First report of Aliarcobacter cryaerophilus in ready-to-cook chicken meat samples from super shops in Bangladesh

Objective: This study aimed to isolate Aliarcobacter cryaerophilus in ready-to-cook poultry meat in Bangladesh. Materials and Methods: Thirty drumstick samples were collected from super shops in Dhaka city (n = 10), Mymensingh city (n = 10), and Patuakhali town (n = 10). After sample processing, they were cultured in Blood agar media with Campylobacter base using a microfilter (0.42 nm). Suspected colonies were subjected to DNA extraction and PCR assay targeting 16SrRNA genes. Then, sequencing was performed for confirmation. Results: Of 30 samples, 3 (10%) were positive for A. cryaerophilus. Phylogenetic analysis shows that our isolate has strong similarities with one of the isolates from China. Conclusion: The presence of this organism in ready-to-cook poultry meat is a significant concern for consumers as it bears zoonotic importance.


Introduction
Aliarcobacter cryaerophilus (previously Arcobacter cryaerophilus) is a foodborne and zoonotic pathogen increasing globally. Aliarcobacter (formerly Arcobacter) is a genus of the family Campylobacteraceae [1,2]. They can be distinguished from Campylobacter sp. by their ability to grow at 15°C. This genus has grown its importance in recent years as its members are considered emergent enteropathogens. The Aliarcobacter genus comprises nine Gram-negative species, which include A. cryaerophilus, A. butzleri, A. cibarius, A. faecis, A. skirrowii, A. lanthieri, A. thereius, A. vitoriensis, and A. trophiarum [1,3,4]. Among these, A. cryaerophilus, A. butzleri, A. skirrowii are considered to be zoonotic and foodborne pathogens that could cause illness in humans. Aliarcobacter species are typically found in poultry, beef, pig, seafood, and aquatic habitats [4][5][6]. Some sources of contamination include animal and human waste, fertilizer runoff, sewage backups, and animal defecation [7]. And A. cryaerophilus has pathogenic effects on humans and animals, which may cause gastroenteritis, bacteremia, sepsis, mastitis, diarrhea, abortion, and reproductive disorders [8].
Till now, they have been found in food of animal origin, particularly in poultry, carcasses and offal, milk, and mussels, as well as in water bodies, sewage, and feces of many animal species. The presence of these organisms can be a significant threat to public health and a primary concern for food safety issues. However, minimal data are available on the prevalence of Aliarcobacters spp. in readyto-cook chicken meat. In addition, there is no standard procedure for the culture and isolation of Aliarcobacters spp. Therefore, this study aimed to isolate and characterize A. cryaerophilus based on the 16SrRNA gene from poultry meat (drumstick) samples.

Ethical approval
The protocols of this study were approved by the Animal Welfare and Experimentation Ethics Committee, Bangladesh Agricultural University, Mymensingh [approval number AWEEC/BAU/2020(36)].

Sample collection
Thirty frozen chicken meat (Drumstick) samples were collected randomly from selective supermarkets in Dhaka city (n = 10), Mymensingh city (n = 10), and Patuakhali town (n = 10) in Bangladesh. Aseptic methods were used to collect the meat samples, which were then transferred adequately to sterile containers. Cool chain was maintained to transport the samples to the Bacteriology Laboratory at the Department of Microbiology and Hygiene, BAU, Mymensingh.

Culture and staining
Aliarcobacter spp. was isolated using the filtration method [9]. Samples were prepared by separating 1 gm of each meat sample suspended in 900 μl of PBS. Blood agar bases, including Campylobacter supplements, were used for isolation, and a 0.45 um filter paper was placed on each agar plate. 100 μl of the suspension was spread onto the surface filters using the drop method, and the drops were permitted to stand for 30 min at room temperature. After 30 min, the filter was removed and incubated at 37°C for 48 h in the anaerobic jar. Selected colonies were subjected to Gram stain.

Molecular detection of Aliarcobacter spp. and nucleotide sequencing
Following the method described by Shahid et al. [10], DNA was extracted from the pure colonies. The genus of Aliarcobacter was confirmed using 16s rRNA gene primers by PCR, as described in the previous study [9].
The PCR reaction used the set of primers 16S9F (Antisense: 5'-GAG TTT GAT CCT GGC TC-3") and 16S1540R (Sense: 5'-AAG GAG GTG ATC CAG CC-3") to amplify a fragment of 1,530 base pairs. The composition of the mix for each reaction with 25 μl of the final volume was: 1 μl (20 pmol) of each primer, 2 ul of (100 ng/ul) of genomic DNA, and 2× of Go Taq Green Master Mix (Promega, USA). The amplification was performed at 47°C annealing temperature for 30 sec. After electrophoresis on 1.2% agarose gel, ethidium bromide staining was performed and subjected to gel documentation under UV light.

Phylogenetic analysis
One PCR product was analyzed by forward and reversed sequencing with a Sanger sequencing technology. Chromas version 2.5 and MEGA X software were used for sequence assembly. The sequence was compared with the GenBank database using the BLASTn [11]. Seven strains of A. cryoaerophilus and one A. skirrowii strain were selected based on percent identity for phylogenetic analysis. Multiple sequence alignment was performed in CLUSTALW [12].
Furthermore, the phylogenetic tree was constructed using the Neighbor-Joining method. The p-distance method was used for evolutionary distance, and the bootstrap value was 1,000 [13]. The nucleotide sequence generated in this study has been deposited in the GenBank.

Occurrence of A. cryaerophilus
Out of 30 chicken meat samples, 3 (10%) were culturally positive (Table 1). They formed grey, flat, and irregularly spreading colonies on Blood agar with Campylobacter agar base. Gram-negative curves were observed under the microscope (Fig. 1). Culturally positive samples were further subjected to PCR targeting 16SrRNA genes, and 1,530bp amplicons were found in gel electrophoresis (Fig. 1).

PCR and nucleotide sequencing
The PCR amplicons were visualized in 1.2% gel under UV light, and 1,530-bp band sizes were found (Fig. 1). After sequencing and completion of forward and reverse sequence alignment, a 1,449-bp length sequence was found for further study. Nucleotide BLAST was performed for sequence validation, and our query sequence produced significant alignment with available A. cryaerophilus ( Table 2) with substantial similarities.

Phylogenetic analysis
A total of eight sequences were downloaded from NCBI, including in-group and out-group, to construct a phylogenetic tree for our isolate ( Table 2). The neighbor-joining tree was constructed, and our sequence shares the same clade with two other isolates of A. cryoaerophilus belonging to China and New Zealand (Fig. 1).

Data availability
16SrRNA gene fragment sequence of A. cryaerophilus KHMN_BAU1 isolate is available in NCBI with the accession number OP748780.

Discussion
Aliarcobacter (previously Arcobacter) prevalence is noticeable in food, especially in poultry worldwide. This concern led us to conduct this study to ensure the presence of this organism in ready-to-cook chicken meat available in super shops in Bangladesh. This study has found a 10% occurrence of Aliarcobacter spp. in chicken meat purchased from super shops of selected regions in Bangladesh. Further, sequencing has confirmed the A. cryaerophilus. This is the first report of A. cryaerophilus in chicken meat(drumstick) in Bangladesh. This incidence rate supports the previous findings in Iran, where it was observed at 8.66% [14]. Besides, the prevalence of Aliarcobacter spp. in chicken meat was reported in India (58%) [15], Japan (60%) [16], Korea (45.8%) [17], and Malaysia (39.2%) [18]. The possible reason for this low prevalence might be the ready-to-cook chicken meat in supermarkets, and multiple washing steps reduce the contamination possibilities of these meats [19].
Approximately 90% of the broiler carcasses are contaminated with arcobacters, particularly with A. butzleri and A. cryaerophilus [20,21]. Several studies have been conducted worldwide, and most of them have reported the presence of this organism on the skin and the slaughter processing unit of poultry [22,23]. It is assumed that the contamination of A. cryaerophilus in ready-to-eat drumsticks might have occurred from the food processing unit or the carcass of those chickens, a significant public health concern and one health issue.
Culture, PCR, and sequencing have been used in this study to isolate and detect this organism. 16s rRNA gene-specific primer of Campylobacter sp. has been used as they share the same family [1,7]. It is challenging to differentiate Campylobacter and Aliarcobacter using cultural and genus-specific PCR assays. Thus, sequencing was performed for the confirmation of A. cryareophilus.
Phylogenetic analysis revealed that our isolate is closely related to isolates of China (CP099556.1). It is relatable that the organism can be derived directly from China as lots of food items and poultry shed utensils are imported every year [24].
Ready-to-cook poultry meat is considered the safest product based on its processing. However, A. cryaerophilus-contaminated meat can infect humans during handling and cooking, which may lead to self-limiting diarrhea [25]. Again, undercooked meat can lead to foodborne illness. Thus, a comprehensive study is required, especially from the slaughterhouse, including their surrounding environmental samples, to elucidate the transmission pattern of these bacteria. Moreover, the antimicrobial resistance profile of these bacteria should be studied to develop the prevention and control of this zoonotic genus.

Conclusion
Aliarcobacter cryaerophilus has been detected in readyto-cook poultry meat (Drumstick) for the first time in Bangladesh. It is suggested to adopt hygienic measures when handling and cooking such food items.