Distribution of Antibiotic Resistance and Antibiotic Resistant Genes in Campylobacter jejuni Isolated from Poultry in North West of Pakistan

Sher Bahadar Khan1, Mumtaz Ali Khan2,*, Hameed Ullah Khan3, Sher Ali Khan4, Shah Fahad5, Faheem Ahmad Khan6, Irshad Ahmad7, Nighat Nawaz8, Sidra Bibi9 and Muhammad Muneeb10

1Department of Animal Health, The University of Agriculture, Peshawar, Pakistan

2Department of Livestock and Dairy Development, Govt. of Khyber Pakhtunkhwa, Peshawar, Pakistan

3Veterinary Research Institute, Peshawar, Pakistan

4Direcotorate General of Agriculture Research, Peshawar, Pakistan

5University of Swabi, Swabi, Khyber Pakhtunkhwa, Pakistan

6Centre for Biomedical Research, Key Lab of Organ Transplantation, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

7Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan

8Department of Chemistry, Islamia College University, Peshawar, Pakistan

9Department of Poultry Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan

10Department of Food Science and Technology, Faculty of Nutrition Sciences, The University of Agriculture, Peshawar, Pakistan

ABSTRACT

Campylobacter species are one of the most important food borne zoonotic pathogens. A total of 1260 poultry meat samples were collected from four different regions of Khyber Pakhtunkhwa province and processed for isolation of campylobacter species. A total of 182 (14%) Campylobacter jejuni were isolated using enrichment and plate media followed by confirmation through multiplex PCR. Isolates were tested for 15 antibiotics using disc diffusion method followed by detection of their respective antimicrobial resistant genes. Overall prevalence of Campylobacter jejuni was 14% being higher in Peshawar division (21%) followed by Bannu division (16%), Malakand division (13%) and Hazara division (8%). Over all highest antibiotic resistance was found against AMX (93%) followed by LIN (88%), AMP (86%), TET (82%), SXT (75), CHL (68%), CLR (65%), STR (50%), GEN (44%), OFX (27%), CIP (25%), LFX (13%) and AZM (11%) while the least resistance was found against GAT (8%) and CRO (9%). 90% isolates were found to have multiple drug resistance. As for as antibiotic resistant genes are concerned, the highest ARG was blaTEM (93%) followed by tetA (82%), sul2 (75%), blaSHV (72%), tetC (71%), strA/strB (50%), sul1 (49%), blaCMY2 and aadA (44%) while the least resistant gene was aadb (9%) followed by sul3 (21%) and aac(3)IV (37%). About 92% isolates were found to have multiple drug resistance genes which is a matter of great concern from human public health perspective.

Article Information

Received 28 August 2019

Revised 23 November 2019

Accepted 20 December 2019

Available online 28 November 2020

Authors’ Contribution

SBK, MAK, SF and IA designed the study. SBK, MAK, HU, SB and MM performed the experiments. SBK, MAK, SAK, FAK and NN analyzed the data and wrote the article.

Key words

Antibiotic resistance, Antimicrobial resistant genes, Campylobacter jejuni, Poultry meat, Zoonotic pathogens.

DOI: https://dx.doi.org/10.17582/journal.pjz/20190828140843

* Corresponding author: dr_mumtazkhan@yahoo.com

0030-9923/2021/0001-0079 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan

Introduction

Campylobacter is one of the most important pathogen implicated in food borne zoonoosis. The pathogen is world widely distributed and have been reported in different countries including European Union, USA and New Zeland (EFSA-ECDC 2015; CDC, 2017; Rapp et al., 2012). Campylobacter jejuni is the most important species responsible for human campylobacteriosisis while Campylobacter coli and C. lari are second and third responsible species (EFSA-ECDC, 2014). These organisms are fastidious, gram negative, bacilli, non spore forming, thermo tolerant, grow in microaerphilic conditions with a wide incubation period of 1-10 days (Gharst et al., 2013). Foods of animal origin are most commonly contaminated by this pathogen and the reason is these organism are commensals of GIT of different animals including cattle, buffalo, sheep, goat, swine and birds (Zhao et al., 2001; Bork and Petersen et al., 2005; Moran et al., 2009; Di Giannatale et al., 2010; Adzitey et al., 2012; Rejab et al., 2012; Wieczorek et al., 2013). Poultry meat is one of the most animal food source of this pathogen responsible for further transmission and cross contamination to other food items (Silva et al., 2011). Utilization of contaminated food items with this pathogen and under cooked meat have been reported for possible human illness. Gastrointestinal tract is mostly involved in human infection characterized by bloody diarrhea, vomiting, abdominal cramps and pyrexia. The disease may lead to further complications including Guillian barre syndrome, arthritis and Miller Fisher syndrome if not properly treated (WHO, 2017).

Antimicrobial resistance is a worldwide problem in all pathogens in general and in campylobacter species in particular. Unnecessary usage of antibiotics in animals feed particularly in poultry feed as a growth promoting factors is the main reason behind this AMR development. Besides this self medication/inappropriate usage of antibiotics in human illness are other contributing factors. Different mechanisms are involved in AMR development including biofilm formation, antibiotic resistant genes, plasmids and transposons.

Study on this pathogen in poultry is very scarce or very limited in KPK. To the best of our research and knowledge this is first study in Khyber Pakhtunkhwa province and therefore it was planned to find out the prevailing situation of AMR in campylobacter species in poultry meat along the supply chain.

 

Materials and Methods

Samples collection

A total of 1260 poultry meat and tissue samples were collected and brought to laboratory under sterile condition. These meat samples were first cultured on preston campylobacter enrichment broth and then on Columbia blood agar under incubation temperature of 42°C for 48 h in microaerophilic atmosphere according to ISO standard. Identification was performed through colonial characteristics, microscopic morphology and rapid biochemical identification system (Oxoid, Basingstoke, UK). For extraction of genomic DNA from the bacterial isolates kit method was used (Omega Bio-Tek, USA). Species specific genes for campylobacter were targeted in genomic DNA through PCR. Specific primers, PCR amplifications and conditions are described in Table I.

Antimicrobial susceptibility testing (AST)

To check antibiotic susceptibility Campylobacter isolates were tested against 15 different antibiotics through disc diffusion method. For interpretation of the antibiotic susceptibility results standard guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Galni et al., 2008). Following 15 different antibiotics were tested in the AST: Lincomycin (LIN, 2 μg), Azithromycin (AZM, 15 μg), Ampicillin (AMP, 10 μg), Suphamethoxazole+Trimethpram (SXT, 25 μg), Ciprofloxacin (CIP, 5ug), Gatifloxacin (GAT, 5ug), Ofloxacin (OFX, 5 μg), Levofloxacin (LVX, 5 μg), Clarithromycin (CLR, 15 μg), Chloramphenicol (CHL, 30 μg), Tetracycline (TET, 30 μg), Strptomycin (STR, 10 μg), Gentamycin (GEN, 10 μg), Amoxicillin (AMX, 20 μg), and Ceftriaxone (CRO, 30 μg). Multidrug resistance (MDR) strains (isolates resistant to three or more than three antibiotics) were determined.

Detection of antibiotic resistance genes (ARGs)

For detecting major resistance genes, a set of multiplex PCRs were used (Kozak et al., 2009). Major ARGs including b-lactamases (blaCMY-2, blaTEM, blaSHV), sulfonamides (sul1, sul2 and sul3), gentamycin (aac(3)IV, aadB),

 

Table I.- Specific primers and PCR conditions for species specificity of Campylobacter.

Specific genes	Primers	Sequence of primers (5′-3′)	Size of products (bp)
C. jejuni 23S rRNA	23S F 23S R	TATACCGGTAAGGAGTGCTGGAG ATCAATTAACCTTCGAGCACCG	650
C. fetus sapB2	CF F CF R	GCAAATATAAATGTAAGCGGAGAG TGCAGCGGCCCCACCTAT	435
C. upsaliensis glyA	CU F CU R	AATTGAAACTCTTGCTATCC TCATACATTTTACCCGAGCT	204
C. lari glyA	CL F CL R	TAGAGAGATAGCAAAAGAGA TACACATAATAATCCCACCC	251
C. coli glyA	CC F CC R	GTAAAACCAAAGCTTATCGTG TCCAGCAATGTGTGCAATG	126
C. jejuni hipO	CJ F CJ R	ACTTCTTTATTGCTTGCTGC GCCACAACAAGTAAAGAAGC	323

 

streptomycin (strA/strB, aadA and (aac(3)IV) and tetracycline [tet(A), tet(B), tet(C)] were targeted. These specific genes were targeted through specific primers. Details of the primers, PCRs amplification and conditions used are given in Table II.

 

Table II.- Zone of inhibition and concentrations of different antibiotics discs.

S. No	Antibiotics	Abbreviation	Disc content	Zone of inhibition (mm)
				Sensitive	Intermediate	Resistance
1.	Azithromycin	AZM	15 µg	>18	14-17	<13
2.	Lincomycin	LIN	2 µg	>21	16-20	<15
3.	Ampicillin	AMP	10 µg	>17	14-16	<13
4.	Sulphamethoxazole + Trimethoprim	SXT	25 µg	>16	11-15	<10
5.	Ciprofloxacin	CIP	5 µg	>31	21-30	<20
6.	Gatifolxacin	GAT	5µg	>18	15-17	<14
7.	Ofloxacin	OFX	5 µg	>31	21-30	<20
8.	Levofloxacin	LVX	5 µg	>31	21-30	<20
9.	Clarithromycin	CLR	15 µg	>18	14-17	<13
10.	Chloramphenicol	CHL	30 µg	>18	13-17	<12
11.	Tetracyclin	TET	30 µg	>15	12-14	<11
12.	Strptomycin	STR	10 µg	>15	12-14	<11
13.	Gentamycin	GEN	10 µg	>15	13-14	<12
14.	Amoxicillin	AMX	20 µg	>17	14-16	<13
15.	Ceftriaxone	CRO	30 µg	> 23	20-22	<19

 

Table III.- Targeted antibiotic resistance genes, their primers and PCR conditions.

mPCR	Targeted genes	Primers	Sequence of primers	Annealing temp (°C)	Product size (bp)
1	blaTM	GKTEMFd	TTAACTGGCGAACTACTTAC	55	247
		GKTEMRd	GTCTATTTCGTTCATCCATA
	blaSHV	SHV-Fj	AGGATTGACTGCCTTTTTG	55	393
		SHV-Rj	ATTTGCTGATTTCGCTCG
	blaCMY-2	CMYFd	GACAGCCTCTTTCTCCACA	55	1000
		CMYRd	GGACACGAAGGCTACGTA
2	aadA	4Fe	GTGGATGGCGGCCTGAAGCC	63	525
		4Re	AATGCCCAGTCGGCAGCG
	strA/strB	strA-Ff	ATGGTGGACCCTAAAACTCT	63	893
		strB-Rf	CGTCTAGGATCGAGACAAAG
	aac(3)IV	aac4-Lg	TGCTGGTCCACAGCTCCTTC	63	653
		aac4-Rg	CGGATGCAGGAAGATCAA
3	aadB	aadB-Li	GAGGAGTTGGACTATGGATT	55	208
		aadB-Ri	CTTCATCGGCATAGTAAAAG
4	tet (A)	TetA-Lc	GGCGGTCTTCTTCATCATGC	63	502
		TetA-Rc	CGGCAGGCAGAGCAAGTAGA
	tet (B)	TetBGK-F2m	CGCCCAGTGCTGTTGTTGTC	63	173
		TetBGK-R2m	CGCGTTGAGAAGCTGAGGTG
	tet (C)	TetC-Lc	GCTGTAGGCATAGGCTTGGT	63	888
		TetC-Rc	GCCGGAAGCGAGAAGAATCA
5	sul1	sul1-Fb	CGGCGTGGGCTACCTGAACG	66	433
		sul1-Bb	GCCGATCGCGTGAAGTTCCG
	Sul2	sulII-Lc	CGGCATCGTCAACATAACCT	66	721
		sulII-Rc	TGTGCGGATGAAGTCAGCTC
	Sul3	sul3-GKa-Fd	CAACGGAAGTGGGCGTTGTGGA	66	244
		sul3-GKa-Rd	GCTGCACCAATTCGCTGAACG

 

Results

Prevalence of Campylobacter jejuni

Broiler meat samples (n=1260) were processed for detection of campylobacter species. All isolates were further confirmed through colony characteristics, morphology, biochemical testing and detection of their specific genes through PCR. The overall prevalence of Camylobacter jejuni was 14% being higher in Peshawar Division (21%) followed by Bannu division (16%), Malakand Division (13%) and Hazara Division (8%). A total of 182 isolates were obtained from four different regions. All the four regions are different in temperature and climatic condition.

Distribution of phenotypic antibiotic resistance

A total of 182 isolates were tested for 15 different antibiotics using disc diffusion method. Over all highest antibiotic resistance was found against AMX (93%) followed by LIN (88%), AMP (86%), TET (82%), SXT (75), CHL (68%), CLR (65%), STR (50%), GEN (44%), OFX (27%), CIP (25%), LFX (13%) and AZM (11%) while the least resistance was found against GAT (8%) and CRO(9%). There was a very obvious and crystal clear difference in distribution of antibiotic resistance in Campylobacter jejuni isolates from four different regions as shown in Table IV. 90% isolates were found to have multiple drug resistance.

 

Table IV.- Antibiotic resistance in Campylobacter jejuni.

S. No.	Antibiotics	No. of resistant isolates
		Total n=182 (%)	Peshawar division n= 65(%)	Bannu division n= 50(%)	Malakand division n=42(%)	Hazara division n= 25(%)
1	LIN	160 (88)	50 (77)	45 (90)	35 (83)	25 (100)
2	AMX	170 (93)	62 (95)	48 (96)	37 (88)	23 (92)
3	TET	150 (82)	55 (84)	40 (80)	35 (83)	20 (80)
4	AMP	157 (86)	60 (92)	45 (90)	33 (78)	19 (76)
5	SXT	136 (75)	48 (74)	40 (80)	30 (71)	18 (72)
6	CHL	124 (68)	42 (65)	39 (78)	28 (67)	15 (60)
7	CLR	118 (65)	40 (61)	38 (76)	30 (71)	10 (40)
8	STR	91 (50)	25 (38)	24 (48)	32 (76)	10 (40)
9	GEN	80 (44)	32 (49)	21(42)	15 (36)	12 (48)
10	OFX	50 (27)	18 (28)	15 (30)	12 (28)	5 (20)
11	CIP	45 (25)	20 (31)	11 (22)	9 (21)	5 (20)
12	LFX	25 (13)	9 (14)	10 (20)	5 (11)	1(4)
13	AZM	20 (11)	8 (12)	7 (14)	5 (11)	0 (0)
14	CRO	15 (8)	6 (9)	5 (10)	4 (9)	0 (0)
15	GAT	10 (5)	5 (8)	4 (8)	1 (2)	0( 0)

 

Table V.- Antibiotic resistant genes (ARGs) in Campylobacter jejuni.

ARGs	Total, n=182 (%)	Peshawar, n= 65(%)	Bannu, n= 50 (%)	Malakand, n=42(%)	Hazara, n= 25(%)
tetA	150 (82)	55 (85)	40 (80)	35 (83)	20 (80)
tetB	87 (48)	40 (61)	30 (60)	10 (24)	7 (25)
tetC	129 (71)	48 (74)	38 (76)	30 (71)	13 (52)
aadA	80 (44)	32 (49)	18 (36)	24 (57)	6 (24)
strA/strB	91 (50)	25 (38)	24 (48)	32 (76)	10 (40)
aac(3)IV	68 (37)	28 (43)	20 (40)	17 (40)	3 (12)
blaTEM	170 (93)	62 (95)	48 (96)	37 (88)	23 (92)
blaSHV	131 (72)	51 (78)	36 (72)	29 (69)	15 (60)
blaCMY-2	80 (44)	30 (46)	17 (34)	20 (48)	13 (52)
Sul1	90 (49)	29 (45)	23 (46)	18 (43)	20 (80)
Sul2	136 (75)	48 (74)	40 (80)	30 (71)	18 (72)
Sul3	38 (21)	12 (18)	17 (34)	10 (24)	1 (4)
aaddB	16 (9)	9 (14)	5 (10)	2 (5)	0 (0)

 

Distribution of antibiotic resistant genes (ARGs)

As for as antibiotic resistant genes are concerned, the highest ARG was blaTEM (93%) followed by tetA (82%), sul2 (75%), blaSHV (72%), tetC (71%), strA/strB (50%), sul1 (49%), blaCMY2 and aadA (44%) while the least resistant gene was aadb (9%) followed by sul3 (21%) and aac(3)IV (37%). All the isolates from four different regions were found to have different distribution of resistant genes as shown in Table V. 92% isolates were found to have multiple antibiotic resistances.

 

Discussion

Campylobacter species are among the most important food borne pathogens causing zoonosis. Mostly this infection is restricted to GIT in human but in severe cases it may lead to other severe syndromes. Different countries have reported different prevalence of campylobacter in poultry meat including 85% in Northern Ireland (Moran et al., 2009), 87% in Poland (Wieczorek et al., 2013), 20.8% in Estonia (Mäesaar et al., 2014), and 73-81% in Italy (Parisi et al, 2007; Pezzotti et al., 2003). These results are a bit higher and not consistent to our study and the reasons could be due to different climatic conditions, different slaughtering techniques, evisceration, and packing processing. Other reasons may due to different types of samples used.

Antibiotic resistance is one of the greatest threat to the world after infections. This study also described the prevailing situation of AMR in Campylobacter jejuni. Here are also different study reports from different countries describing different scenario of AMR in Campylobacter. Ledergerber et al. (2003) have reported 28.7% resistance to ciprofloxacin, 12.6% to tetracycline, 11.8% to sulphonamide, and 10.3% to ampicillin in a study conducted in Switzerland. Mattheus et al. (2012) have conducted a study in Belgium poultry in which he found resistance of Campylobacter species to AMP (47.4%), CIP (42.1%), Erythromycin (12.1%), GEN (25.6%), nalidixic acid (46.4%) and TET (45.3%). Miflin et al. (2007) have conducted a study on Campylobacter jejuni in Queensland region and found 18.4% resistance for tetracycline and 17.6% for ampicillin. Bester et al. (2008) have reported highest resistance for tetracycline (98.2%) and ceftriaxone (96.4%) in a study conducted in broiler in South Africa. Obeng et al. (2012) have observed extensive resistance of campylobacter to lincomycin (51-100%), ampicillin (33·3-60·2%) and tetracycline (5·6-40·7%). Wieczorek et al. (2018) conducted a study in Poland in poultry and found resistance to ciprofloxacin (92.5%), followed by nalidixic acid (88.9%) and tetracycline (68.4%). Another study conducted in Poland by Wysok et al. (2017), where he reported 52.7% resistance to ciprofloxacin, 56% to nalidixic acid and 61.3% to doxycycline.

Nguyen et al. (2016) have found high rate of resistance to nalidixic acid, tetracycline and ciprofloxacin of 77.4, 71.0 and 71.0%, respectively. Low resistance (25.8%) was detected for gentamicin and chloramphenicol. Gupta et al. (2004) have conducted a study on AMR in USA from 1998-2001. They observed that ciprofloxacin-resistant Campylobacter have increased from 13% to 19%. No increase was observed in erythromycin resistance which remains the same at 2% from 1998-2001. Senok et al. (2007) have discovered highest resistance of Campylobacter to CIP (88.8%) and 32.6% to TET in a study conducted in Kingdom of Bahrain. Similarly, a study conducted in China by Xia et al. (2010) have reported 98% resistance of Campylobacter to nalidixic acid, ciprofloxacin, enrofloxacin, tetracyclines and doxycycline. These studies are a clear indication of extensive AMR in Campylobacter around the world. The difference in the results could be due to different geographical locations, different climatic conditions and usage of different antibiotics in animal feeds.

To the best of our search, knowledge and understanding this is the first study that we conducted on the detection of ARGs in Campylobacter in Pakistan. Our study have reported the highest ARG blaTEM (93%) followed by tetA (82%), sul2 (75%), blaSHV (72%), tetC (71%), strA/strB (50%), sul1 (49%), blaCMY2 and aadA (44%) while the least resistant gene was aadb (9%) followed by sul3 (21%) and aac(3)IV (37%) which is consistent to phenotypic data. Different countries have reported different ARGs in Campylobacter. Abdi-Hachesoo et al. (2014) have tested Campylobacter species for TET genes in Iran and found that 18% isolates were positive for TET (A) gene. Obeng et al. (2012) have found different antibiotic resistance genes including bla (OXA-61) (82·6-92·7%), cmeB (80·3-89%) and tet(O) (22·3-30·9%) in C. coli isolates from pigs, while C. jejuni from chickens were found to harbor bla(OXA-61) (59-65·4%) and tet(O) (19·2-40·7%). Similarly, Reddy and Zishiri (2017) have tested Campylobacter species for gyrA, blaOXA-61, and TET genes. 68% isolates were found positive for tetO gene which was the most prevalent. Quinolone resistance was highly associated with gyrA genes. Gleisz et al. (2006) have tested campylobacter for AMR in Austria and found that 21% were resistant to tetracycline, 18% for AMP and 11% for STR and all isolates were found positive for tetO gene. Again results of ARGs are also in disagreement and possible reasons could be due to different geographical locations, usage of different antibiotics and testing of different targeted ARGs.

 

Conclusion

Campylobacter jejuni 90% have multiple drug resistance while more than 92% have multiple ARGs. This is an alarming situation of AMR in Campylobacter jejuni in Khyber Pakhtunkhwa province of the country which needs prompt attention of the concerned veterinary and public health authorities since the diseases is zoonotic that could pose potential health threat.

 

Acknowledgment

The study was supported by the Microbiology Laboratory of Department of Animal Health, The University of Agriculture, Peshawar, Directorate General Extension and Directorate General of Research, Veterinary Research Institute, Peshawar.

 

Statement of conflict of interest

There is no conflict of interest.

 

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