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Prevalence of Antibiotic Resistant Genes in Staphylococcus aureus Isolated from Bovine Mastitis

PJZ_54_5_2239-2244

Prevalence of Antibiotic Resistant Genes in Staphylococcus aureus Isolated from Bovine Mastitis

Sher Bahadar Khan1,*, Mumtaz Ali Khan2, Irshad Ahmad3, Faheem Ahmad Khan4, Hamayun Khan1 and Sher Ali Khan5

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

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

3Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan

4The Centre for Biomedical Research, Key Laboratory of Organ Transplantation, Ministry of Education, Ministry of Health, Tongi Medical College, Huazhong University of Science and Technology, China

5Sericulture Agrifood Research Institute, Guangdong Academy of Agricultural Sciences, China

ABSTRACT

A total of 280 Staph. aureus strains from a total of 1250 milk samples from buffaloes were tested for 15 antibiotics using disc diffusion method followed by detection of their respective antimicrobial resistant genes through PCR. Among them, the highest prevalence of Staph. aureus was found in Peshawar-Mardan division (30%), followed by Malakand (28.5%), Bannu-Dera Ismail khan division (25%) and Hazara division (16%). Over all the high resistance was found against Lin (96.25%) followed by AMX (82.5%), TET (63.75%), AMP (58.75%), SXT (50%), CHL (48.7%), CLR (36.25%), STR (25%), GEN (17.5%), OFX (15%), LFX (12.5%), AZM (8.75%) while least resistance against GAT (3.375%) and CRO (6.25%). Over all the highest prevalent gene was blaTEM (179) followed by tetA (147), tetB (144), blaCMY-2 (142), sul1 (139), sul3 (137), tetC (130), aadA (121), sul2 (118), strA/strB (117) while the least resistant gene was aaddB (12) and aac(3)IV (16).


Article Information

Received 03 April 2020

Revised 30 May 2020

Accepted 16 June 2021

Available online 16 November 2021

(early access)

Published 08 June 2022

Authors’ Contributions

SBK, MAK, IA and FAK designed the study. SBK, MAK, SAK executed the expserimental work and analyzed the samples. SBK, MAK, HK, SAK helped in data analysis and article drafting.

Key words

Antibiotic resistance, Antibiotic resistant genes, Bovine, Mastitis, Staphylococcus aureus.

DOI: https://dx.doi.org/10.17582/journal.pjz/20200403170447

* Corresponding author: sbvetdr@yahoo.com

0030-9923/2022/0005-2239 $ 9.00/0

Copyright 2022 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).



INTRODUCTION

Mastitis is one of the most important economic diseases of dairy animals. It causes huge economic losses to the national exchequer in terms of morbidity, drop in milk production, reduction of milk quality and veterinary services cost. Different countries have reported different economic losses due to disease including UK, USA and Holland (Hillerton et al., 2005; Huijps et al., 2008; Viguier et al., 2009). There are reports of more than 140 species of different microbes responsible for bovine mastitis. Staphylococci, coliforms and streptococci are most frequently isolated microbes (Watts, 1998; Tenhagen et al., 2006; Piepers et al., 2007; Malinowski et al., 2010; Smulski et al., 2011). Staphylococcus aureus associated mastitis is more dangerous and complex than others microbes as the cure rates are comparatively lower. This complexity of Staph. aureus is because of their frequent acquisition of antibiotic resistance and biofilm formation (Cramton et al., 1999). It is thought that biofilm production is the major reason behind recurrent mastitis in dairy animals (Melchior et al., 2006). A rapid increase in spreading of antibiotic resistant staphylococci and other microorganism is caused by merciless and indiscriminate use of antibiotics in animal feed and veterinary practice. An appropriate and proper usage of these antibiotics could minimize this malady of antibiotic resistance. There are certain factors including antibiotic resistant genes responsible for resistance to antibiotics.

Proper and appropriate usage of antibiotics is the need of the hour to overcome this malady of antimicrobial resistance (AMR). Discovery and development of new antibiotics is another alternative to tackle this issue. The prime purpose of the present study was to uncover the prevalence of antibiotic resistance and antibiotic resistant genes in Staph. aureus strains isolated from clinically positive animals suffering from mastitis in North West Pakistan.

Materials and methods

A total of 1250 milk samples from buffaloes clinically positive for mastitis were collected. Samples were brought to laboratory under hygienic condition at 4°C. Upon arrival to the Laboratory these samples were processed for culturing on tryptose agar followed by identification through colonial, microscopic morphology and tube tests for coagulase and catalse activity. For extraction of genomic DNA, bacterial DNA extraction kit (E.Z.Nce.A, Omega Bio-Tek, USA) was used. Thermostable gene (nuc), mecA and blaZ specific for S. aureus were targeted in genomic DNA. PCR conditions and primer sequences are given in Table I.

Fifteen different antibiotics namely Chloramphenicol (CHL) 30µg, Clarithromycin (CLR) 15µg, Levofloxacin (LVX) 5µg, Ofloxacin (OFX) 5µg, Gatifolxacin (GAT) 5µg, Ciprofloxacin (CIP) 5µg, Sulphamethoxazole+Trimethoprim (SXT) 25µg, Ampicillin (AMP) 10µg, Lincomycin (LIN) 2µg, Azithromycin (AZM) 15µg, Ceftriaxone (CRO) 30µg, Amoxicillin (AMX) 20µg, Gentamycin (GEN) 10µg,

 

Table I.- Targeted genes, their specific primers and PCR conditions.

Name of gene

Name of

primer

Primer sequence

Primer concentration (µM)

Annealing Temp. (°C)

Size of product (bp)

nuc

nucF5′

GCGATAGATGGTGATACGGTT

0.1

55

270

nucR5′

AGCCAAGCCTTGACGAACTAAAGC

0.1

55

mecA

mec1 5′

AAAATCGATGGTAAAGGTTGG

0.25

55

533

mec2 5′

AGTTCTGCAGTACCGGATTTGC

0.25

55

blaZ

blaZ15′

AAGAGATTTGCCTATGCTTC

0.20

54

517

blaZ25′

GCTTGACCACTTTTATCAGC

0.20

blaTM

GKTEMFd

TTAACTGGCGAACTACTTAC

0.2

55

247

GKTEMRd

GTCTATTTCGTTCATCCATA

0.2

blaSHV

SHV-Fj

AGGATTGACTGCCTTTTTG

0.4

55

393

SHV-Rj

ATTTGCTGATTTCGCTCG

0.4

blaCMY-2

CMYFd

GACAGCCTCTTTCTCCACA

0.2

55

1000

CMYRd

GGACACGAAGGCTACGTA

0.2

aadA

4Fe

GTGGATGGCGGCCTGAAGCC

0.1

63

525

4Re

AATGCCCAGTCGGCAGCG

0.1

strA/strB

strA-Ff

ATGGTGGACCCTAAAACTCT

0.4

63

893

strB-Rf

CGTCTAGGATCGAGACAAAG

0.4

aac(3)IV

aac4-Lg

TGCTGGTCCACAGCTCCTTC

0.2

63

653

aac4-Rg

CGGATGCAGGAAGATCAA

0.2

aadA

4Fe

GTGGATGGCGGCCTGAAGCC

0.1

63

525

4Re

AATGCCCAGTCGGCAGCG

0.1

tet (A)

TetA-Lc

GGCGGTCTTCTTCATCATGC

0.1

63

502

TetA-Rc

CGGCAGGCAGAGCAAGTAGA

0.1

tet (B)

TetBGK-F2m

CGCCCAGTGCTGTTGTTGTC

0.2

63

173

TetBGK-R2m

CGCGTTGAGAAGCTGAGGTG

0.2

tet (C)

TetC-Lc

GCTGTAGGCATAGGCTTGGT

0.5

63

888

TetC-Rc

GCCGGAAGCGAGAAGAATCA

0.5

strA/strB

strA-Ff

ATGGTGGACCCTAAAACTCT

0.4

63

893

strB-Rf

CGTCTAGGATCGAGACAAAG

0.4

aac(3)IV

aac4-Lg

TGCTGGTCCACAGCTCCTTC

0.2

63

653

aac4-Rg

CGGATGCAGGAAGATCAA

0.2

aadB

aadB-Li

GAGGAGTTGGACTATGGATT

0.2

55

208

aadB-Ri

CTTCATCGGCATAGTAAAAG

0.2

sul1

sul1-Fb

CGGCGTGGGCTACCTGAACG

0.2

66

433

sul1-Bb

GCCGATCGCGTGAAGTTCCG

0.2

Sul2

sulII-Lc

CGGCATCGTCAACATAACCT

0.3

66

721

sulII-Rc

TGTGCGGATGAAGTCAGCTC

0.3

Sul3

sul3-GKa-Fd

CAACGGAAGTGGGCGTTGTGGA

0.2

66

244

sul3-GKa-Rd

GCTGCACCAATTCGCTGAACG

0.2

 

Strptomycin (STR) 10µg and Tetracyclin (TET) 30µg were used to test sensitivity and resistance in Staph. aureus isolates according to disc diffusion method as already described (Galani et al., 2008). Strains resistant to two or more than two antibiotics are considered multi drug resistant (MDR).

Specific antibiotic resistant genes (ARGs) responsible for or conferring resistance to these antibiotics were targeted using multiplex PCR according to the method already described (Kozak et al., 2009). Details of these ARGs, their primers specifications and PCR conditions are given in Table I.

Results and discussion

A total of 280 (22.4%, 280/1250) Staph. aureus strains were isolated from the four different divisions of Khyber Pakhtunkhwa province. Among them, the highest prevalence of Staph. aureus was found in Peshawar-Mardan division (30%, 85/280), followed by Malakand division (28.5%, 80/280), Bannu- Dera Ismail khan division (25%,70/280 ) and Hazara division (16%, 45/280) (Table II). A total of 280 Staph. aureus strains were isolated which were tested for 15 antibiotics using disc diffusion method. Overall the high resistance was found against Lin (96.25%) followed by AMX, TET, AMP, SXT, CHL, CLR, STR, GEN, OFX, LFX , AZM while least resistance against GAT (3.375%) and CRO (6.25%) (Table II). About 80% Staph aureus were found to have multiple drug resistance. The drugs of choice were GAT and CRO. As for as antibiotic resistant genes are concerned, over all the highest prevalent gene was blaTEM followed by tetA, tetB, blaCMY-2, sul1, sul3, tetC, aadA, sul2, strA/strB while the least resistant gene was aaddB and aac(3)IV (Table III). It was observed that tetA gene were more associated with TET antibiotic followed by tetB and tetC. Similarly for beta- lactams antibiotic resistance blaTEM was found the highest followed by blaCMY-2 and blaSHV. For sulpha drugs sul1 was found the highest followed by sul3 and sul2. For streptomycin, the highest ARG was aadA followed by strA/strB and aac(3)IV.

Antimicrobial resistance is one of the global and greatest issues after infection. There are reports of different countries regarding antimicrobial resistance in Staph. aureus. Malinowski et al. (2008) have reported 62.3% resistance to penicillin, 41.7% to tetracycline, 39.4% to lincomycin and 20% to bacitracin and cephalexin. In Turkey, Turutoglu et al. (2006) have reported resistance to penicillin, ampicillin and amoxicillin that were 62.1%, 56.3% and 45.6%, respectively. Resistance to gentamicin (56.3%) and trimethoprim/sulfa-methoxazole (45.6%) was also reported in the same study. Kalmus et al. (2011) have reported resistance to ampicillin (59.5%) and penicillin (61.4%) in Estonia. In Lithuania, Klimiene et al. (2012) have also found resistance to penicillin (76.7%), ampicillin (78.4%) and amoxicillin (81.3%). In China, Gao et al. (2012) have reported 96.3% resistance to penicillin and 98.1% to tetracycline, and 100% sensitivity to oxacillin, cefazolin and ciprofloxacin. In Ethiopia, 82.4%

 

Table II.- Prevalence of antibiotic resistance in Staph. aureus.

S. No.

Antimicrobials

No. of isolates resistant in different regions

Total

n= 280 (22.4%)

Malakand division

n= 80 (28.5%)

Hazara division

n= 45 (16%)

Bannu- DIkhan

n= 70 (25%)

Peshawar -Mardan

n= 85 (30%)

1

LIN

277(96.25)

80(100)

45(100)

70(100)

70(85)

2

AMX

266(82.5)

78(95)

44(95)

60(80)

60(60)

3

TET

180(63.75)

50(65)

40(90)

37(55)

40(45)

4

AMP

170(58.75)

49(65)

26(70)

34(45)

47(55)

5

SXT

140(50)

32(40)

32(80)

32(40)

36(40)

6

CHL

120(48.75)

48(60)

30(65)

32(40)

25(30)

7

CLR

110(36.25)

45(55)

9(20)

35(50)

18(20)

8

STR

70(25)

20(25)

14(30)

20(30)

13(15)

9

GEN

28(17.5)

10(10)

14(30)

3(10)

18(20)

10

OFX

22(15)

5(5)

15(35)

5(15)

4(5)

11

CIP

22(15)

5(5)

14(30)

5(15)

9(10)

12

LVX

15(12.5)

5(5)

14(30)

3(10)

4(5)

13

AZM

10(8.75)

16(15)

3(5)

0(0)

13(15)

14

CRO

8(6.25)

0(0)

9(20)

0(0)

4(5)

15

GAT

3(3.75)

0(0)

0(0)

3(10)

4(5)

 

LIN, Lincomycin; AMX, Amoxicillin; TET, Tetracyclin; AMP, Amipicillin; SXT, Sulphamethoxazole-Trimethoprim; CHL, Chloramphinicol; CLR, Clarithromycin; STR, Streptomycin; GEN, Gentamycin; OFX, Ofloxacin; CIP, Ciprofloxacin; LVX, Levofloxacin; AZM, Azithromycin; CRO, Ceftrioxone; GAT, Gatifloxacin.

 

Table III.- Prevalence of antibiotic resistant genes (ARGs) in Staph. aureus.

ARGs

Overall

n=280 (%)

Malakand division

n=80 (%)

Hazara division

n=45 (%)

Bannu-DIKhan

n=70 (%)

Peshawar -Mardan

n=85 (%)

tetA

52.5

52.5

77.7

52.8

47

tetB

51.4

52.5

75.5

47.1

31.7

tetC

46.4

50

57.7

34.2

49.4

aadA

43.2

31.2

57.7

34.2

30.5

strA/strB

41.7

31.2

53.3

20

28.2

aac(3)IV

5.7

13.7

46.6

18.5

12.9

blaTEM

63.9

100

44

28.5

92.9

blaSHV

42.1

16.2

28.8

38.5

29.4

blaCMY-2

50.7

57.5

84.4

0

32.9

Sul1

49.6

35

80

38.5

32.9

Sul2

42.1

28.7

60

18.5

17.6

Sul3

48.9

35

80

25.7

17.6

aaddB

4.2

0

0

15.7

9.4

 

resistance to pencillin, 88.2% to clindamycin and 58.8% to erythromycin while sensitivity to chloramphenicol (58.8%) and nalidixic acid (82.4%) was reported by Haftu et al. (2012). In India Kumar et al. (2011) have found resistance to streptomycin (36.4%), oxytetracycline (33.6%), gentamicin and ampicillin (29.9%), penicillin (28.9%) and chloramphenicol, pristinamycin and ciprofloxacin (26.2%). Resistance to tetracyclin in France (3.1%) and Switzerland (5.3%) has been reported by Sakwinska et al. (2011). Very low antibiotic resistance (3%) has been reported in Sweden to kanamycin, tetracyclin and penicillin by Persson et al. (2011). The difference in antibiotic resistance in the different countries may be due to use of different antibiotics, difference in antibiotic concentration and geographical variation. The high prevalence of antibiotic resistance to beta-lactams worldwide could be due to their worldwide application against staphylococcal mastitis. It was found that tetA gene was more associated with TET antibiotic followed by tetB and tetC which is in close agreement to the previous study conducted by Olowe et al. (2013). Similarly for beta-lactams antibiotic resistance blaTEM was found the highest followed by blaCMY-2 and blaSHV which is partially in agreement and partially in disagreement with the previous study conducted by Nambram et al. (2018). For sulpha drugs sul1 was found the highest followed by sul3 and sul2 which is closely related to the study conducted by Patrícia et al. (2005). For streptomycin, the highest AMRG was aadA followed by strA/strB and aac(3)IV which is a little disagreement with the previous study conducted by Ramirez and Tolmasky (2010).

conclusion

In conclusion, 80% S. aureus strains have multiple drug resistance and antibiotic resistant genes which is a matter of great concern. The drugs of choice against Staph aureus are CRO and GAT followed by AZM, LFX and OFX. It is the need of the hour to develop alternatives antibiotics and ban unnecessary use of antibiotics to overcome this alarming and challenging situation of antimicrobial resistance.

Statement of conflict of interest

All the authors declare no conflict of interest

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