an observational study in Maputo, Mozambique Sara Lino Faife UKZN - - PowerPoint PPT Presentation

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ESBL-producing Enterobacteriaceae in poultry: an observational study in Maputo, Mozambique

Sara Lino Faife

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• 1. Introduction
• Antibiotic resistance is considered a global health problem

for many decades (CDC, 2010)

• One of the most studied and clinically relevant resistance

mechanism in bacteria is extended-spectrum beta- lactamase (ESBL) - producing Enterobacteriaceae.

• ESBL-producing bacteria are not isolated only in humans

but also in food-borne bacteria (Plateel et al. 2013)

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• 1. Introduction
• Food animals, including chickens, turkeys, pigs and cattle,

may be colonized by ESBL and/or plasmid-mediated AmpC (pAmpC)-producing Escherichia coli (Carttoli, 2008).

• The most frequent ESBL genes associated with this

resistance among food producing animals and companion animals encode various CTX-M enzymes, followed by blaTEM-52 and blaSHV-12 (Ewers et al, 2011)

• Chicken is one of the most consumed foods by the

population in Mozambique.

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• 1. Introduction

Reseach question

• Are there ESBL/AmpC-producing Enterobacteriaceae in

imported and locally produced chicken?

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• 2. Objectives
• Aim
• To investigate the presence of the ESBL/AmpC-producing

Enterobacteriaceae in imported and locally produced chicken and perform relevant phenotypic and genotypic characterization of resistant isolates.

• Objectives
• Screen for ESBL-producing isolates by culturing samples

from thaw liquid and carcass from poultry produced in Mozambique, South Africa and Brazil.

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• 2. Objectives
• To identify bacterial isolates from imported and

domestically produced chickens/poultry to species level

• To ascertain the antibiotic susceptibility of isolates against

an appropriate panel of antibiotics by standardized agar disc diffusion and/or minimum inhibitory concentration (MIC) determination

• To phenotypically confirm the production of:

– ESBLs using the double-disc synergy test, – AmpC β-lactamase using the cefoxitin disc sensitivity test, and,

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• 2. Objectives
• To identify the EBSL and/or AmpC genes by PCR and

sequencing

• To ascertain similarities of isolates using ERIC PCR
• To compare the phenotypic and genotypic differences

between imported and domestically produced chickens/ poultry

• To disseminate the information to policy-makers to

improve/inform agricultural practice related to growth promoters in poultry

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• 3. Methods
• Ethical Considerations
• Ethical clearance was received from Biomedical Research

Ethics Committee of University of KwaZulu-Natal

• Sampling
• In the study were included 99 frozen chickens imported

from South Africa (33) and Brazil (33), as well as chickens produced in Mozambique and they were acquired in 3 different supermarkets.

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Sampling

• In each chicken were obtained 2 samples from:
• (1) thaw liquid and
• (2) swab of the internal part of the carcass, totaling 198

samples from 99 chickens

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Strains iden+fica+on

• Four plates per sample of chicken were used (1) 2

MacConkey plates to prove growth of Enterobacteriaceae, and (2) 2 Macconkey with 2mg/l of ceftriaxone to select for ESBL-producers.

• The 2 samples were from (1) thaw liquid and (2) swab of

the internal part of the carcass.

• Colonies were biochemically tested for bacteria

identification.

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Susceptibility test and phenotical evaluation antimicrobial susceptibility and resistance testing based on agar disc diffusion:

• according to CLSI guidelines (2015) recommended panel
• f antibiotics.
• phenotypical evaluation for the presence of ESBL/pAmpC

– disk sinergy test (CLSI, 2015).

• ESBL confirmation - double-disc diffusion test following the

ROSCO diagnostic (Kits for detection of resistance mechanism) ( EUCAST, 2013).

• During all procedures will be guaranteed quality control of

the Medias and discs.

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DNA extraction, PCR and Electrophoresis

• The DNA extraction was done using Gram-Negative

Bacteria Gene JET Genomic DNA Purification Kit (Thermo Scientific, USA).

• Clonality was done using Enterobacterial Repetitive

Intergenic Concensus-PCR analysis

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The PCR amplification and Sequence

• The PCR was done using Phusion Flash High-Fidelity PCR

Mater Mix (Thermo Scientific, USA) protocol and primers already described with different annealing temperatures

• The amplicons obtained were visualized by electrophoresis
• The sequences were analyzed using Basic Local Alignment

Search Tool

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• 4. Results

Table 1: bacterias isolated by place of provenience

Nr. Bacterias name

• Nr. Of positive

samples Provenience 1 Enterobacter Cloacae 1 Mozambique 2 Enterobacter aglomerans 1 South Africa 3 Citrobacter divresus 1 South Africa 4 Citrobacter freundii 3 South Africa (2) Brazil (1) 5 Escherichia coli 21 South Africa (15) Mozambique (4) Brazil (2)

UKZN INSPIRING GREATNESS Table: 2-Number of samples with ESBLA, AmpC and Fluoroquinolone resistance

• Nr

ESBL AmpC Nr of samples 23 SHV-85% CTX-M-62% TEM-44% 10 (CMY, FOX, MOX and DHA)-below 50%

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South Africa

rep-PCR_Sara

100 80 60 40 20

rep-PCR_Sara A25C A29C A17C A26D A26C A23C A27C A25D B18C A32D M27D M32C A30C B17C A33D B23D M27C A30D A13D A20C A12C M30C A10C A10D

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Preliminary findings

• South African chickens yieded the most bacteria
• Complexity and diversity of resistance was evident
• Sequence analysis and correlation of all results is

pending

UKZN INSPIRING GREATNESS REFERENCES

• 1. Centers for Disease Control and Prevention (2010) Get smart: know when antibiotics
• work. Available from: www.cdc.gov/Features/GetSmart. Accessed: 18 May 2015.
• 2. Platteel TN, Leverstain-van Hall MA, Cohen Stuart JW, Voets GM, Van Den Munckhof

MP, Scharringa J, Van de Sande N, Fluit AC, Bonten MJ and behalf of the ESBL Nation Surveillance Working Group (2013) Differences in antibiotic susceptibility of human Escherichia coli with poultry associated and non-poultry associated extended spectrum beta-lactamases. Eur. J. Clin. Microbiol. Infect. Dis. 32: 1091-1095.

• 3. Carattoli A (2008) Animal reservoirs for extended spectrum beta-lactamase producers.

Clin Microbiol Infect 14 Suppl 1: 117–123.

• 4. Ewers C, Grobbel M, Bethe A, Wieer H, Guenther S (2011) Extended-spectrum β-

lactamase-producing gram negative bacteria in companion animals: action is early warranted! Berl Munch TierarztlWochenschr 124: 10-17.

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Acknowlegments

• My Supervisors:

Prof Sabiha Essak, Sundsfjord Tomas and

• UKZN stuff-John Osei Sekyere
• ISCISA
• NORAD Project

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• Obrigada