Introduction

Campylobacter is considered as a principal cause of gastroenteritis in humans responsible for approximately 166 million diarrheal cases and 37,600 deaths per year globally.¹ In addition to gastrointestinal infections, campylobacteriosis is the best predisposing factor of Guillain-Barré syndrome, a serious demyelinating neuropathy, which roughly occurs in 3/10 000 cases of Campylobacter infections.² Thermotolerant Campylobacter which has a clinical significance due to the consumption of meat and meat products are C.jejuni and its closely connected C.coli represents more than 90% of human infections.³ The natural reservoirs of Campylobacter spp. are intestinal tracts of domesticated and wild birds and mammals. The consumption or the mishandling with raw or undercooked meat in particular poultry meat is considered to be the major risk factors for human campylobacteriosis.⁴ In general, self-limiting infection with Campylobacter does not necessitate therapeutic involvement, but in patients with severe cases or insufficiencies in the immunity response medical cure should be considered.² The drugs of choice used in the medical cure of campylobacteriosis are azithromycin, erythromycin, and ciprofloxacin.⁴ While tetracyclines could be considered an alternative option in the cure of Campylobacter infection.^4,5

Campylobacter becomes extra resistant to antibiotics and several strains sophisticated MDR to various medications.⁶ Multi-resistant Campylobacter exclusively against quinolones and erythromycin, has increased universally and has generated global alarms since these are the chief particles for the treatment of infection by Campylobacter.^{7, 8} Contaminated foods of animal origin with resistant Campylobacter strains to antibiotics harbour a significant hazard to public health.⁴

As the incidence of the infection has increased, there is an urgent need to take measures to identify the source of the bacterium.⁹ Biotyping methods aid in categorization of strains recovered from human, birds and on bird produces and permit to evaluate these strains at the species and subspecies points. Documentation of these strains offers researchers the capability toward analyze pathogenesis of contagions, perceive as well as scrutinize epidemics, support surveillance then avoidance infection in humans.⁹

In our country, poultry meat is measured as the best prevalent food in most residents and as far as we know, there are no available documents on the presence of Campylobacter spp. in Wasit poultry products, so the current study was undertaken to investigate the prevalence, antimicrobial resistance, and biotyping of C. jejuni and C. coli in two kinds of poultry products (chicken meat and turkey meat) which would assist in the microbiological and epidemiological evaluation of these vended products at consumer level in our markets.

Materials and Methods

Ethical Approval

Not essential for this style of study. Poultry meat were vended from the markets.

Collection and Treating of Poultry Meat

From August to December 2018, a total of 85 samples of poultry meat, comprising chicken thighs (n = 45) and turkey thighs (n = 40), were collected randomly from numerous superstores and retail supplies. The samples were collected in sterilized bag and transported to the laboratory with ice packets in 3 h. All samples were thawed in a refrigerator overnight and treated in 3 h.

Isolation and Identification of Campylobacter

Isolation of Campylobacter spp. was performed in accordance with the standard microbiological protocols,^10,11 with some modifications. Briefly samples were defrosted at 4°C for 18 h, then treated aseptically by weighing 20 g into a sterilized stomacher bag, and then 180 ml of Preston enrichment broth using the next preparation [Nutrient broth No.2 (Oxoid,CM0067); Campylobacter selective antibiotics (Oxoid,SR0204E); Campylobacter development supplement (Oxoid,SR0232E); lysed horse blood (Oxoid,SR0048C)] was added and stomached for 2 min. Following selective enhancement at 42°C for 18 h, 20 µl of enrichment broth was streaked on to plates of modified Charcoal Deoxycholate agar (mCCDA)(Oxoid,CM 739) enhanced with mCCDA antibiotic (Oxoid, SR155), and raised under microaerophilic condition (O₂ 5 %, CO₂ 10 %, N₂ 85 %) inside an anaerobic jar at 42°C for 72 h. Colonies demonstrating typical morphology of Campylobacter on mCCDA (greyish, smooth and moistened, spreading trend, film like transparent growth) were purified through culturing onto mCCDA agar base deprived of enhancement, then conserved in Trypton Soya Broth (Oxoid,CM0129) supplemented by 20% (v/v) pure glycerin at deep freezing.¹² Further identification based on biochemical reactions (wet mount slide test, oxidase activity and, microaerobic growing at diverse temperatures) was performed.^10,11 For the identification of thermotolerant Campylobacter, the bioMérieux API^® identification kit API CAMPY (BIOMERIEUX, 20800) was adopted. Biotyping of the isolates was performed using Lior scheme,¹³ based on rapid H₂S production in a semisolid agar complemented via using Campylobacter growing enhancement (Oxoid, SR 0232E), DNase test and, hippurrate hydrolysis test.¹³

Antibiotic Resistance of Thermotolerant Campylobacter

The disc diffusion technique on Mueller-Hinton agar (Oxoid, CM0337) complemented by 5% lysed horse blood (SR0048C), was used to detect the sensitivity to antibiotics in Campylobacter isolates.¹⁴ Interpretation of results was done according to the Clinical and Laboratory Standards Institute (CLSI).¹⁵ In summary, bacterial growth recovered from freezing cultures were grown on mCCDA base deprived of enhancement for 24 h at 42°C under microaerophilic conditions. A method was implemented in which the inoculum was made through direct suspending of isolated colonies in broth. This approach has been recommended to check demanding bacteria for instance Campylobacter.¹⁴ Sterilized swabs were exploited to uniformly distribute the inoculum on agar plates. The selected antimicrobials were nalidixic acid (ND) 30 μg, ciprofloxacin (CIP) 5 μg, erythromycin (E) 15 μg, tetracycline (T) 30 μg, gentamicin (GM) 10 μg, ofloxacin (OFL) 5 μg, oxacillin (OX) 1 μg and vancomycin (VAN) 30 μg.The petri dishes were raised in microaerobic conditions at 42° C overnight.¹⁴

Multiple Antibiotic Resistance (MAR index)

The MAR index of isolates was detected as the proportion between the numeral of multiple antibiotics to which the recovered isolates are resistant to the numeral of multiple antibiotics to which the specific isolates are exposed.¹⁶

Statistics

Data analysis were performed by MedCalc Software bvba version 18 (BE,USA). Two samples Chi-square (χ²) between proportions was used to compare significance between proportions with a 5% significant level https://www.medcalc.org/.

Results

Our study carried out to inspect the prevalence of thermotolerant Campylobacter in poultry meat vended in Wasit marketplaces. The results (Table 1) showed that the prevalence of thermotolerant Campylobacter in poultry meat was (63.5%) of those (68.5% and 31.5%) were identified as C.jejuni and C.coli respectively. Moreover, with regard to the Campylobacter species, chicken meat had the highest prevalence for C.jejuni (81.5%), whereas turkey meat had the highest prevalence for C.coli (44.4%). Statistically, there is no significant effect (p>0.05) on the prevalence of Campylobacter based on sample type (χ² = 0.508, p=0.476), but with regard to the Campylobacter species, there is a significant effect (p<0.05) on the prevalence of C.jejuni and C.coli based on sample type (χ² = 0.600, p=0.0102).

Table 1: Prevalence of Campylobacter spp. in poultry meat vended in Wasit markets.

n=number of positive samples, N= number of tested samples, %= percentage.

Antibiotic Resistance

The results (Table 2) showed that high proportion of the experienced isolates displayed resistance to OX,T,VAN and, E with prevalence of (94.4%, 85.2%,74.1% and 72.2%), respectively. While the resistance against fluoroquinolones (ND, CIP and, OFL) is moderate up to 50% by which C. coli isolates presented a high prevalence of resistance (up to 80%) than C. jejuni against these antibiotics. On the other hand, our results showed that GM had the lowest prevalence (29.6%) of resistance in the tested isolates. Moreover, based on Campylobacter species and regardless of the type of samples, our results presented great prevalence of resistance in C. coli than in C. jejuni for entirely scrutinized antibiotics (Figure 1), but taken into consideration the type of sample, our results publicized high prevalence of resistance against the screened antibiotics in turkey Campylobacter isolates than in chicken Campylobacter isolates (Figure 2). Statistically and according to the Campylobacter species (C. jejuni and C. coli), there is a significant effect (p <0.05) in the level of resistance observed only towards the CIP (χ² = 4.1143, p = 0.041). According to the type of sample, there is no significant effect (p> 0.05) for the sample type on the prevalence of resistance to the selected antibiotics (p = 0.175, 0.414, 0.414, 0.763, 0.448, 0.237, 0.556 and, 0.538) for (ND, CIP, OFL, E, T, GM, OX and, VAN), respectively.

Figure 1: Prevalence of resistance in Campylobacter isolates based on species Click here to View figure

Figure 2: Prevalence of resistance in Campylobacter isolates based on type of sample. Click here to View figure

Table 2: Prevalence of antibiotic resistance in Campylobacter recovered from poultry meat vended in Wasit markets.

n/N = The number of resistant isolates /the number of tested isolates, %= percentage

Antibiotic resistance patterns (ARP) and MAR index of C. jejuni and C. coli isolates were surveyed and the results are presented in (Table 3). The obtainable results showed that 51(94.4%) of the tested isolates displayed resistance to one or more antimicrobials by which these isolates demonstrated 15 ARP. The results also showed that the vast majority (90.7%) of the experienced isolates demonstrated MDR toward as a minimum three antibiotics with MDR of (NA CIP OFL E T GM OX VAN) is the chief resistance model which apparent in 27.8% of the tested isolates. One of the most important observations of this study is that the prevalence of MDR with seven and eight antibiotics is more common in turkey Campylobacter isolates, counting the MDR model (NA CIP OFL ET GM OX VAN) as the only model reported in 9 (16.7%) of 54 isolates.

Table 3: Antibiotic resistance patterns and MAR index of thermotolerant Campylobacter recovered from poultry meat vended in Wasit markets.

NA= nalidixic acid, CIP= ciprofloxacin, OFL= ofloxacin, E= erythromycin, T= tetracycline, GM= gentamycin, OX= oxacillin, VAN= vancomycin, MAR index= multiple- drug resistance index, n= number of resistant isolates, ARP= Antibiotic resistance patterns.

Additionally, prevalence of Campylobacter isolates recorded MAR index 0.1, 0.3, 0.4,0.5,0.6,0.8,0.9 and 1 were 3.7%,11.1%, 11.1%, 14.8%, 7.4%, 5.6%, 13% and 27.8%, respectively (Table 3).

Biotyping of Campylobacter

Biotyping of thermotolerant Campylobacter recovered from poultry meat was experienced, and the results offered in (Table 4). Campylobacter jejuni and C. coli isolates showed a broad prevalence of biotype I (70.2% and, 76.5%), respectively. While the prevalence of biotype II, III and IV in C. jejuni isolates was (16.2%, 8.1% and, 5.4%), respectively. In addition, four (23.5%) of 17 C. coli isolates displayed a prevalence of biotype II.

Table 4: Biotyping of thermotolerant Campylobacter recovered from poultry meat vended in Wasit markets.

n = number of positive isolates; N= number of tested isolates, %= percentage.

Discussion

Pollution of poultry carcasses and then poultry meat at market outlets chiefly occurred throughout evisceration as well through scalding.¹⁷ During treatment, the intestinal gut possibly leakage or rupture and the contents moved to the skin of the carcass, which delivers a proper environment aimed at the existence of Campylobacter spp. and later to cross contamination.¹⁸ In addition, under frozen or storing conditions at refrigerator temperature, Campylobacter spp. can still be recovered and continued in chicken meat.¹⁹ There are many opportunities for how this could happen. Though bacterial development is detained throughout low temperatures and part of them can be eliminated, but a proportion of these bacteria can persist or suffer serious injury,²⁰ and could be restored to become a viable but non-culturable pathogen (VBNC).²¹ This may perhaps be the reply towards why Campylobacter was recovered from frozen meat at current study. Unfortunately, if poultry meat taken towards marketplaces is previously polluted or tainted throughout the practices previously being stored at low temperatures, the likelihoods of Campylobacter to persevere are still there. Although food products in supermarkets appear to be highly sanitized, the practices previously packaging can be pathogen-infested and the packaging conveniences themselves may be in a reduced situation. In another word, there is truly an opportunity of Campylobacter arising in this category of foodstuff as a result of cross adulteration. Rob et al., found that the existing rate of C. jejuni is 15 times better at 2°C than at 20°C.²² So when we realize that chiller temperature is ranging from 4-16°C, so it possibly enhance the attendance of C.jejuni and C.coli then rise the opportunity of pollution.

The results of our study (Table 1) showed that 63.5% of poultry meat was positive for thermotolerant Campylobacter of those (68.5% and 31.5%) were identified as C.jejuni and C.coli, respectively. Similarly, Garin et al.,²³ and Kovalenko et al.,²⁴ detected Campylobacter in 65% and 60% of poultry samples, respectively. Moreover, they recovered C. jejuni from 48.3% of the surveyed samples, that is lower than 68.5% achieved in the current study. Additionally, MAĆKIW et al.,²⁵ in Poland and Chokboonmongkol et al.,²⁶ in Thailand detected Campylobacter in 51.7% and 51% of raw poultry meat and broiler skin samples, respectively which are closely comparable with isolation percentage acquired in the existing investigation.

Lower prevalence rates than that described in our investigation were earlier achieved by Mäesaar et al.,²⁷ in Estonian who found that 20.8% of the trade poultry meat presented positive results to Campylobacter by which C. jejuni and C. coli were found at a proportion of 43% and 13%, respectively. Awadallah et al.,²⁸ in Egypt recovered Campylobacter spp. from 25.9% and 47.5% of the inspected breast and thigh poultry samples, respectively.

The higher prevalence of this fastidious pathogen in our study may be a mirror to higher preliminary microbial counts and chromosomal variances among isolates which cause persistence and resistance to heat stress of Campylobacter spp. in food through storage.^29,30

The higher prevalence rate of Campylobacter spp. from poultry products than that reported in the current study was formerly described in Argentina (83%),¹⁸ UK (83.3%)³¹ and, Nigeria (81.9%).³² Additionally, previous studies conducted in Iraq found that the detection rates for Campylobacter spp. and C.jejuni in frozen chicken meat samples were (75% and 93.75 %) in provinces of Baghdad and Al-Muthanna, respectively.^33,34

Our results indicated a large prevalence of Campylobacter in turkey than in chicken meat (Table1).This finding was in accordance with Luangtongkum et al.,³⁵ who accounted prevalence rate of Campylobacter spp. as (83.1%) and (65.8%) in turkeys and broilers, respectively. This study also displayed that C. jejuni was more prevalent than C. coli which detected in 68.5% of the positive samples. Numerous scientists noted the higher presence of C. jejuni than C. coli as Taylor³⁶ in USA, Rajendran et al.,³⁷ in India, and Deckert et al.,³⁸ in Canada and Mikulić et al.,³⁹ in Croatia. Reverse that, several workers including Awadallah et al.,²⁸ Kanaan and Khashan,³³ Kurinčič et al.,⁴⁰ and Rawat et al.,⁴¹ reported the higher presence of C. coli than C. jejuni.

The variation in the prevalence of C. jejuni and C. coli could be due to the difference in the age of birds, the sampling season, the use of antibiotics, the variations in speciation approaches to discriminate these two pathogens and the incapability of some isolates to inhabit poultry have been shown to briefly choice for which of the two pathogens in some poultry herds.³⁶

In addition, our outcomes showed a greater prevalence of C.coli (44.4%) in turkeys compared to chicken meat. The possible explanation for this result may be related to the growth period of the turkeys (18 weeks) compared to the chickens (6-8 weeks) and when the turkey is more tolerable in unfavorable environments than the chicken, which makes it a suitable host for C.coli since this pathogen is more resistant to the critical conditions than C. jejuni.⁴² Furthermore, the use of β-lactams as growth supporters and the increase of antimicrobial resistance in C.coli may also be motivated by this prevalence.⁶ Notwithstanding this variance, it is essential to realize as an entire superiority that thermotolerant Campylobacter in particular C. jejuni and C. coli are measured vital mediators of diarrhea and that the infected meat of poultry is predictable as a chief source of infection.⁴³

It has been well documented that resistant Campylobacter was detected in animal species and in the food chain. The presence of resistant strains of Campylobacter to antibiotics in poultry may initiate its manifestation in poultry meat and their products, representing a threat to human wellbeing.⁴⁴

The high prevalence of resistance toward antibiotics possibly resulted from mishandling of antibiotics in the growth period of poultry, particularly as growth complements and to avoid contagions.⁴⁵

In the present study, Campylobacter isolated from retail poultry meat was highly resistant towards OX. In Thailand, 93% of Campylobacter isolates from vegetable farms and retail markets were found to be resistant to beta-lactams, which in accordance with our results.⁴⁶ This phenomenon may be related to the intrinsic resistance in Campylobacter to many beta-lactam drugs that make the use of these drugs not optimal, especially in severe infections.¹⁶

In the existing study, it has seen that Campylobacter isolates from retail poultry meat were highly resistant towards T, VAN and E. The expansive usage of T in human and veterinary medicines and as feed complements for poultry may be credited to the rise of high resistant organisms.⁴⁷ The selection of E resistance may be related to frequently usage of spiramycin for growth raise in poultry production.⁴⁸ Additionally, the multi-resistant bacteria inhabiting the poultry gut such as Enterococci spp. display resistance to several antibiotics through conveying numerous resistance genes, which can allocate resistance to Campylobacter.⁴⁸ Hence, poultry meat can be exposed to such resistant bacteria particularly vancomycin-resistant Enterococci (VRE). A study conducted in Turkey described that poultry meat was more frequently tainted with VRE among all samples of food with the prevalence of 57.1%.⁴⁹

Our results were in accordance with other studies that presented high resistance of Campylobacter towards T up to 82%, 66.2% and 57.6% in Thailand, USA and Poland, respectively.^46,50,51 High level of resistance in Campylobacter isolates recovered from retail markets and chicken meat against VAN and E up to 86.7% was previously reported.^16,52 On the other hand, these findings are contradicted with other studies that demonstrate low levels of resistance to E, T and beta- lactams up to (9.4%,40.6%, and 31.2%), respectively.^26,40 Throughout the breeding period, poultry was routinely in contact with antimicrobials such as enrofloxacin and sarafloxacin, which could explain the emergence of quinolone resistance.⁴⁸

Our results demonstrate a moderate resistance to quinolone that is consistent with previous USA result, they displayed that resistance to ND and CIP among Campylobacter isolates is up to (41% and 35%), respectively.⁵⁰ These results were contradictory with the Malaysian findings, they described that the resistance to enrofloxacin, norfloxacin and CIP among isolates of Campylobacter in retail markets is very low up to (1%).¹⁶ They attributed low resistance to these antibiotics to small-scale vegetable production. On the other hand, the high prevalence of resistance in Campylobacter isolated from retail poultry and from raw meat against quinolones ranged from (86.6% -99%) based on other investigators.^{51, 53} The use of untreated chicken dung as fertilizer has also been measured one of the factors contributing to the high resistance to quinolone in Campylobacter isolates.¹⁶

Apramycin has widely been used in a veterinary cure that may be related to the emergence of resistance to GM in Campylobacters.⁴⁸ Our results publicized a low level of resistance among Campylobacter isolates to GM up to 29.6%. This finding in agreement with previous results documented in Iraq and Malaysia that reported a similar level of resistance in Campylobacter isolated from broiler meat to GM up to 26.7% and 22.4%, respectively.^33,54 On the other hand, a very low level of resistance to GM ranged from (0- 2%) was previously acquired.^16,50,51

Based on our results, the resistance of these isolates varies relating to species of organisms and the origin of isolation with which the C. coli isolates display greater resistance proportions to the selected antibiotics than C. jejuni. On the other hand, isolates of Campylobacter from the turkey showed higher resistance rates than chicken isolates (Figure 1 and 2). This possibly linked to longer raising period for turkeys compared to chickens. Furthermore, since turkeys are further profitable than chickens, breeders are motivated to administer turkeys antibiotics for cure and avoidance of diseases and as growth complement.⁵⁰ These findings were in accordance with the previous results acquired in USA.⁵⁰

Multidrug resistance (MDR) was identified as an isolate demonstrating resistance toward at least two antimicrobials concurrently.⁵⁵ The evolving of multi-resistance perhaps reveal gaining of single or diverse resistance factors on the similar DNA particle, such as multidrug pumps, that specify efflux activity against diverse antimicrobials.⁵⁶ The modes of genetic resistance might be chromosomal or plasmid-borne, and represent a combination of endogenous and picked up genes.⁴⁵ The resistance to two or more classes of antimicrobials has been perceived by other researchers.^16,45,51,55 Overall, MDR phenomenon to seven and eight antibiotics tended to be more ubiquitous in turkey Campylobacter isolates. These findings were in accordance with other researchers.^50,55 The detection of MDR Campylobacter especially towards CIP, E and GM in poultry meat had generated worldwide alarms as these particles are generally utilized in cure of man infections with Campylobacter.⁵⁰

This study proposed that there are dissimilarities in husbandry practices used in the production period of these animals. This elucidates the dissimilarities in the MAR index between Campylobacter isolates found in poultry meat. As most antimicrobials administered through feed or water are not entirely absorbed in the intestine of the birds and up to 90% of the directed amount of particular drugs can be defecated in the faeces, so raw waste can be a vital resource of antimicrobial residues once utilized as fertilizer.⁵⁷ Thus, low MAR index would indicate that these isolates were recovered from meat were from low dangers of animal waste contamination.¹⁶ And when these products were imported from various countries and from various origins so the farmers in these countries might be implemented different husbandry practices that postulate the dissimilarities in MAR index fluctuating from (0.1-1).

The results of our study presented a large prevalence of C.jejuni and C.coli biotype I. The identification of this biotype as the principal biotype is compatible with the preceding results reported in Nigeria and South of Chile found a large prevalence of biotype I in C. jejuni and C. coli isolates recovered from poultry meat and dairy cattle up to (60% and 68%), respectively.^32,58 It is clear from our results that isolates of poultry meat in this study displayed high prevailing of biotype I that is nearer to results acquired from humans by some authors.^13,59 Thus when we realize that biotype I is the most public biotype in human and biotype II is public in animals, so we will recognize the postulated function of these products as reservoirs of contagion to man. This fact is an alarm for the public health inferences.

In conclusion our data demonstrated that most tested isolates presented resistance to E and/or CIP with increase resistance towards GM. And since the consumption of diseased poultry meat may account for most human campylobacteriosis cases, this information is alarming when realizing that these antibiotics are considered first–choice drugs for human infections. Our results proposed that the poultry industry could be the cause of a serious public health problem through the spread of pathogenic Campylobacter and resistance to antibiotics. These results highlight the necessity to monitor the occurrence and the MDR event of Campylobacter in animals, humans as well as in the food chain. Therefore, it is strongly recommended to implement particular control measures from farm to fork to enhance public fortification against campylobacteriosis.

Acknowledgments

This study did not receive any fund or grant. The manuscript was written and supported by the authors.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors declare they do not have any conflict of interest.

References

1. Oh E., Katelyn, Andrews J., & Jeon B. Enhanced Biofilm Formation by Ferrous and Ferric Iron Through Oxidative Stress in Campylobacter jejuni. Front Microbiol. 2018 ; 9 (1204): 1-9.
   CrossRef
2. Skarp C. P. A., Hänninen M. L., & Rautelin H. I. K. Campylobacteriosis: the role of poultry meat. Clin Microbiol Infect. 2016; 22(2): 103-109.
   CrossRef
3. Mikulić M., Humski A., Njari B., Ostović M., Duvnjak S.,& Cvetnić Z. Prevalence of Thermotolerant Campylobacter spp. in Chicken Meat in Croatia and Multi locus Sequence Typing of a Small Subset of Campylobacter jejuni and Campylobacter coli Isolates. Food Technol Biotechnol. 2016; 54(4): 475-481.
   CrossRef
4. Szczepanska B., Andrzejewska M., Spica D., & Klawe J. J. Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from children and environmental sources in urban and suburban areas. BMC Microbiol.2017; 17(80):1-9.
   CrossRef
5. Wimalarathna HM, Richardson JF, Lawson AJ, Elson R, Meldrum R, Little CL, Maiden M. C.J., McCarthy N. D., & Sheppard S. K. Widespread acquisition of antimicrobial resistance among Campylobacter isolates from UK retail poultry and evidence for clonal expansion of resistant lineages. BMC Microbiol. 2013; 13:160.
   CrossRef
6. Bouhamed R., Bouayad L., Messad S., Zenia S., Naïm M., & Hamdi T. M. Sources of contamination, prevalence, and antimicrobial resistance of thermophilic Campylobacter isolated from turkeys. Vet World. 2018; 11(8):1074.
   CrossRef
7. Mansouri N.L., Saleha A.A.,& Wai S.S. Prevalence of multidrug resistance Campylobacter jejuni and Campylobacter coli in chickens slaughtered in selected markets, Malaysia. Trop Biomed. 2012; 29 (2): 231-8.
8. Zhou J., Zhang M., Yang W., Fang Y., Wang G., & Hou F. A seventeen-year observation of the antimi­crobial susceptibility of clinical Campylobacter jejuni and the molecular mechanisms of erythromycin-resistant iso­lates in Beijing, China. Int. J. Infect. Dis.2016; 42 (1): 28-33.
   CrossRef
9. Eberle K.N., & Kiess A.S. Phenotypic and genotypic methods for typing Campylobacter jejuni and Campylobacter coli in poultry. Poult Sci. 2012; 91(1): 255-264.
   CrossRef
10. International Organization for Standardization (ISO). Microbiology of food and animal feeding stuffs – Horizontal method for the detection and enumeration of Campylobacter spp. Part 1: Detection method; Part 2: Colony count technique. Geneva 20, Switzerland: Central Secretariat, 1 rue de Varembé, Case Postale ; 2006. 56, CH-1211/10272-1 AND 10272-2.
11. World Organization for Animal Health (OIE).Campylobacter jejuni and Campylobacter coli. In Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris: international – standard – setting / terrestrial- manual; 2012. 7^th (1)1-8/ISBN978-92-9044-878-5/http://www.oie.int/en/standard-setting/terrestrial-manual. 2016.
12. Donnison A. Isolation of Thermotolerant Campylobacter. Review and Methods for New Zealand Laboratories. Client Report /Enteric Zoonotic Disease research in New Zealand/ Ministry of Health; 2003.25-26/ New Zealand Laboratories.
13. Lior H.E.R.M.Y. New, extended biotyping scheme for Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis. J Clin Microbiol.1984;20 (4),636-640.
14. Quinn P.J., Carter M.E., Markey B.,& Carter G.R. Clinical Veterinary Microbiology. 2^nd ed., Mosby Int., USA. R. (2000) J Agric Food Chem. 2004; 48 : 1155-1159.
15. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement. CLSI document M100-S25. Wayne: Clinical and Laboratory Standards Institute. 2015.
16. Tang J. Y. H., Khalid M. I., Aimi S., Abu-Bakar C. A., & Radu S. Antibiotic resistance profile and RAPD analysis of Campylobacter jejuni isolated from vegetables farms and retail markets. Asian Pac J Trop Biomed. 2016; 6 (1): 71-75.
    CrossRef
17. Ellis-Iversen J., Pritchard G. C., Wooldridge M., & Nielen M. Risk factors for Campylobacter jejuni and Campylobacter coli in young cattle on English and Welsh farms. Prev Vet Med. 2009; 88(1):42-48.
    CrossRef
18. Zbrun M. V., Romero-Scharpen A., Olivero C., Rossler E., Soto L. P., Rosmini M. R., Sequeira G.J., Signorini M.L., & Frizzo L. S. Occurrence of thermotolerant Campylobacter spp. at different stages of the poultry meat supply chain in Argentina. N Z Vet J. 2013; 61(6):337-343.
    CrossRef
19. Eideh A. A. M., & Al‐Qadiri H. M. Effect of refrigerated and frozen storage on the survival of Campylobacter jejuni in cooked chicken meat breast. J Food Sci. 2011; 76(1): M17-M21.
    CrossRef
20. Ilida M. N., & Faridah M. S. Prevalence of Campylobacter jejuni in chicken meat and chicken-based products. J. Trop. Agric. and Fd. Sc. 2012; 40(1): 63-9.
21. Thomas C., Hill D., & Mabey M. Culturability, Injury and Morphological Dynamics of Thermophilic Campylobacter spp. Within a Laboratory-based Aquatic Model System. J Appl Microbiol. 2002; 92(3) :433-442.
    CrossRef
22. Rob L., Andrew H., Peter C., & Gerhard N. Risk profile: Campylobacter jejuni/coli in poultry (whole and pieces). Report of New Zealand Food Safety Authority. 2003; 61.
23. Garin B., Gouali M., Wouafo M., Perchec A. M., Thu P. M., Ravaonindrina N., Urbès F., Gay M., Diawara A., Leclercq A., & Rocourt J. Prevalence, quantification and antimicrobial resistance of Campylobacter spp. on chicken neck-skins at points of slaughter in 5 major cities located on 4 continents. Int J Food Microbiol. 2012;157(1): 102-107.
    CrossRef
24. Kovalenko K., Roasto M., Liepinš E., Mäesaar M., & Hörman A. High occurrence of Campylobacter spp. in Latvian broiler chicken production. Food control. 2013; 29(1): 188-191.
    CrossRef
25. MAĆKIW E., Rzewuska K., STOŚ K., JAROSZ M., & Korsak D. Occurrence of Campylobacter spp. in poultry and poultry products for sale on the Polish retail market. J Food Prot. 2011; 74(6): 986-989.
    CrossRef
26. Chokboonmongkol C., Patchanee P., Gölz G., Zessin K. H., & Alter T. Prevalence, quantitative load, and antimicrobial resistance of Campylobacter spp. from broiler ceca and broiler skin samples in Thailand. Poult Sci. 2013; 92(2): 462-467.
    CrossRef
27. Mäesaar M., Praakle K., Meremäe K., Kramarenko T., Sõgel J., Viltrop A., Muutra K., Kovalenko K., Matt D., Hörman A., & Hänninen M. L. Prevalence and counts of Campylobacter spp. in poultry meat at retail level in Estonia. Food control. 2014; 44(2014): 72-77.
    CrossRef
28. Awadallah M. A. I., Ahmed H. A., El-Gedawy A. A., & Saad A. M. Molecular identification of C. jejuni and C. coli in chicken and humans, at Zagazig, Egypt, with reference to the survival of C. jejuni in chicken meat at refrigeration and freezing temperatures. INT FOOD RES J. 2014; 21(5): 1801-1812.
29. Pearson A., Greenwood M., Felthman R., Healing T., Donaldson J., Jones D., & Colwell R. Microbial ecology of Campylobacter jejuni in a United Kingdom chicken supply chain: intermittent common sources, vertical transmission, and amplification by flock propagation. Appl Environ Microbiol. 1996; 62(12): 4614-20.
30. Sampers I., Habib I., Berkvens D., Dumoulin A., Zutter L. D., & Uyttendaele M. Processing practices contributing to Campylobacter contamination in Belgian chicken meat preparations. Int J Food Microbiol.2008; 128(2): 297-303.
    CrossRef
31. Kramer J. M., Frost J. A., Bolton F. J., & Wareing D. R. Campylobacter contamination of raw meat and poultry at retail sale: identification of multiple types and comparison with isolates from human infection. J Food Prot. 2000; 63(12):1654-1659.
    CrossRef
32. Salihu M. D., Junaidu A. U., Magaji A. A., Abubakar M. B., Adamu A. Y., & Yakubu A. S. Prevalence of Campylobacter in poultry meat in Sokoto, North-western Nigeria. JPHE. 2009;1(2):041-045.
33. Kanaan M.H. & Khashan H.T. Prevalence of multidrug resistant thermotolerant species of Campylobacter in Retail Frozen Chicken meat in Baghdad Province. Curr Res Microbiol Biotechnol. 2018; 6 (1): 1431-1440.
34. Abd, M. T., & Al-Nasrawi H. A.. Identification of Campylobacter jejuni in poultry products by Real-Time PCR in Al-Muthanna province. AL-Qadisiya Journal of Vet. Med. Sci. 2015; 14 (2): 1-6.‏
35. Luangtongkum T., Morishita T. Y., Ison A. J., Huang S., McDermott P. F., & Zhang Q. Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry. Appl Environ Microbiol. 2006; 72(5): 3600-3607.
    CrossRef
36. Taylor W. J. Isolation, Antibiotic Resistance, and Molecular Characterization of Campylobacter from Poultry, Swine and Dairy Cows. Doctoral Dissertation University of Tennessee, Knoxville/USA.2012.
37. Rajendran P., Babji S., George A. T., Rajan D. P., Kang G., & Ajjampur S. S. Detection and species identification of Campylobacter in stool samples of children and animals from Vellore, south India. Indian J Med Microbiol. 2012; 30 (1): 85.
    CrossRef
38. Deckert A., Valdivieso-Garcia A., Reid-Smith R., Tamblyn S., Seliske P., Irwin R., Dewey C., Boerlin P., & McEwen S.A. Prevalence and antimicrobial resistance in Campylobacter spp. Isolated from retail chicken in two health units in Ontario. J Food Prot. 2010; 73(7):1317-1312.
    CrossRef
39. Mikulić M., Humski A., Njari B., Ostović M., Duvnjak S., & Cvetnić Ž. Prevalence of Thermotolerant Campylobacter spp. in Chicken Meat in Croatia and Multi locus Sequence Typing of a Small Subset of Campylobacter jejuni and Campylobacter coli Isolates. Food Technol Biotechnol. 2016; 54(4): 475-481.
    CrossRef
40. Kurinčič M., Berce I., Zorman T., & Smole Možina S. The prevalence of multiple antibiotic resistance in Campylobacter spp. from retail poultry meat. Food Technol Biotechnol. 2005;43(2):157-163.
41. Rawat N., Maansi- Kumar D., & Upadhyay A. K. Virulence typing and antibiotic susceptibility profiling of thermophilic Campylobacters isolated from poultry, animal, and human species. Vet World. 2018; 11(12):1698-1705.
    CrossRef
42. Tresierra-Ayala A., Ruiz R., Bendayan M., & Fernández H. Survival times of Campylobacter coli in sterilized buffalo milk. Zentralbl Veterinarmed B. 1999; 46 (2): 141-144.
    CrossRef
43. Fernández H. Thermotolerant Campylobacter species associated with human diarrhea in Latin America. J Braz Ass Adv Sci. (Ciência e Cultura). 1992; 44: 39-43.
44. Garcia-Migura L., Hendriksen R. S., Fraile L., & Aarestrup F. M. Antimicrobial resistance of zoonotic and commensal bacteria in Europe: the missing link between consumption and resistance in veterinary medicine. Vet Microbiol. 2014; 170 (1-2):1-9.
    CrossRef
45. Nguyen T. N. M., Hotzel H., Njeru J., Mwituria J., El‑Adawy H.,Tomaso H., Neubauer H., &Hafez H. M. Antimicrobial resistance of Campylobacter isolates from small scale and backyard chicken in Kenya, Gut Pathog. 2016;8 (39) : 1-9.
    CrossRef
46. Padungtod P., Kaneene J. B., Hanson R., Morita Y., & Boonmar S. Antimicrobial resistance in Campylobacter isolated from food animals and humans in northern Thailand. FEMS Immunol Med Microbiol. 2006;47(2): 217-225.
    CrossRef
47. Hassanain N. A. Antimicrobial Resistant Campylobacter jejuni Isolated from Humans and Animals in Egypt. Global Veterinarian. 2011; 6 (2):195-200.
48. Kanaan M. H. Antibacterial effect of ozonated water against methicillin-resistant Staphylococcus aureus contaminating chicken meat in Wasit Province, Iraq. Vet world. 2018; 11(10):1445.
    CrossRef
49. Elmal M., & Can H. Y. The prevalence, vancomycin resistance and virulence gene profiles of Enterococcus species recovered from different foods of animal origin. Vet Arhiv. 2018; 88(1):111-124.
    CrossRef
50. Ge B., Wang F., Sjoelund-Karlsson M., & McDermott P. F. Antimicrobial resistance in Campylobacter: susceptibility testing methods and resistance trends. J Microbiol Methods. 2013;95(1):57-67.
    CrossRef
51. Wieczorek K., Szewczyk R., & Osek J. Prevalence, antimicrobial resistance, and molecular characterization of Campylobacter jejuni and C. coli isolated from retail raw meat in Poland. Veterinarni Medicina. 2012;57(6): 293-299.
    CrossRef
52. Rodrigo S., Adesiyun A., Asgarali Z., & Swanston W. Antimicrobial resistance of Campylobacter spp. isolated from broilers in small poultry processing operations in Trinidad. Food Control. 2007;18(4):321-325.
    CrossRef
53. Qin S.S., Wu C.M. &Wang Y. Antimicrobial resistance in C. coli isolated from pigs in two provinces of China. Int J Food Microbiol.2011;146(1): 94-98.
    CrossRef
54. Saleha A. A. Isolation and characterization of Campylobacter jejuni from broiler chickens in Malaysia. Int J Poult Sci. 2002;1(4): 94-97.
    CrossRef
55. Thakur S., Zhao S., McDermott P. F., Harbottle H., Abbott J., English L., English L., Gebreyes W.A., & White D. G. Antimicrobial resistance, virulence, and genotypic profile comparison of Campylobacter jejuni and Campylobacter coli isolated from humans and retail meats. Foodborne Pathog Dis. 2010; 7(7):835-844.
    CrossRef
56. Levy S. B. Factors impacting on the problem of antibiotic resistance. J Antimicrob Chemother. 2002; 49(1): 25-30.
    CrossRef
57. Furtula V., Farrell E. G., Diarrassouba F., Rempel H., Pritchard J., & Diarra M. S.Veterinary pharmaceuticals and antibiotic resistance of Escherichia coli isolates in poultry litter from commercial farms and controlled feeding trials. Poult Sci. 2010;89(1): 180-188.
    CrossRef
58. Fernández H., & Hitschfeld M. Occurrence of Campylobacter jejuni and Campylobacter coli and their biotypes in beef and dairy cattle from the south of Chile. Braz J Microbiol. 2009;40(3):450-454.
    CrossRef
59. Pezzotti G., Serafin A., Luzzi I., Mioni R., Milan M., & Perin R. Occurrence and resistance to antibiotics of Campylobacter jejuni and Campylobacter coli in animals and meat in northeastern Italy. Int J Food Microbiol. 2003;82(3):281-287.
    CrossRef