60th NMC – National Mastitis Council

Association between antimicrobial use and antimicrobial resistance of Streptococcus uberis

causing clinical mastitis Larissa Martins¹, Renata de Freitas Leite1, Juliano Leonel Gonçalves1, Tiago Tomazi2,

Marcos Veiga dos Santos1

1 School of Veterinary Medicine and Animal Science, University of Sao Paulo2Department of Population Medicine and Diagnostic Sciences, Cornell University

January, 2021

Presentation outline

1. Introduction

2. Objectives

3. Material and Methods

4. Results

5. Conclusions

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1. Introduction

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1. Introduction

Streptococcus spp.

• Pathogens often isolated frommastitis cases

✓ Strep. agalactiae✓ Strep. canis✓ Strep. dysgalactiae✓ Strep. uberis

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Antimicrobials

Clinical mastitis

MONITORING

1. Introduction

Antimicrobial use (AMU) and Antimicrobial resistance (AMR)

• Saini et al., 2012: Staphylococcus aureus

• Saini et al., 2013: Gram-negative

• Nobrega et al., 2018: non-aureus staphylococci

Streptococcus uberis

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2. Objectives

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2. Objectives

➢The aim of this study was to investigate theassociation between AMU and AMR evaluatingthe susceptibility profile of Strep. uberis toantimicrobials commonly used in CM treatment

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3.Material and

Methods

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3. Material and Methods

Streptococcus uberis (Tomazi et al., 2018)

• 83 isolates from CM• 17 dairy herds (Southeast Brazil)• Microbiological procedures• Cryopreserved (BHI + 20% glycerol)

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Streptococcus uberis(score ≥ 2.0)

3. Material and Methods

Minimum inhibitory concentrationBroth microdilution methodology - CLSI Guideline

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• Aminoglycosides: gentamicin

• Cephalosporins: cephalexin and ceftiofur

• Lincosamides: lincomycin

• Macrolides: erythromycin

• Penicillins: amoxicillin, ampicillin, penicillin G

• Quinolones: ciprofloxacin and enrofloxacin

• Tetracyclines: oxytetracycline and tetracycline

64 – 0.03 µg/mL

Muelller Hinton broth

+ equine serum

3. Material and Methods

Minimum inhibitory concentration (MIC*)

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1:10

MTT

✓ broth sterility✓ isolate growth✓ Staphylococcus aureus ATCC 29213✓ Streptococcus pneumoniae ATCC 49169

Controls:

Thiazolyl Blue Tetrazolium Bromide

3. Material and Methods

Antimicrobial consumption data (17 dairy herds)

• Diagnosis of CM cases:

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✓Cow´s identification✓Affected mammary quarter✓Date✓Antimicrobial commercial name✓Number of administrations✓Description of protocol changes during

the treatment

3. Material and Methods

Antimicrobial consumption data (17 dairy herds)

• Number of defined daily doses (DDD)

• Antimicrobial Treatment Incidence (ATI)

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✓ Intramammary and Systemic antimicrobials

ATI = Total amount of active substance (mg) used in a givenmonth

DDD X number of lactating cows in a givenmonth x days in a monthx 1,000

✓ Estimated for each farm and expressed as the number of DDD used for CM treatmentper 1,000 lactating cow-days at risk

3. Material and Methods

Statistical Analysis

• MIC Assays

• AMU and AMR

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✓ Susceptible or Resistant✓ PROC FREQ– SAS✓ P<0.05

✓ Association between the overall use of antimicrobials and resistance of isolates toeach antimicrobial used in the susceptibility test

✓ Association between antimicrobial use stratified by class and the antimicrobialresistance of the isolates to the same antimicrobial class

✓ PROC GLIMMIX - SAS✓ P<0.05

4. Results

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4. Results

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Antimicrobial Susceptible2 Resistant3

Gentamicin 15.68% 84.32%

Cefalexin 71.09% 28.91%

Ceftiofour 89.15% 10.85%

Lincomicin 26.49% 73.51%

Erythromycin 19.28% 80.72%

Amoxicillin 79.52% 20.48%

Ampicillin 79.51% 20.49%

Penicillin 36.14% 63.86%

Ciprofloxacin 68.67% 31.33%

Enrofloxacin 93.97% 6.03%

Oxitetracycline 4.84% 95.16%

Tetracycline 48.17% 51.83%

Percentage of Streptococcus uberis classified as susceptible orresistant by antimicrobial tested¹

1In vitro tests considering (n = 83) Streptococcus uberis isolates2Susceptible, high probability of therapeutic success according to the standard dosing related to the pathogen3Resistant, high probability of therapeutic fail even with adjustments in the exposure of the pathogen to the antimicrobial

4. Results

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Percentage of Streptococcus uberis classified as susceptible orresistant by antimicrobial classes¹

1In vitro tests considering (n = 83) Streptococcus uberis isolates2Susceptible, high probability of therapeutic success according to the standard dosing related to the pathogen3Resistant, high probability of therapeutic fail even with adjustments in the exposure of the pathogen to the antimicrobial

≥3 antimicrobial classes

Antimicrobial class Susceptible2 Resistant3

Tetracyclines 26.51 73.49

Quinolones 62.72 37.28

Cephalosporins 79.76 20.24

Macrolides 19.05 80.95

Aminoglycosides 19.05 80.95

Penicillins 69.4 30.96

Lincosamides 26.49 73.51

85%Multi-resistant

4. Results

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Antimicrobial class Number of herdsATI (DDD x 1,000 )

Average SD

Tetracyclines 15 5.0 5.8

Quinolones 14 4.7 6.0

Cephalosporins 16 3.8 6.0

Macrolides 7 3.2 6.3

Aminoglycoside 8 2.4 4.8

Penicillins 16 1.8 3.6

Antimicrobial treatment incidence (ATI) and frequency of herdswith reported use of antimicrobial classes administered byintramammary and systemic routes in 17 dairy herds fromSoutheastern Brazil

4. Results

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Antimicrobial class Number of herdsATI (DDD x 1,000 )

Average SD

Tetracyclines 15 5.0 5.8

Quinolones 14 4.7 6.0

Cephalosporins 16 3.8 6.0

Macrolides 7 3.2 6.3

Aminoglycoside 8 2.4 4.8

Penicillins 16 1.8 3.6

Antimicrobial treatment incidence (ATI) and frequency of herdswith reported use of antimicrobial classes administered byintramammary and systemic routes in 17 dairy herds fromSoutheastern Brazil

• ATI = 23.7 DDD x 1,000 lactating dairy cows

5 – 55.4

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Antimicrobials tested Antimicrobial class Estimate SE1 P-value OR2 95% CI3

Ampicillin Penicillins 0.94 1.31 0.47 2.57 0.19 35.15

Cephalexin Cephalosporins 0.02 1.21 0.98 1.02 0.09 11.51

Ceftiofur Cephalosporins -0.46 1.16 0.69 0.63 0.06 6.41

Enrofloxacin Fluoroquinolones -0.95 0.83 0.26 0.39 0.07 2.05

Erythromycin Macrolides 0.37 2.72 0.89 1.45 0.01 331.44

Gentamicin Aminoglycosides -1.57 1.3 0.23 0.21 0.02 2.78

Penicillin Penicillins -1.47 1.46 0.32 0.23 0.01 4.2

Tetracycline Tetracyclines 1.81 0.51 0.001 6.1 2.23 16.74

Association between overall antimicrobial treatment incidence stratified by class and resistance ofStreptococcus uberis to antimicrobials used for susceptibility testing

The overall use of antimicrobial was not associated with the overall resistance of Strep. uberis

BUT...

5. Conlusions

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5. Conclusions

✓ Cephalosporins and penicillins were the most common antimicrobialsused by the 17 herds evaluated in the present study

✓ Although some isolates presented resistance to antimicrobials, theoverall use of antimicrobials was not associated to the overallresistance of Strep. uberis

✓ However, when the data were stratified according to the antimicrobialclasses there was an increase of Strep. uberis resistance to tetracyclines

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Acknowledgments

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Process number 2018/23040-1

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Thanks for

attending!

renata.leite@usp.br