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. 2021 Feb 10;16(2):e0246755. doi: 10.1371/journal.pone.0246755

Isolation, identification, and antimicrobial susceptibility pattern of Campylobacter jejuni and Campylobacter coli from cattle, goat, and chicken meats in Mekelle, Ethiopia

Yohans Hagos 1,#, Getachew Gugsa 2,*,#, Nesibu Awol 2,#, Meselu Ahmed 2,#, Yisehak Tsegaye 3,#, Nigus Abebe 3,#, Abrha Bsrat 3,#
Editor: Kumar Venkitanarayanan4
PMCID: PMC7875392  PMID: 33566816

Abstract

Campylobacter jejuni and Campylobacter coli are globally recognized as a major cause of bacterial foodborne gastroenteritis. A cross-sectional study was conducted from October 2015 to May 2016 in Mekelle city to isolate, identify, and estimate the prevalence of C. jejuni and C. coli in raw meat samples and to determine their antibiotic susceptibility pattern. A total of 384 raw meat samples were randomly collected from bovine (n = 210), goat (n = 108), and chicken (n = 66), and isolation and identification of Campylobacter spp. were performed using standard bacteriological techniques and PCR. Antibiotic susceptibility test was performed using disc diffusion method. Of the total 384 raw meat samples, 64 (16.67%) were found positive for Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (43.93%) followed by bovine meat (11.90%) and goat meat (9.25%). The most prevalent Campylobacter spp. isolated from meat samples was C. jejuni (81.25%). The overall prevalence of Campylobacter in restaurants, butcher shops, and abattoir was 43.93%, 18.30%, and 9.30%, respectively. 96.8%, 81.25%, 75%, and 71% of the Campylobacter spp. isolates were sensitive to norfloxacin, erythromycin, chloramphenicol, and sulphamethoxazole-trimethoprim, respectively. However, 96.9%, 85.9%, and 50% of the isolates were resistant to ampicillin, amoxicillin, and streptomycin, respectively. Strains that developed multi-drug resistant were 68.7%. The result of this study revealed the occurrence of Campylobacter in bovine, goat, and chicken meats. Hence, there is a chance of acquiring infection via consumption of raw or undercooked meat. Thus, implementation of hygienic practices from a slaughterhouse to the retailers, proper handling and cooking of foods of meat are very important in preventing Campylobacter infection.

Introduction

Foodborne diseases occur as a result of the consumption of contaminated foodstuffs especially from animal products such as meat from infected animals or carcasses contaminated with pathogenic bacteria [1, 2]. Food-producing animals are the major reservoirs for many foodborne pathogens such as Campylobacter species, non-Typhi serotypes of Salmonella enterica, Shiga toxin-producing strains of Escherichia coli, and Listeria monocytogenes. Foodborne pathogens cause millions of cases of sporadic illness and chronic complications, as well as large and challenging outbreaks in many countries and between countries [3].

Worldwide, pathogenic Campylobacter species are the leading cause of bacterial-derived foodborne disease and are responsible for the cause of over 400–500 million infections cases each year [4–6]. Campylobacter species are normally carried in the intestinal tracts of many domestic livestock such as poultry, cattle, sheep, goat, pigs, as well as wild animals and birds [7–10]. Fecal matter is a major source of contamination and could reach carcasses through direct deposition [11]. Animal food products can become contaminated by this pathogen during slaughtering and carcass dressing [12]. Humans are infected by ingestion of undercooked or decontaminated meat, or handling of raw products or cross-contamination of raw to cooked foods, swimming in natural waters, direct contact with contaminated animals or animal carcasses, and traveling [13–15].

Pathogenic Campylobacter spp. known to be implicated in human infections include C. jejuni, C. concisus, C. rectus, C. hyointestinalis, C. insulaenigrae, C. sputorum, C. helveticus, C. lari, C. fetus, C. mucosalis, C. coli, C. upsaliensis, and C. ureolyticus [6]. Of these,C.jejuniand C.coli are considered the most commonly reported zoonosis in humans and recognized as the most common causative agents of bacterial gastroenteritis in the world [16–19].

Moreover, Campylobacter with resistance to antimicrobial agents has also been implicated worldwide [4, 20, 21]. The use of antimicrobial agents in food animals has resulted in the emergence and dissemination of antimicrobial-resistant bacteria including antimicrobial-resistant Campylobacter, which has a potentially serious impact on food safety in both animal and human health. The situation seems to deteriorate more rapidly in developing countries where there is widespread and uncontrolled use of antibiotics [22].

In Ethiopia, few studies were conducted on the prevalence and antimicrobial susceptibility of enteric Campylobacteriosis of human beings [23–25] and food of animal origins [9, 20, 26–28]. The absence of a national surveillance program, limited routine culture availability for the isolation of Campylobacter spp. in clinical and research settings, and the need for selective media and unique growth atmosphere make it difficult to give an accurate picture of the burden of the disease in Ethiopia. This fact indicates that Campylobacter as a zoonosis is not given appropriate weight and consideration, particularly in the current study area. As a result, the objectives of this study were to isolate and identify C. jejuni and C. coli from the meat of cattle, goat, and chicken collected from an abattoir, butcher shops, and restaurants in Mekelle City, estimate their prevalence and determine the antibiotic susceptibility pattern of C.jejuniand C. coli isolates.

Material and methods

Ethics approval

This study was reviewed and approved by the Research Ethics Committee of the College of Veterinary Sciences, Mekelle University.

Study area

The study was conducted from October 2015 to May 2016 at an abattoir, butcher shops, and restaurants of Mekelle City. Mekelle is the capital city of Tigray National Regional State of Ethiopia where thousands of cattle and goats are accessible from different districts of the region and the neighboring regions of the country for slaughter. Mekelle is found at 39°29’ East and 13°30’ North of the equator which is 783 kilometers away from Addis Ababa, which is the capital city of Ethiopia. The altitude of the area ranges from 2000–2200 meters above sea level. The mean annual rainfall of the area is 628.8 mm and an annual average temperature of 21°C. The city has seven sub-cities and a total population of 215,546 [29], 308 cafeterias, 292 restaurants, 258 supermarkets, and an active urban-rural exchange of goods which has 30000 micro-and small enterprises [30].

Study design

A cross-sectional study was employed from October 2015 to May 2016 to isolate, identify, and estimate the prevalence and antibiotic susceptibility patterns of C.jejuni and C. coli from bovine, goat, and chicken meat samples collected from the abattoir, butcher shops, and restaurants.

Sample size and sample collection

A total of 384 raw ready-to-eat meat samples comprising of cattle (n = 210), goat (n = 108),and chicken (n = 66) meats were collected from the abattoir (n = 258), butcher shops (n = 60), and restaurants (n = 66) of the study area. All samples were placed in polyethylene plastic bags to prevent spilling and cross-contamination and immediately transported to the Molecular Biology Laboratory of the College of Veterinary Sciences, Mekelle University using an icebox with ice packs.

Bacteriological isolation and identification of Campylobacter species

Approximately 10 grams of raw meat sample was aseptically collected using sterile forceps and scissor and placed into 90ml of buffered peptone water in a sterile plastic bag and homogenized for 1 minute using a stomacher (Lab Blender 400, Seward Medical, London, England)and incubated at 37°C for 48h in the microaerophilic atmosphere (gas mix of 5% O2, 10% CO2, and 85% N2). Then a 0.1ml of the enriched sample was streaked onto Karmali Campylobacter Agar Base (HiMedia Laboratories, Mumbai, India)(Blood free Campylobacter selective agar base medium containing Campylobacter selective supplement comprising cefoperazone, amphotericin B (CCDA selective supplement SR0155E)) [31] and kept in a gas jar containing Campylobacter gas pack systems to maintain the microaerophilic condition and was incubated at a temperature of 37°C for 48h. The colonies were provisionally identified based on staining reaction with Gram’s stain, cellular morphology [32], catalase test, and oxidase test [33], and growth appearance on 5% sheep blood agar (Oxoid Ltd., Basingstoke, Hampshire, England) at 37°C after 24 h [34].

All the thermophilic Campylobacters isolates were tested for Hippurate hydrolysis, H2S production, and susceptibility to Nalidixic acid and Cephalothin as proposed by [34]. Susceptibility tests to Nalidixic acid (30 μg) and Cephalothin (30 μg) were performed using the standard agar disc diffusion method as recommended by Clinical and Laboratory Standards Institutions (CLSI) and isolates were categorized as sensitive and/or resistant according to the interpretation table of the [35]. The presumed Campylobacter isolates were preserved in brain heart infusion broth supplemented with15% glycerol in Eppendorf tubes at -20°C for further analysis. Bacterial strains that were used as quality control organisms in this study were standard strains of S. aureus, S. agalactiae, and E. coli obtained from the National Veterinary Institute (NVI), Debre-Zeit.

Polymerase chain reaction for detection of mapA and ceuE genes

The genomic DNA of the pheno typically resembled isolates of Campylobacter was extracted using the Phenol Chloroform method (Phenol: Chloroform: Isoamyl alcohol mixture (24:25:1)) according to [36]. Then 20μl of each extracted genomic DNA sample was run in an agarose gel electrophoresis and visualized under UV-light gel doc. Then after, a genome-based polymerase chain reaction (PCR) was done as described by [37] using the following species-specific primers: F-5’CTA TTT TAT TTT TGA GTG CTT GTG3’ and R-5’GCT TTA TTT GCC ATTT GTT TTA TTA3’ was used to amplify the mapA gene of C. jejuni(589 bp), and F-5’ATT TGA AAA TTG CTC CAA CTA TG3’ and R-5’TGA TTT TAT TAT TTG TAG CAG CG3’ were used to amplify the ceuE gene of C. coli(462 bp). Each PCR reaction mixture was performed in a 50μl total volume containing 10μl of template DNA,5μl of 5X PC buffer, 5μl of MgCl2, 1μl of each of the primers, 0.75μl of 10 mM of each dNTPs, 0.15μl of Taq DNA polymerase, and27.1μl nuclease-free distilled water. Amplification was carried out with thermal cycling conditions of an initial denaturation at 95°C for 5 min followed by 45 cycles of denaturation at 94°C for 35s, annealing at 54°C for 35s and extension at 72°C for 35s, and with a final extension at 72°C for 6 min. Finally, the PCR products were separated by running on a 1.5% (w/v) agarose gel containing 0.3mg/ml ethidium bromide. Electrophoresis was conducted in a horizontal equipment system for 120 min at 90 V using 1X TAE buffer (40 mM Tris, 1 mM EDTA, and 20 mM glacial acetic acid, pH 8.0). The amplicons were visualized under UV-light gel doc and their molecular weights were estimated by comparing with100bp DNA molecular weight marker(Solis BioDyne, Tartu, Estonia).

Antimicrobial susceptibility testing

The Campylobacter spp. isolates were screened for in vitro antimicrobial susceptibility using the standard agar disc diffusion method as recommended by Clinical and Laboratory Standards Institutions (CLSI) on Mueller-Hinton agar supplemented with 5% sheep blood (Oxoid Ltd., Basingstoke, Hampshire, England). The following nine different antibiotic discs, with their concentrations given in parentheses, were used in the antibiogram testing: Amoxicillin (AML)(10μg), Ampicillin (AMP)(10μg), Chloramphenicol (C)(30μg), Erythromycin (E)(15μg), Gentamycin (CN)(10μg), Norfloxacin (NOR)(10μg), Streptomycin (S)(10μg), Tetracycline (TE)(30μg), and Sulfamethoxazole-trimethoprim (SXT)(25μg) (Oxoid Company, Hampshire, England). After 48h of microaerophilic incubation at 37°C, the clear zones (inhibition zones of bacterial growth around the antibiotic discs(including the discs)diameter for individual antimicrobial agents were measured and then translated into Sensitive (S), Intermediate (I), and Resistant (R) categories according to the interpretation table of the Clinical and Laboratory Standard Institute [35].

Data storage and statistical analysis

All collected data were entered into Microsoft Excel Sheet (Microsoft Corp., Redmond, WA, USA) and analyzed using SPSS version 20 statistical computer software program. Chi-square (χ2) test and Logistic regression were applied to assess the associations. For all tests, a p-value of less than 0.05 was considered statistically significant.

Results

The overall prevalence of Campylobacter species

Out of the total of 384 collected meat samples, 64(16.67%) were positive for the two Campylobacter spp. The highest (43.93%) and lowest (9.25%) prevalence of Campylobacter spp. were recorded in samples taken from chicken (found to be 7.68 times more likely to have Campylobacter contamination compared to other sample types) and goat, respectively. Whereas, the highest (43.93%) and lowest (9.25%) prevalence were recorded in meat samples collected from restaurants (found to be four times more likely to have Campylobacter contamination compared to other sample sources) and abattoir, respectively. Both sample types and sources had significant differences (p = 0.00; χ2 = 43.04or OR = 7.68, CI = 3.40–17.30,and p = 0.00; χ2 = 45.53 or OR = 7.64, CI = 4.01–14.52, respectively) in the prevalence of the two Campylobacter spp. as it is shown in Tables 1 and 2 below.

Table 1. Prevalence of Campylobacter spp. among different sample types and sources.

Risk Factors No. of Examined No. of Positive (%) χ2 P-value
Type of meat
Goat meat 108 10(9.25) 43.04 0.000
Cattle meat 210 25(11.90)
Chicken meat 66 29(43.93)
Total 384 64(16.67)
Sources of meat
Abattoir 258 24(9.30) 45.53 0.000
Butcher 60 11(18.33)
Restaurants 66 29(43.93)
Total 384 64(16.67)
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Table 2. Logistic regression analysis results of sample types and sample sources.

Types of sample No of examined No of positive (%) OR(95%CI) P-value
Goat meat* 108 10(9.25) 1
Cattle meat 210 25(11.90) 1.32(0.61–2.86) 0.476
Chicken meat 66 29(43.93) 7.68(3.40–17.30) 0.000
Total 384 64(16.67)
Abattoir* 258 24(9.30) 1
Butcher 60 11(18.33) 2.18(1.00–4.76) 0.048
Restaurants 66 29(43.93) 7.64(4.01–14.52) 0.000
Total 384 64(16.67)
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OR = Odd ratio; CI = Confidence interval.

Contamination rate of C.jejuni and C. coli in the different sample types

Of the two Campylobacter spp. isolated and identified from cattle, goat, and chicken meat samples C. jejuni and C. coli accounted for 81.25% and 18.75%, respectively. The prevalence of C.jejuni and C.coli in cattle, goat, and chicken meat samples were found to be 76% and 24%, 80% and 20%, and 86.21%and 13.79%, respectively (Table 3).

Table 3. The contamination rate of C. jejuni and C. coli among different sample types.

Sample Type Prevalence Campylobacter spp.
C.jejuni C.coli
Cattle meat (n = 25) 19(76%) 6(24%)
Goat meat (n = 10) 8(80%) 2(20%)
Chicken meat (n = 29) 25(86.21%) 4(13.79%)
Total (n = 64) 52(81.25%) 12(18.75%)
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PCR amplification results of Campylobacter spp. isolates

Besides the phenotypic characterization, PCR amplification of the 64 samples revealed that 52(81.25%) of the isolates were C.jejuni(having a molecular weight of 589 bp) and the remaining 12(18.75%) isolates were C.coli (having a molecular weight of462 bp) to the targeted genes.

Antimicrobial susceptibility pattern of Campylobacter spp. isolates

Campylobacter spp. isolated from the different sample types and sources were susceptible to Norfloxacin (96.8%), Erythromycin (81.25%), Chloramphenicol (75%), and Gentamycin (75%). However, the isolates had shown resistance to Ampicillin (96.9%) and Amoxicillin (85.9%) (Table 4). Moreover, 96.8% of the isolates developed resistance for two or more than two drugs as it is shown in Fig 1.

Table 4. In vitro antimicrobial sensitivity pattern of Campylobacter spp. isolates.

Type of antibiotics Interpretations
Susceptible (%) Intermediate (%) Resistant (%)
Ampicillin 0(0) 2(3.1) 62(96.9)
Amoxicillin 3(4.7) 6(9.4) 55(85.9)
Chloramphenicol 48(75) 5(7.8) 11(17.2)
Erythromycin 52(81.25) 1(1.6) 11(17.2)
Gentamycin 48(75) 8(12.5) 8(12.5)
Norfloxacin 62(96.8) 1(1.6) 1(1.6)
Streptomycin 25(39.06) 7(10.9) 32(50)
Tetracycline 42(65.6) 6(9.4) 16(25)
Sulfamethoxazole-Trimethoprim 46(71.8) 4(6.25) 14(21.9)
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Fig 1. Percentage of drug resistance of Campylobacter spp.

Fig 1

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Species based antibiogram result of the isolates revealed that the highest level of sensitivity of both C. jejuni (98.1%) and C. coli (91.7%)was observed against Norfloxacin. Whereas both C.jejuni(96.2%) and C. coli(100%) were showed the highest level of resistance against Ampicillin as it is shown in Table 5.

Table 5. In vitro antimicrobial sensitivity pattern of C. jejuni and C. coli isolates.

Antibiotic Discs Interpretations
C. jejuni (N = 52) C. coli (N = 12)
S (%) I (%) R (%) S (%) I (%) R (%)
Ampicillin - 2(3.8) 50(96.2) - - 12(100)
Amoxicillin 3(5.8) 4(7.7) 45(86.5) - 2(16.7) 10(83.3)
Chloramphenicol 40(76.9) 4(7.7) 8(15.4) 8(66.7) 1(8.3) 3(25)
Erythromycin 43(82.7) - 9(17.3) 9(75) 1(8.3) 2(16.7)
Gentamycin 41(78.8) 6(11.5) 5(9.6) 7(58.3) 2(16.7) 3(25)
Norfloxacin 51(98.1) - 1(1.9) 11(91.7) 1(8.3) -
Streptomycin 22(42.5) 7(13.5) 23(44.2) 3(33.3) - 9(66.7)
Tetracycline 38(73.1) 6(11.5) 8(15.4) 4(41.7) - 8(58.3)
Sulfamethoxazole- Trimethoprim 39(75.5) 3(5.8) 10(19.2) 7(58.3) 1(8.3) 4(33.3)
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S = Susceptible, I = Intermediate, R = Resistant.

Discussion

In the current study, the overall prevalence of Campylobacter spp. was found to be 16.67%. The highest prevalence was found in chicken meat samples (43.93%). Chicken meats were found to be 7.68 times more likely to have Campylobacter when compared to goat and cattle meat. The difference in the prevalence of Campylobacter between different types of meat samples was found to be statistically significant (p<0.05) (OR = 7.68, CI = 3.40–17.30). The prevalence of Campylobacter spp. in chicken meat samples was 43.93% which was comparable with those reported by [38], 44%, [39], 56.1%,and [40], 48.02% in Iran. This was higher than the report of [41] who reported a prevalence of 1.93%. However, the present finding was lower than studies conducted by [42–44], and [45] who reported the prevalence of 61.7% and 70.7% and 65% and 81.3% of Campylobacter spp. in Ahvaz, Iran; Washington; Northern Ireland, and Northern Italy, respectively. It is a well-known fact that poultry appeared to be a significant source of Campylobacter and chicken was found to be heavily intestinal carriers of Campylobacter when compared with other food animals [8]. Wide variation (0–90%) in the prevalence of Campylobacter in fresh poultry meat had been reported in different countries [46–48]. These variations in Campylobacter spp. prevalence might be due to differences in hygienic conditions, cross-contamination that may occur during de-feathering, eviscerating, and some other environmental factor such as the temperature of water in the scalding tank.

In this study, the prevalence of Campylobacter spp. in bovine meat was 11.90%. This was comparable to the finding reported from a previous study done by [10] (12.9%) in Nigeria, and [49] (10%) in Iran. However, it was higher than the findings reported by [9] (6.2%) in Ethiopia [50]; (5.6%) in Morogoro, Tanzania; and [51] (0.8%)in Australia. Food of animal origin has been incriminated for being the main source of Campylobacter infection in humans [52]. Since raw meat from beef is widely consumed in the country; the occurrence of Campylobacter in meat increases the likelihood of the pathogen transmission to humans. The present finding was lower than studies conducted by [10] and [53] who reported a prevalence of 69.1% and 22%, respectively. One of the most likely hypotheses to explain the discrepancies is the differences in protocols used for the detection of thermophilic Campylobacter, and especially the absence of an enrichment step for the isolation of thermophilic Campylobacter in [54] work. In general, these variations might be due to approaches of sample collection, the difference in isolation and identification techniques, and differences in sample size.

The prevalence of Campylobacter spp. in goat meat was found to be 9.25%. This finding was in agreement with the reports of [9] and [55], who reported 7.6% and 6.4%, respectively. But it slightly higher than the report of [42] who reported 4.4%. However, the present study was lower than the findings of [5] and [26] who reported 41.2% and 27.5%, respectively.

The meat samples collected from restaurants had the highest prevalence (43.93%). The meat samples collected from restaurants were found to be four times more likely to have Campylobacter compared to meat collected from Butcher and eight times more likely to have Campylobacter compared to meat collected from the abattoir. The difference in the prevalence of Campylobacter between sources of meat samples was found to be statistically significant (P<0.05) (OR = 7.64, CI = 4.01–14.52). This might be due to an extra chance of acquiring contamination from individuals who are working in restaurants during handling or cross-contamination among different carcasses.

In the current study, the bacteriological and PCR characterization of Campylobacter isolates revealed that the prevalence of C.jejuni was higher than C.coli. C. jejuni has been reported to be the most frequent species recovered from the food of animal origin especially chicken meat [48, 56–58]. The prevalence of C. jejuni and C. coli in bovine, goat, and chicken meat were found to be 76% and 24%; 80% and 20%; and 86.3% and 13.7%, respectively. These findings were in agreement with the findings of [9] who reported 78% C.jejuni and 18% C.coli [28]; who reported 78% C. jejuni and 22% C.coli [27]; who reported 93.3% C. jejuni and 6.7% C. coli [26]; who reported 72.5% C.jejuni and 27.5% C. coli in Ethiopia. The prevalence of C.jejuni in raw meat was in agreement with the reports from other countries [48, 57, 59, 60].

Antibiotic resistance in Campylobacter is emerging globally and has already been described by several authors and recognized by the WHO, as a problem of public health importance [61–63]. Campylobacter spp. resistance to antibiotics (C.jejuni and C.coli) can be transferred from different sources to humans. This situation, alarmingly, announces the need to perform an antimicrobial sensitivity test for Campylobacter. Macrolides and Fluoroquinolones are usually considered the drugs of choice for the treatment of foodborne Campylobacteriosis [64–66]. Antibiotic susceptibility patterns have been determined in previous studies conducted in Ethiopia where the 80%-100% of isolates from food animals were sensitive to these antimicrobial agents [9, 20]. However, there are pieces of evidence from different parts of the world that antimicrobial resistance in food animals and human isolates is increasing.

In the current study, 52 C.jejuni and 12 C.coli isolates were investigated for their antimicrobial susceptibility pattern. The percentage of ampicillin and amoxicillin resistant Campylobacter isolates were 96.9% and 85.9%, respectively. This was in agreement with the report of [28], who reported 97.2% and 83.3% for ampicillin and amoxicillin, respectively. Moreover, [67] reported a resistance level of 100% for C. coli. On the other hand, C. coli isolates are generally more resistant than C. jejuni strains [68]. In general, several studies have reported resistance to beta-lactam antibiotics is high in food animals [69–71]. The resistance rate of Campylobacter isolates (25%) to tetracycline in the present study was comparable with the findings of [27] (20.8%)but higher than that of [9] (10%) and [72] (6%). However, it was lower than the report of [73] (77.94%). The resistance level to streptomycin in the current study was 50%, which was higher than reports from Ireland and Thailand by [73] and [74].

Multi-drug resistance isolates always remained susceptible to norfloxacin and erythromycin and chloramphenicol. In the present study, multi-drug resistance to more than two antimicrobial agents was 68.7% which was comparable to the findings of [69] in Belgium, 60% [75]; in Estonia, 60% [49]; in Iran, 75%;and [76] in Korea, 93.4%. However, the current multidrug resistance finding was higher than the report from Addis Ababa and Debre Zeit, Ethiopia, by [9] (20%). Despite global commitments to reduce antimicrobial resistance and protect the effectiveness of antimicrobials, most countries have not yet started implementing government policies to reduce their overuse and misuse of antimicrobials [77]. Hence, the current multidrug resistance finding might be since antibiotics can be bought for human or animal use without a prescription, and similarly, in countries like Ethiopia without standard regulation and treatment guidelines, antibiotics are often over-prescribed by health workers and veterinarians and over-used by the public. Moreover, new resistance mechanisms are emerging and spreading globally. Hence, antibiotic resistance is rising to dangerously high levels in all parts of the world.

Conclusion and recommendations

The present study revealed the occurrence of Campylobacter in bovine, goat, and chicken raw meat samples collected from different sites of the study area. Hence, they can serve as a potential vehicle for transmitting Campylobacter spp. and risk of infection to humans through the consumption of raw or undercooked meat. Therefore, retailers can act as a major source of cross-contamination. Moreover, the current study was revealed the development of antimicrobial resistance by the isolated Campylobacter spp. for certain drugs which is an alert for the concerned bodies. Hence, coordinated actions are needed to reduce or eliminate the risks posed by these pathogens at various stages in the food chain. Moreover, controlled and careful use of antibiotics, both in veterinary and human treatment regimes should be practiced. Finally, further nationwide molecular epidemiology and phenotypic and molecular characterization of the disease should be undertaken.

Acknowledgments

The authors acknowledged owners, managers, and workers of the different abattoir, butcher shops, and restaurants of the study site for their keen interest and cooperation during the collection of the meat samples. We would like to extend our acknowledgement to the College of Veterinary Science Staff members’ who were directly or indirectly helping us during the research period.

Data Availability

All relevant data are within the paper.

Funding Statement

This research work was funded by College of Veterinary Sciences, Mekelle University.

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Decision Letter 0

Kumar Venkitanarayanan

11 Nov 2020

PONE-D-20-31123

Isolation, Identification, and Antimicrobial Susceptibility Pattern of Campylobacter jejuni and Campylobacter coli from Cattle, Goat, and Chicken Meats in Mekelle, Ethiopia

PLOS ONE

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Reviewer #1: Summary

In this manuscript by Hagos et. al., authors isolated Campylobacter spp. from cattle, goat and chicken meat sources in the city of Mekell of Ethiopia, and their antibiotic resistance profiling was performed. This study is significant since the Campylobacter infection is a major foodborne bacterial pathogen and their prevalence in various sources result in foodborne outbreaks. Also, these sources also can act as major hub for antibiotic resistance transfer. However, my major concern is that the selection of sample size for different meat type is not systematic or not defined well. And different meat types were having huge variation in sample sizes, provided, the study was conducted as a cross sectional study by actively collecting various meat types.

Major comments

1. Different meat types have different number of sample sizes, what was the basis of choosing the sample sizes for each category? Abstract says that the samples were collected from abattoir, butcher shops and restaurants, it should be clarified that how many samples are collected from each sourced for each meat type.

2. The ladder for PCR is not well defined and bands are not clear. Is there any reason why both C. jejuni and C. coli have different number of samples? Were they all collected from same meat type?

3. Authors discuss that the difference in prevalence of Campylobacter could be due to the difference in hygienic conditions in the chicken processing. Did the authors notice significant difference in hygienic conditions in processing various meats, if so, what were the differences?

Minor comments

1. The highest prevalence of Campylobcter is found in chicken meat, could it because of lower sample size of chicken meat? Was there any species difference between different meat types?

2. Some sentences are incomplete especially when quoting published articles, needs to double checked.

3. Reference formatting is not consistent. For example, page number in reference 52 compared to others.

Reviewer #2: Summary: The study investigated the prevalence of Campylobacter spp. in meat samples from various species followed by their antibiotic resistance profiling. The study is straightforward. Improvement is needed in the result and discussion sections. Please revise the manuscript based on my comments below.

Title: ok

Abstract: ok

Introduction: ok

Study area: ok

Study design: ok

Line 104-106: Sample size: Since it is known that chicken meat primarily harbors Campylobacter, why is the sample size of chicken meat less than cattle or goat. How was the sample size decided?

144: Typo- Mm or mM?

Results:

Question: How is the flow of meat till it reaches the restaurant? Is it Bucher to abattoir to restaurant? Why is butcher and abattoir a different category? Can the author comment on the significance of having butcher and abattoir separate? I am interested to see if during the supply chain, there is any change in the prevalence of Campylobacter. Interested to know what the authors think about this.

Figure 2: The quality of the figure is not publication standard. The 462 and 589 bp are too close to discern and the ladder is not well spread out. I recommend that the authors run the gel again. Maybe a thicker gel (2%) will help spread the bands out. It is also not clear if the c. coli or C. jejuni are randomly selected to run on the gel among the various strains available. Please explain.

Table 4 needs revision since some of the words are cut off from the manuscript. Also, please explain the numbers outside of the bracket. For example in Ampicillin resistant 2(96.9) what is 2?

Discussion:

Line 228-234: Can the authors comment on the sample size of studies where a higher Campylobacter prevalence was observed. How does the sample size affect the prevalence estimations?

Campylobacter in Cattle and goat meat: What is the potential source of Campylobacter in cattle and goat meat?

Line 258-263: Can the authors elaborate on why such a difference is observed in prevalence?

Line 264-272: The authors need to provide justification on the selection of the two genes to differentiate jejuni and coli. How are the two genes different? Where are they located on the genome? What is their function? Why are they separated based on species? What specific role do they play in the two species? Are they present on mobile genetic elements or DNA that can be exchanged between the species? These information needs to be added to the discussion.

Line 283-298: In its current form, the discussion on MDR or resistance in Campylobacter is very basic. Authors should include information on the timeline of antibiotic resistance analysis by the various studies and if there is any temporal changes observed. Of course, being from different geographical regions, a temporal correlation would be difficult but still an attempt should be made to observe trends. Also, the authors should comment on the potential route of exposure of Campylobacter to antibiotics in the various regions. IF there are regulations in place to reduce use of antibiotics and if that is bringing any change.

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Reviewer #2: No

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PLoS One. 2021 Feb 10;16(2):e0246755. doi: 10.1371/journal.pone.0246755.r002

Author response to Decision Letter 0

26 Nov 2020

Manuscript PONE-D-20-31123

Response to Reviewers

Dear Publisher

Thank you for allowing us to submit a revised draft of the manuscript “Isolation, Identification, and Antimicrobial Susceptibility Pattern of Campylobacter jejuni and Campylobacter coli from Cattle, Goat, and Chicken Meats in Mekelle, Ethiopia” with an opportunity to address the reviewers’ comments for publication in your esteemed journal, PLOS ONE.

Moreover, we would like to say You and both Reviewers many thanks for your invaluable time, critical and professional evaluation, as well as dedication to providing feedback on our manuscript, and are grateful for the insightful comments on and valuable improvements to our manuscript. We have a great appreciation for the corrections given by the reviewers and found them more valuable and professional for the betterment of our manuscript.

Before we proceed to the response to the reviewers, let us explain the overall content of our manuscript and Journal requirement issues.

1. Initially, we have prepared our manuscript according to the PLOS ONE style templates.

2. We didn’t take Figure 1 (Administrative Map of Mekelle City) from other sources rather it is generated by us. Similarly, the image of Figure 2 (PCR Products: M-DNA ladder; Lane 1-4 C. coli (462bp); Lane 6-11 C. jejuni (589bp); and Lanes 5 and 12-Negative control) was taken by us.

3. In the revised manuscript, we have included/referred to Table 5 in the text.

We have incorporated most of the suggestions made by the reviewers though we have seen that the majority of comments require how and why explanations. Those changes are highlighted within the Revised Manuscript with Track Changes. Please see below, for a point-by-point response to the reviewers’ comments and concerns:

1. We have made some grammatical corrections as Reviewer 2 mentioned.

2. As Reviewer 1 stated the samples that were collected from the different meat types seem to be non-systematic and have a great variation among them. The reason for the variation in the number of samples collected from the different species of animals is that due to the accessibility/availability of the respected samples. Hence, the majority of bovine are slaughtered at abattoirs. However, the majority of goat and chicken are slaughtered at the home level without the recognition of the concerned bodies of the City and are most of the time inaccessible for researchers. Reviewer 1 also suggested us to indicate the numbers of samples collected from each sour, hence we have included under the sample size and sample collection section of the manuscript.

3. Reviewer 1 mentioned that the ladder for PCR is not well defined and bands are not clear. It is quite right that the bands' resolution is not that much, however, we have mentioned under PCR section of the methodology as each band of the ladder has a molecular weight difference of 100bp. The samples were collected from different sources and we run the whole 64 phenotypically positive samples, hence, Figure 2 is a representative.

4. There were differences in hygienic conditions and evisceration of the internal organs which lead to cross-contamination.

5. Reviewer 1 mentioned why the highest prevalence is found in poultry? It is a well-known fact that poultry appeared to be a significant source of Campylobacter and chicken were found to be heavily intestinal carriers of Campylobacter when compared with other food animals in addition to what the reviewer suggested, sample size.

6. As per Reviewer 1’s comment, we have made corrections in the reference section.

7. Reviewer 2 needs an explanation for the small sample size of chicken meat which is also raised by Reviewer 1. So, we have indicated above in no. 2.

8. Reviewer 2 indicated that as there is an editorial problem in measurement, we have corrected it as per the given comment.

9. Reviewer 2 needs an explanation for the flow of meat till it reaches the restaurant. As per our Country’s practice, the animals are first transported to the abattoir to be slaughtered, and then the meat is either directly taken to the restaurant or the butcher shops depending upon the customer. As it is presented in Table 1, the highest prevalence was recorded in meat samples collected from restaurants.

10. Reviewer 2 indicated that some words and numbers are cut off. So, we have corrected them. However, related to the number outside of the bracket it is not 2 rather it is 62, which means out of the 64 isolates 62 (96.9) had shown resistance against Ampicillin.

11. Reviewer 2 needs an explanation of how sample size variation affects the prevalence estimation. In epidemiologic studies, the sample size has an important role to detect an effect and to achieve the desired precision in estimates of the parameter of interest. A small sample size will not provide a precise estimate and reliable answers to the study hypothesis. Hence, sample size variation may lead to a foregone conclusion in the prevalence of a disease.

12. Reviewer 2 has also asked the potential sources of Campylobacter in cattle and goat meat. It is known that Campylobacter species are normally carried in the intestinal tracts of many domestic livestock such as poultry, cattle, sheep, goat, pigs, as well as wild animals and birds. Fecal matter is a major source of contamination and could reach carcasses through a direct deposition. The meat of cattle and goat origin can become contaminated by this pathogen during slaughtering and carcass dressing.

13. Reviewer 2 has requested us to elaborate on the possible reasons for variations in the prevalence of Campylobacter in the different sources of samples. It is an interesting comment. This might be because of an extra chance of acquiring contamination in restaurants due to mishandling of meat or cross-contamination among different carcasses or individuals who are working in restaurants might serve as a source of contamination.

14. Reviewer 2 requested us to explain about the two genes. Several multiplex PCR assays have been used to detect Campylobacter spp., C. coli, and C. jejuni. Most of these assays have used a variety of species-specific genes such as omp50, 16S rRNA, 23S rRNA, hipO, mapA, ceuE or putative aspartokinase for identification of C. coli and C. jejuni. The two commonly used species differentiation markers are mapA and ceuE . A species-specific 24-kDa membrane-associated protein of C. jejuni, named MAPA for membrane-associated protein is encoded by the mapA gene, which is useful for genetic identification of C. jejuni by PCR. ceuE encodes a 34.5 to 36.2 kDa lipoprotein component of a binding-protein-dependent transport system for the siderophore enterochelin. In the current study, for C. jejuni, the mapA gene was used and for C. coli, the gene ceuE was used.

15. Reviewer 2 has recommended us to include the timeline of antibiotic resistance as well as the potential route of exposure of Campylobacter to antibiotics in the various regions. Since we didn’t see the current study as per the given comment, we can’t do anything.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (16.9KB, docx)

Decision Letter 1

Kumar Venkitanarayanan

29 Dec 2020

PONE-D-20-31123R1

Isolation, Identification, and Antimicrobial Susceptibility Pattern of Campylobacter jejuni and Campylobacter coli from Cattle, Goat, and Chicken Meats in Mekelle, Ethiopia

PLOS ONE

Dear Dr. Gugsa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:

Please make the following revisions.

Line 69, Page

The situation seems to deteriorate more rapidly in developing countries where there is the widespread and uncontrolled use of antibiotics [22].

Delete “the” before widespread..

Line 79

goat, and chicken collected from the abattoir, butcher shops, and restaurants in Mekelle City.

Replace “the” with “an” if samples were collected from a single abattoir.

Line 87

The study was conducted from October 2015 to May 2016 at the abattoir, butcher shops, and

See previous comment.

Line 191-192

Contamination rate of C. jejuni and C. coli in the different samples  types (sub-heading)

Replace “samples types” with “sample types”.

Figure 1 PCR products

Separation of bands not clear. Replace the figure with a new figure showing band separation.

219-223

Species based antibiogram results of the isolates revealed that the high level of sensitivity of both  C. jejuni and C. coli was observed against Norfloxacin and Erythromycin, were 98.1% ………..as it is shown in Table 5.

Revise the statements for proper grammar.

270-273

This might be because of an extra chance of acquiring contamination in restaurants due to mishandling of meat or cross-contamination among different  carcasses or individuals who are working in restaurants might serve as a source of contamination

Revise the statements for proper grammar.

Reviewer 2 comment “Line 283-298: In its current form, the discussion on MDR or resistance in Campylobacter is very basic. Authors should include information on the timeline of antibiotic resistance analysis by the various studies and if there is any temporal changes observed. Of course, being from different geographical regions, a temporal correlation would be difficult but still an attempt should be made to observe trends. Also, the authors should comment on the potential route of exposure of Campylobacter to antibiotics in the various regions. IF there are regulations in place to reduce use of antibiotics and if that is bringing any change.

The authors have not responded adequately to the above comment and failed to revise the discussion. The authors are requested to respond/revise to the comments other than the timeline.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Kumar Venkitanarayanan, DVM, Ph.D.

Academic Editor

PLOS ONE

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Feb 10;16(2):e0246755. doi: 10.1371/journal.pone.0246755.r004

Author response to Decision Letter 1

5 Jan 2021

Manuscript PONE-D-20-31123

Response to Reviewers

Dear Publisher

Thank you for allowing us to submit the revised draft of the manuscript “Isolation, Identification, and Antimicrobial Susceptibility Pattern of Campylobacter jejuni and Campylobacter coli from Cattle, Goat, and Chicken Meats in Mekelle, Ethiopia” with an opportunity to address the reviewers’ comments for publication in your esteemed journal, PLOS ONE, for the second time.

Moreover, we would like to say You and both Reviewers many thanks for your invaluable time, critical and professional evaluation, as well as dedication to providing feedback on our manuscript, and are grateful for the insightful comments on and valuable improvements to our manuscript. We have a great appreciation for the corrections given by the reviewers and found them more valuable and professional for the betterment of our manuscript.

We have incorporated almost all of the comments given by the reviewers. Those changes are highlighted within the Revised Manuscript with Track Changes. Please see below, for a point-by-point response to the reviewers’ comments and concerns:

1. We have made the corrections given at Line 69, Line 79, Line 87, Line 191-192, Line 219-223, Line 270-273, and Line 283-298 as per the comments.

2. Regarding the comment related to the image in Figure 1, we have agreed with the reviewers’ idea. However, as we have already indicated in our previous response to reviewers, we can’t replace the figure with a new one since by the time the gel-doc was not working properly and we had taken the picture using our mobile phone’s camera. So, we have decided either to keep it as it is or to remove it from the manuscript. If you believe that as it is better to remove it, we don’t have a reservation on it. Hence, you can do that.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (13.6KB, docx)

Decision Letter 2

Kumar Venkitanarayanan

26 Jan 2021

Isolation, Identification, and Antimicrobial Susceptibility Pattern of Campylobacter jejuni and Campylobacter coli from Cattle, Goat, and Chicken Meats in Mekelle, Ethiopia

PONE-D-20-31123R2

Dear Dr. Gugsa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kumar Venkitanarayanan, DVM, Ph.D.

Academic Editor

PLOS ONE

Acceptance letter

Kumar Venkitanarayanan

1 Feb 2021

PONE-D-20-31123R2

Isolation, Identification, and Antimicrobial Susceptibility Pattern of Campylobacter jejuni and Campylobacter coli from Cattle, Goat, and Chicken Meats in Mekelle, Ethiopia

Dear Dr. Gugsa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kumar Venkitanarayanan

Academic Editor

PLOS ONE

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    Submitted filename: Response to Reviewers.docx

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