules, CA). Each PCR run was performed using DNA from the reference CCM (Czech Collection of Microorganisms) 4826A. butzleri and CCM 3933A. cryaerophilus strains as positive controls and ATCC 33560Campylobacter jejuni as a negative control.


### 2.4. Identification of Virulence-Associated Genes

A. butzleri andA. cryaerophilus isolates were PCR-screened for the presence of nine virulence-associated genes, such ascadF, cj1349,ciaB,mviN,pldA,tlyA,irgA,hecA, andhecB, as described by Douidah et al. [17]. The 10 μL aliquots of PCR products were resolved on 1.5% agarose gel and documented with a GelDoc XR documentation system (BioRad, Hercules, USA).


### 2.5. Genotyping ofA. butzleri andA. cryaerophilus Isolates

A survey of the genetic diversity of theA. butzleri andA. cryaerophilus isolates was performed using ERIC PCR primers ERIC2 5′-AAGTAAGTGACTGGGGTGAGCG-3′ and ERIC1R 5′-ATGTAAGCTCCTGGGGATTCAC-3′ as described by Houf et al. [26]. The PCR was performed in a mixture containing 20 mmol/L Tris-HCl, pH 8.4, 3 mmol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L of each deoxyribonucleotide, 1.25 U of Taq DNA polymerase (Thermo Scientific, Poland), 50 pmole of each primer (Genomed, Warsaw, Poland), and 2 μL of DNA solution. Thirty-six PCR cycles were carried out according to the following scheme: denaturation at 94°C for 30 s, annealing at 35°C for 30 s, and elongation at 72°C for 120 s. The denaturation/annealing and annealing/elongation ramping rates were set to 5 minutes [27]. Amplicons were separated on 3% agarose gel at 170 V for 3 h. The gels were stained with ethidium bromide, visualized on a UV transilluminator, and photographed. The resulting profiles were analyzed using the software available on http://insilico.ehu.es/dice_upgma/ to generate dendrograms by UPGMA clustering, using Dice correlation.


### 2.6. Statistical Analysis

Mann-WhitneyU test was applied to assess statistical significance of obtained data. Calculations were performed using Statistica 9.1 (StatSoft, Poland). p values below 0.05 were considered to be significant.


## 2.1. Bacterial Strains

Seventy-nineA. butzleri and 6A. cryaerophilus cultures were isolated from meat samples, purchased from retail market in Lower Silesia region (Poland) as described previously [22]. Among 79A. butzleri isolates, 58, 11, and 10 were obtained from chicken, beef, and pork samples, respectively. WithinA. cryaerophilus cultures, 3 isolates were from beef, 2 were from chicken, and one was from pork meat.Arcobacter spp. isolates were cultured according to Houf et al. [23] with modifications, inArcobacter broth (Oxoid) with selective supplement containing cefoperazone, amphotericin B, and teicoplanin (CAT, Oxoid). Additionally, novobiocin (32 mg/L), 5-fluorouracil (100 mg/L), and trimethoprim (64 mg/L) (Sigma) were added to the broth. After 48-hour incubation in aerobic atmosphere, at 30°C, the bacteria were subcultured onArcobacter agar plates (supplemented with chemotherapeutics mentioned above) and in parallel on agar plates with defibrinated sheep blood (Oxoid). Phenotypically suspected colonies were transferred to blood agar plates and incubated in aerobic conditions for 48 h at 30°C. OneArcobacter spp. isolate per sample was taken for further characterization. The isolates were preserved by freezing in Cryobank (Mast Diagnostics) at −80°C.


## 2.2. Preparation of Bacterial DNA

Total DNA was isolated as described by Agersborg et al. [24]. Briefly, the bacteria from 1 mL overnight culture were pelleted by centrifugation and suspended in 200 μL of distilled water containing 1% Triton X-100. The mixture was boiled for 10 min, and then the tubes were centrifuged for 5 min at 13 000 ×g. The supernatant containing DNA was used in PCR.


## 2.3. Species Identification of Arcobacter Isolates

The isolates were identified at species level using multiplex PCR according to Houf et al. [25]. Amplification products were resolved in 1.5% agarose containing 0.5 μg/mL ethidium bromide and documented using GelDocXR System (BioRad, Hercules, CA). Each PCR run was performed using DNA from the reference CCM (Czech Collection of Microorganisms) 4826A. butzleri and CCM 3933A. cryaerophilus strains as positive controls and ATCC 33560Campylobacter jejuni as a negative control.


## 2.4. Identification of Virulence-Associated Genes

A. butzleri andA. cryaerophilus isolates were PCR-screened for the presence of nine virulence-associated genes, such ascadF, cj1349,ciaB,mviN,pldA,tlyA,irgA,hecA, andhecB, as described by Douidah et al. [17]. The 10 μL aliquots of PCR products were resolved on 1.5% agarose gel and documented with a GelDoc XR documentation system (BioRad, Hercules, USA).


## 2.5. Genotyping ofA. butzleri andA. cryaerophilus Isolates

A survey of the genetic diversity of theA. butzleri andA. cryaerophilus isolates was performed using ERIC PCR primers ERIC2 5′-AAGTAAGTGACTGGGGTGAGCG-3′ and ERIC1R 5′-ATGTAAGCTCCTGGGGATTCAC-3′ as described by Houf et al. [26]. The PCR was performed in a mixture containing 20 mmol/L Tris-HCl, pH 8.4, 3 mmol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L of each deoxyribonucleotide, 1.25 U of Taq DNA polymerase (Thermo Scientific, Poland), 50 pmole of each primer (Genomed, Warsaw, Poland), and 2 μL of DNA solution. Thirty-six PCR cycles were carried out according to the following scheme: denaturation at 94°C for 30 s, annealing at 35°C for 30 s, and elongation at 72°C for 120 s. The denaturation/annealing and annealing/elongation ramping rates were set to 5 minutes [27]. Amplicons were separated on 3% agarose gel at 170 V for 3 h. The gels were stained with ethidium bromide, visualized on a UV transilluminator, and photographed. The resulting profiles were analyzed using the software available on http://insilico.ehu.es/dice_upgma/ to generate dendrograms by UPGMA clustering, using Dice correlation.


## 2.6. Statistical Analysis

Mann-WhitneyU test was applied to assess statistical significance of obtained data. Calculations were performed using Statistica 9.1 (StatSoft, Poland). p values below 0.05 were considered to be significant.


## 3. Results

### 3.1. Prevalence of Virulence-Associated Genes inA. butzleri andA. cryaerophilus Isolates

Screening of 79A. butzleri isolates revealed that all of them harboured thetlyA gene. Most of theA. butzleri isolates carriedciaB (97%),mviN (97%),pldA (92%),cadF (89%), andcj1349 (66%), whilehecB (48%),irgA (46%), andhecA (30%) genes were less frequent (Table 1).ciaB was found to occur with higher frequency in poultry if compared with pork (p
=
0.0007), whileirgA was more frequent in poultry than in beef (p
=
0.007).Table 1
Carriage of virulence genes byA. butzleri and A. cryaerophilus isolates.
Species
Origin/number of isolates
Percent of isolates harbouring virulence genes possessing specific gene
cadF
cj1349
ciaB
irgA
hecA
hecB
pldA
mviN
tlyA
A. butzleri
Chicken meat/58
90
71
100
53
34
48
93
98
100
Pork/10
90
60
80
40
10
60
100
90
100
Beef/11
82
45
100
9
27
36
82
100
100
Total/79
89
66
97
46
30
48
92
97
100
A. cryaerophilus
Chicken meat/2
0
0
100
0
0
0
0
0
0
Pork/1
100
0
100
0
0
0
0
0
0
Beef/3
0
0
100
0
0
0
0
100
100
Total/6
17
0
100
0
0
0
0
50
50Ten percent (8 out of 79) of theA. butzleri isolates harboured all nine genes, and most of them (7 isolates) derived from chicken meat. The carriage of 8 and 7 virulence-associated genes was reported in 44% (35 out of 79) ofA. butzleri isolates. Most of them were found in chicken meat (28 isolates), followed by pork (4 isolates) and beef (3 isolates). Seventeen (22%), 14 (18%), and 2 (3%)A. butzleri isolates possessed 6, 5, and 4 virulence-associated genes, respectively (Figure 1). The isolates harbouring 3 (2 isolates) and 2 genes (1 isolate) were only reported inA. butzleri from beef and pork, respectively.Figure 1
Dendrogram generated by UPGMA clustering based on PCR fingerprinting using ERIC primers performed on 79A. butzleri isolates. Repertoire of virulence-associated genes of each isolate was presented in left panel. Isolate origin was indicated as C, P, and B for chicken, pork, and beef meat, respectively.AllA. cryaerophilus isolates possessed theciaB gene, whilemviN andtlyA were detected in 3 out of the 6 isolates. Only one isolate carried thecadF gene.cj1349,hecA,hecB,irgA, andpldA genes were not detected in this population. All the 3 beef-derivedA. cryaerophilus isolates carriedciaB,mviN, andtlyA genes. TwoA. cryaerophilus isolates from chicken meat harbouredciaB gene only. The pork-derived isolate harbouredciaB andcadF genes (Table 1).


### 3.2. Genotypes ofA. butzleri andA. cryaerophilus

Seventy-four genotypes were distinguished within 79A. butzleri isolates (Figure 1). These isolates could be grouped into 8 clusters using 90% cut-off similarity. Nineteen from 21 isolates derived from beef and pork were found to be closely related toA. butzleri from chicken meat. Genotypes of 6 isolates from beef and 6 from pork shared >90% similarity to chicken isolates, when >80% similarity to chicken genotypes was found in 4 isolates from beef and 3 isolates from pork. Only two genotypes, one shared by beef (isolate 204) and one by pork isolate (isolate 60), were found to be distant from chicken genotypes. No relation betweenA. butzleri genotype and incidence of virulence genes could be found.Each of the 6A. cryaerophilus isolates was found to have unique genotype (Figure 2). Over 80% genotype similarity was only found within two beef and two porkA. cryaerophilus isolates but not between isolates from beef, pork, and chicken meat.Figure 2
Dendrogram generated by UPGMA clustering based on PCR fingerprinting using ERIC primers performed on 6A. cryaerophilus isolates. Repertoire of virulence-associated genes of each isolate was presented in left panel. Isolate origin was indicated as C, P, and B for chicken, pork, and beef meat, respectively.


## 3.1. Prevalence of Virulence-Associated Genes inA. butzleri andA. cryaerophilus Isolates

Screening of 79A. butzleri isolates revealed that all of them harboured thetlyA gene. Most of theA. butzleri isolates carriedciaB (97%),mviN (97%),pldA (92%),cadF (89%), andcj1349 (66%), whilehecB (48%),irgA (46%), andhecA (30%) genes were less frequent (Table 1).ciaB was found to occur with higher frequency in poultry if compared with pork (p
=
0.0007), whileirgA was more frequent in poultry than in beef (p
=
0.007).Table 1
Carriage of virulence genes byA. butzleri and A. cryaerophilus isolates.
Species
Origin/number of isolates
Percent of isolates harbouring virulence genes possessing specific gene
cadF
cj1349
ciaB
irgA
hecA
hecB
pldA
mviN
tlyA
A. butzleri
Chicken meat/58
90
71
100
53
34
48
93
98
100
Pork/10
90
60
80
40
10
60
100
90
100
Beef/11
82
45
100
9
27
36
82
100
100
Total/79
89
66
97
46
30
48
92
97
100
A. cryaerophilus
Chicken meat/2
0
0
100
0
0
0
0
0
0
Pork/1
100
0
100
0
0
0
0
0
0
Beef/3
0
0
100
0
0
0
0
100
100
Total/6
17
0
100
0
0
0
0
50
50Ten percent (8 out of 79) of theA. butzleri isolates harboured all nine genes, and most of them (7 isolates) derived from chicken meat. The carriage of 8 and 7 virulence-associated genes was reported in 44% (35 out of 79) ofA. butzleri isolates. Most of them were found in chicken meat (28 isolates), followed by pork (4 isolates) and beef (3 isolates). Seventeen (22%), 14 (18%), and 2 (3%)A. butzleri isolates possessed 6, 5, and 4 virulence-associated genes, respectively (Figure 1). The isolates harbouring 3 (2 isolates) and 2 genes (1 isolate) were only reported inA. butzleri from beef and pork, respectively.Figure 1
Dendrogram generated by UPGMA clustering based on PCR fingerprinting using ERIC primers performed on 79A. butzleri isolates. Repertoire of virulence-associated genes of each isolate was presented in left panel. Isolate origin was indicated as C, P, and B for chicken, pork, and beef meat, respectively.AllA. cryaerophilus isolates possessed theciaB gene, whilemviN andtlyA were detected in 3 out of the 6 isolates. Only one isolate carried thecadF gene.cj1349,hecA,hecB,irgA, andpldA genes were not detected in this population. All the 3 beef-derivedA. cryaerophilus isolates carriedciaB,mviN, andtlyA genes. TwoA. cryaerophilus isolates from chicken meat harbouredciaB gene only. The pork-derived isolate harbouredciaB andcadF genes (Table 1).


## 3.2. Genotypes ofA. butzleri andA. cryaerophilus

Seventy-four genotypes were distinguished within 79A. butzleri isolates (Figure 1). These isolates could be grouped into 8 clusters using 90% cut-off similarity. Nineteen from 21 isolates derived from beef and pork were found to be closely related toA. butzleri from chicken meat. Genotypes of 6 isolates from beef and 6 from pork shared >90% similarity to chicken isolates, when >80% similarity to chicken genotypes was found in 4 isolates from beef and 3 isolates from pork. Only two genotypes, one shared by beef (isolate 204) and one by pork isolate (isolate 60), were found to be distant from chicken genotypes. No relation betweenA. butzleri genotype and incidence of virulence genes could be found.Each of the 6A. cryaerophilus isolates was found to have unique genotype (Figure 2). Over 80% genotype similarity was only found within two beef and two porkA. cryaerophilus isolates but not between isolates from beef, pork, and chicken meat.Figure 2
Dendrogram generated by UPGMA clustering based on PCR fingerprinting using ERIC primers performed on 6A. cryaerophilus isolates. Repertoire of virulence-associated genes of each isolate was presented in left panel. Isolate origin was indicated as C, P, and B for chicken, pork, and beef meat, respectively.


## 4. Discussion

A. butzleri andA. cryaerophilus are considered as potential food-borne pathogens [28], but the knowledge on the presence of virulence-associated factors and genetic diversity of food-derived isolates is still limited. Thus, we examined the prevalence of virulence-associated genes inA. butzleri andA. cryaerophilus populations recovered from retail pork, beef, and chicken meat. ThetlyA,cj1349,mviN,pldA,cadF, andciaB genes were found to occur most frequently inA. butzleri isolates. Similar results were obtained by Collado et al. [18], who showed that at least 73% ofA. butzleri isolates from shellfish in Chile harbouredcadF,ciaB,mviN,pldA, andtlyA genes, although only 31% of the isolates possessedcj1349 gene. In turn, in Belgium [17], Germany [29], and Iran [21], these six genes were found in allA. butzleri isolates recovered from various sources, including meat. A high occurrence of these genes was also reported in Spain, where all isolates from meat, mussels, and sewage possessedciaB gene, when 92% of them harboredcadF andcj1349 genes [30]. Consistent to other reports, we showedhecA,hecB, andirgA genes to occur most rarely withinA. butzleri population [17, 18, 29, 30].Our results indicate that 14% ofA. butzleri isolates harboured all nine virulence-associated genes. These results are similar to that obtained by Douidah et al. [17], who found all these genes in 15% ofA. butzleri isolates, and Karadas et al. [29], who reported 13% ofA. butzleri isolates carrying all mentioned virulence-associated genes.Consistent with previous reports [18, 21, 30], we noted a higher incidence of virulence-associated genes inA. butzleri as compared toA. cryaerophilus. In our study, onlyciaB gene was detected in all the sixA. cryaerophilus isolates,mviN andtlyA were detected in three isolates, andcadF gene was detected in one isolate. A high frequency of virulence genes was observed in beef-derived isolates, since all the three isolates possessedciaB,mviN, andtlyA genes. A high incidence ofmviN (100%) andtlyA (62%) genes was already reported inA. cryaerophilus derived from cattle [21]. Collado et al. [18] showed thatmviN andtlyA genes commonly occur inA. cryaerophilus from molluscs, when Douidah et al. [17] reportedmviN gene to dominate inA. cryaerophilus from human and animals. In contrast toA. butzleri, noA. cryaerophilus isolates harboured multiple virulence genes, which is consistent with results obtained by other authors [17, 18, 21]. According to Douidah et al. [17], this difference could be explained by a different pathogenic behaviour of these species or due to a high heterogeneity of their genomes.A high diversity of virulence genes pattern was found inA. butzleri isolates from each source, exceptciaB andirgA genes found to be more frequent inA. butzleri from poultry.Relatively little is known on genetic structure ofArcobacter spp. population and even less on impact of genetic background on distribution of virulence-associated genes. Data obtained in this study demonstrate no relation between genotype and virulence genes repertoire in studiedArcobacter species. According to our data, even closely related isolates may carry different virulence genes. Similar observation was already made by Lehmann et al. [19] studyingArcobacter spp. genotypes from food in Germany.Irrespective of genotypic method used, a large genetic heterogeneity ofArcobacter spp. population is frequently reported [20, 31–33]. However, some reports point out that closely relatedArcobacter spp. genotypes can be isolated from some nonoverlapping sources like beef, lamb, pork, poultry, and fish meat [9, 19]. Our data also argue that genotypes closely related toA. butzleri from poultry meat can also be found within populations from pork and beef meat. InA. cryaerophilus isolates studied here, low genetic relatedness between pork, beef, and chicken meat isolates was noted, but general conclusions cannot be drawn here due to limited number of isolates.


## 5. Conclusions

Repertoire of virulence-associated genes inA. butzleri andA. cryaerophilus isolates from retail meat in Poland is similar to that reported in other countries. It seems that closely related genotypes can spread within pork, beef, and chicken meat populations ofA. butzleri but notA. cryaerophilus.



---

*Source: 956507-2015-10-11.xml*