Abstract
Arcobacter is an emerging foodborne pathogen having zoonotic significance. Enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive sequence-based PCR (rep-PCR) analysis of a total of 41 Arcobacter isolates revealed a greater degree of genetic diversity. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power for ERIC and rep-PCR was found to be 0.997 and 0.996, respectively. Close clustering between isolates of animal and human origin are indicative of probable zoonotic significance.
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Keywords: Arcobacter, Discriminatory power, ERIC-PCR, Genetic diversity, rep-PCR
Arcobacter is an emerging food borne pathogen, of which A. butzleri, A. cryaerophilus and A. skirrowii have been frequently isolated from different livestock species, foods of animal origin as well as humans [1]. PCR based genotyping techniques such as enterobacterial repetitive intergenic consensus (ERIC)-PCR and repetitive sequence-based PCR (rep-PCR) have been successfully applied in various epidemiological studies for the characterization of genetic diversity of Arcobacters below species level [2–4]. The present study was carried out to assess the genetic diversity of 41 Arcobacter isolates (16 A. butzleri, 13 A. cryaerophilus and 12 A. skirrowii) recovered from diverse sources like faecal swabs of livestock (21), raw foods of animal origin (13) and diarrhoeic humans (7) in Andhra Pradesh (India). The reference strain of A. butzleri (ATCC 49616) as well as positive DNA standards of A. cryaerophilus and A. skirrowii used in the present study were obtained from Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, India.
ERIC-PCR fingerprinting was carried out using the primer pair ERIC-1 (5′-ATG TAA GCT CCT GGG GAT TCA C-3′) and ERIC-2 (5′-AAG TAA GTG ACT GGG GTG AGC G-3′) in 25 µl optimized reaction mixture under the following standardized thermal cycling conditions: initial denaturation at 95 °C for 5 min, 40 cycles of denaturation at 94 °C for 1 min, annealing at 25 °C for 1 min, elongation at 72 °C for 2 min, final elongation at 72 °C for 10 min [4]. Rep-PCR fingerprinting was carried out using single oligonucleotide primer (GTG)5 (5′-GTG GTG GTG GTG GTG-3′) in 25 μl optimized reaction mixture under the following standardized thermal cycling conditions: initial denaturation at 95 °C for 5 min, 40 cycles of denaturation at 94 °C for 45 s, annealing at 40 °C for 1 min, elongation at 65 °C for 10 min, final elongation at 65 °C for 20 min [2]. Gel electrophoresis was carried out and banding patterns were visualized using BIO-RAD Gel Documentation system (USA). Presence of a particular band in a strain was coded as 1 and the absence of that particular band was coded as 0 in a binary matrix. The binary data was analysed using dollop programme of phylip version 3.6 software and dendrograms were constructed. Numerical index of discrimination was calculated by Simpson’s index of diversity [5].
Of the 16 A. butzleri isolates analyzed, 14 ERIC-PCR patterns (E1 to E14) and 15 rep-PCR patterns (R1 to R15) were obtained. A pair of A. butzleri isolates that had identical ERIC-PCR pattern (E3) also had indistinguishable rep-PCR pattern (R3). However, a pair of A. butzleri isolates that had identical ERIC-PCR pattern (E7) were distinguished by rep-PCR into two different genotypes (R7 and R8). The two A. butzleri isolates sharing identical ERIC-PCR and rep-PCR pattern (E3 and R3) were recovered from chicken meat samples (CM 13 and CM 18) of different birds slaughtered in the same chicken retail shop, indicating the possibility of cross-contamination of carcasses during slaughter process. Cluster analysis of ERIC and rep-PCR profiles differentiated A. butzleri isolates from different sources into two clusters (cluster I, with isolates of human origin and cluster II, with isolates of animal origin) (Supplementary Fig. 1 and 2). However, under rep-PCR cluster analysis, an isolate recovered from cattle faeces clustered into human group and isolates from a pig farm worker and a diarrhoeic human clustered into animal group (Supplementary Fig. 2), indicating the possibility of zoonotic nature of Arcobacters.
Of the 13 A. cryaerophilus isolates analyzed, 13 ERIC-PCR patterns (E1 to E13) and 12 rep-PCR patterns (R1 to R12) were obtained. A pair of A. cryaerophilus isolates that had same rep-PCR pattern (R6) were distinguished by ERIC-PCR into two different genotypes (E6 to E7). Cluster analysis of ERIC and rep-PCR profiles differentiated A. cryaerophilus isolates from different sources into two clusters (cluster I, with isolates of foods of animal origin and cluster II, with isolates of animal faecal origin) (Supplementary Fig. 3 and 4). Of the 12 A. skirrowii isolates analyzed, 12 ERIC-PCR patterns (E1 to E12) and 11 rep-PCR patterns (R1 to R11) were obtained. A pair of A. skirrowii isolates that had same rep-PCR pattern (R7) were distinguished by ERIC-PCR into two different genotypes (E7 to E8). Cluster analysis of ERIC and rep-PCR profiles differentiated A. skirrowii isolates from different sources into three clusters (cluster I with isolates of sheep faecal origin and mutton samples, cluster II with isolates of pig faecal origin and cluster III with isolates of poultry faecal origin) (Supplementary Fig. 5 and 6).
A greater degree of heterogeneity was observed among 41 Arcobacter isolates from different sources as revealed by the presence of 39 and 38 different genotypes under ERIC-PCR and rep-PCR analysis, respectively (Table 1). In a study from Belgium using ERIC-PCR, 3, 81 and 4 genotypes were distinguished among 5, 164 and 13 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively [6]. Other studies reported 248 genotypes out of 275 A. butzleri isolates and 60 genotypes out of 63 A. cryaerophilus isolates [7] and 9 genotypes out of 10 Arcobacter isolates [8]. In the present study, the discriminatory power of the two typing methods was found almost identical i.e. 0.997 and 0.996, for ERIC-PCR and rep-PCR, respectively. This was in accordance with earlier studies [2, 3] and discriminatory powers above 0.90 are considered highly significant [5]. The present study reporting the genotyping and genetic diversity of Arcobacter species adds to the heterogeneity reports among Arcobacter species world-wide. The rep-PCR dendrogram analysis also indicated the genetic similarity among A. butzleri isolates recovered from livestock farm workers, diarrhoeic humans and healthy animals, revealing the possibility of zoonotic transmission.
Table 1.
Genetic diversity of Arcobacter isolates from different sources
| Species | Source | Number of isolates | Number of ERIC-types | Number of rep-types |
|---|---|---|---|---|
| Arcobacter butzleri (n = 16) | Poultry faeces | 2 | 2 | 2 |
| Pig faeces | 2 | 1 | 2 | |
| Cattle faeces | 1 | 1 | 1 | |
| Chicken meat | 2 | 1 | 1 | |
| Pork | 1 | 1 | 1 | |
| Milk | 1 | 1 | 1 | |
| Veterinary students | 2 | 2 | 2 | |
| Farm workers | 3 | 3 | 3 | |
| Diarrhoeic humans | 2 | 2 | 2 | |
| Total | 16 | 14 | 15 | |
| Arcobacter cryaerophilus (n = 13) | Poultry faeces | 2 | 2 | 2 |
| Pig faeces | 3 | 3 | 2 | |
| Cattle faeces | 1 | 1 | 1 | |
| Chicken meat | 3 | 3 | 3 | |
| Pork | 3 | 3 | 3 | |
| Milk | 1 | 1 | 1 | |
| Total | 13 | 13 | 12 | |
| Arcobacter skirrowii (n = 12) | Poultry faeces | 5 | 5 | 5 |
| Pig faeces | 3 | 3 | 2 | |
| Sheep faeces | 2 | 2 | 2 | |
| Mutton | 2 | 2 | 2 | |
| Total | 12 | 12 | 11 | |
| Grand total | 41 | 39 | 38 | |
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Acknowledgements
Thankful to Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh, for supporting this research.
Footnotes
Electronic supplementary material
The online version of this article (doi:10.1007/s12088-017-0649-1) contains supplementary material, which is available to authorized users.
REFERENCES
- 1.Collado L, Figueras MJ. Taxonomy, epidemiology, and clinical relevance of the genus Arcobacter. Clin Microbiol Rev. 2011;24:174–192. doi: 10.1128/CMR.00034-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Phasipol P, Chokesajjawatee N, Luangtongkum T. Comparison of repetitive sequence-based polymerase chain reaction (rep-PCR) and pulsed-field gel electrophoresis (PFGE) for genetic characterization of Arcobacter spp. Thai J Vet Med. 2013;43:307. [Google Scholar]
- 3.Ramees TP, Rathore RS, Bagalkot PS, Sailo B, Mohan HV, Kumar A, Dhama K, Singh RK. Genotyping and genetic diversity of Arcobacter butzleri and Arcobacter cryaerophilus isolated from different sources by using ERIC-PCR from India. Vet Q. 2014;34:211–217. doi: 10.1080/01652176.2014.979511. [DOI] [PubMed] [Google Scholar]
- 4.Houf K, De Zutter L, Van Hoof J, Vandamme P. Assessment of the genetic diversity among arcobacters isolated from poultry products by using two PCR-based typing methods. Appl Environ Microbiol. 2002;68:2172–2178. doi: 10.1128/AEM.68.5.2172-2178.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol. 1988;26:2465–2466. doi: 10.1128/jcm.26.11.2465-2466.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Van Driessche E, Houf K, Vangroenweghe F, De Zutter L, Van Hoof J. Prevalence, enumeration and strain variation of Arcobacter species in the faeces of healthy cattle in Belgium. Vet Microbiol. 2005;105:149–154. doi: 10.1016/j.vetmic.2004.11.002. [DOI] [PubMed] [Google Scholar]
- 7.Collado L, Kasimir G, Perez U, Bosch A, Pinto R, Saucedo G, Huguet JM, Figueras MJ. Occurrence and diversity of Arcobacter spp. along the Llobregat River catchment, at sewage effluents and in a drinking water treatment plant. Water Res. 2010;44:3696–3702. doi: 10.1016/j.watres.2010.04.002. [DOI] [PubMed] [Google Scholar]
- 8.Suelam IA. Isolation and identification of Arcobacter species recovered from rabbits in Zagazig, Egypt. Int J Microbiol Res. 2012;3:87–92. [Google Scholar]
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Supplementary Materials
Supplementary material 1 (TIFF 30544 kb)
Supplementary material 2 (TIFF 31405 kb)
Supplementary material 3 (TIFF 30564 kb)
Supplementary material 4 (TIFF 30625 kb)
Supplementary material 5 (TIFF 30149 kb)
Supplementary material 6 (TIFF 29778 kb)