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Phenotypic and genotypic methods for identification of slime layer production, efflux pump activity, and antimicrobial resistance genes as potential causes of the antimicrobial resistance of some mastitis pathogens from farms in Menoufia, Egypt

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Abstract

Mastitis caused by multi- or pan-drug resistant bacteria is a growing health concern. A total of 110 milk samples were collected: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Escherichia coli were present in 54/110 (49.09%), 37/110 (33.63%), 25/110 (22.72%), 7/110 (6.36%), and 50/110 (45.45%) samples, respectively. A total of 20 methicillin-resistant S. aureus (MRSA) isolates, 19 Streptococcus sp. isolates, and 15 E. coli isolates were selected, and 100% were positive for (coagulase and hemolysins), streptokinase, and hemolytic activity, respectively. A number of 11 E. coli isolates were serotyped, and the serotypes were: O26, O55, O111, O119, O124, O125, O127, and O158. The antimicrobial resistance index ranges for MRSA, Streptococcus sp., and E. coli were 0.49–0.83, 0.39–0.83, and 0.56–1, respectively. The most effective antimicrobials on Gram-positive isolates were cephradine, ciprofloxacin, doxycycline, norfloxacin, and vancomycin, while doxycycline and norfloxacin were effective on E. coli serotypes. All of the selected isolates exhibited slime layer production. The efflux pumps of the 12 MRSA, 12 Streptococcus sp., and 11 E. coli isolates exhibited activity with ethidium bromide concentrations of 1, 1.5, and 0.5 µg/ml, respectively. There was a simultaneous antimicrobial activity of the efflux pump inhibitor chlorpromazine with amoxicillin/clavulanic acid, erythromycin, and oxacillin, to which the isolates were resistant. The 12 MRSA isolates harboured the methicillin resistance genes mec(A,A1, and A2), mecA1, and mecC at frequencies of 9/12 (75%), 9/12 (75%), and 8/12 (66.7%), respectively, and the penicillin resistance gene BlaZ was present at a frequency of 5/12 (41.7%). The distributions of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 8/12 (66.7%), 5/12 (41.7%), 12/12 (100%), 2/12 (16.7%), 0/12 (0.0%), and 8/12 (66.7%), respectively. The 12 Streptococcus sp. isolates harboured mec(A, A1, and A2), mecA1, mecC, and blaZ at rates of 4/12 (33.33%), 4/12 (33.33%), 5/12 (41.7%), and 4/12 (33.33%), respectively. The frequencies of erm(A) and erm(F) were 4/12 (33.33%), and 9/12 (75%), respectively. The 11 E. coli isolates harboured the extended-spectrum β-lactamases integrase1, integrase2, blaCTX-M, blaCTX-M-1, and blaTEM at frequencies of 10/11 (90.90%), 11/11 (100%), 9/11 (81.81%), 6/11 (54.54%), and 10/11 (90.90%), respectively. Moreover, the frequencies of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 7/11 (63.63%), 4/11 (36.36%), 4/11 (36.36%), 5/11 (45.45%), 10/11 (90.90%), and 10/11 (90.90%), respectively. Our results demonstrated the high antimicrobial resistance of the investigated isolates and confirmed the existence of multiple mechanisms underlying multidrug resistance.

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Acknowledgements

The authors express their gratitude to the veterinary administration at Menoufia, farmers, and farm owners for helping in sample collection.

Funding

The authors declare that there was no funding source.

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Authors and Affiliations

  1. Department of Bacteriology, Mycology, and Immunology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Menoufia, Egypt

    Mohamed Sabry Abd Elraheam Elsayed & Reda Tarabees

  2. Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Menoufia, Egypt

    Tamer Roshdey & Ahmed Salah

  3. Department of Bacteriology, Mycology, and Immunology, Faculty of Veterinary Medicine, University of Mansoura, Mansoura, Egypt

    Gamal Younis

  4. Veterinary Administration, Ashmoun, Menoufia, Egypt

    Doaa Eldeep

Authors
  1. Mohamed Sabry Abd Elraheam Elsayed
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  2. Tamer Roshdey
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  3. Ahmed Salah
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  4. Reda Tarabees
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  5. Gamal Younis
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  6. Doaa Eldeep
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Contributions

Conceptualization: (MSE, TMR, AS, RT, GA, DE), Data curation: (MSE, TMR, AS, DE), Formal analysis: (MSE, TMR, AS), Investigation: (MSE, TMR, AS, RT, GA), Methodology: (MSE, TMR, AS, DE), Project administration: (MSE), Supervision: (MSE), Writing the original draft: (MSE), Review & editing: (MSE, TMR, AS). All authors have read and approved the manuscript.

Corresponding author

Correspondence to Mohamed Sabry Abd Elraheam Elsayed.

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Conflict of interest

The authors declare that there are no competing interests.

Ethical approval

The protocol was approved by the institutional animal care and use committee (IACUC) at the Faculty of Veterinary Medicine, University of Sadat City. Furthermore, our manuscript reporting adheres to the ARRIVE guidelines. The authors obtained written informed consent to use the animals in their study from the owner(s) of the animals.

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Elsayed, M.S.A.E., Roshdey, T., Salah, A. et al. Phenotypic and genotypic methods for identification of slime layer production, efflux pump activity, and antimicrobial resistance genes as potential causes of the antimicrobial resistance of some mastitis pathogens from farms in Menoufia, Egypt. Mol Biol Rep 46, 6533–6546 (2019). https://doi.org/10.1007/s11033-019-05099-6

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