Experience of Transitioning from Phenotype to Genotype using Whole Genome Sequencing for

Antimicrobial Resistance Surveillance.

Dr. Muna Anjum

Molecular Lead: Antimicrobial Resistance and Enteric Pathogens, Dept. of Bacteriology,

Animal and Plant Health Agency,

Background

• The gut microflora is a complex community of microorganisms that live in the

digestive tract, with the gut microbiota having the largest numbers of bacteria and

greatest diversity of species.

• Health and nutritional status of animals is interlinked with the gastrointestinal

microflora.

• The gut microflora is thought to be relatively unstable and can easily be disturbed

by various factors such as pathogenic challenges, resulting in disease in animals.

Presence of zoonotic bacteria may also have health implications for humans.

• An added complication is the rise in bacterial pathogens and commensal indicator

bacteria harbouring antibiotic resistance, which can cause problems in treatment of

disease in both animals and humans.

One Health: Dissemination of pathogens and AMR from humans, animals and the Environment.

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Courtesy of The European Food Information Council

Development of research capacity and tools for accurate detection and characterisation of threat arising in livestock.

Surveillance activities at the APHA

• This includes provision of a range of National and International Reference Centres and delivery of National Control Programmes (NCP) for zoonoses to control public health risk of Salmonella in livestock.

• Campylobacter control in chickens is a high priority due to the human disease burden and APHA provides national monitoring of broiler chicken.

• For AMR APHA is involved in anonymised surveillance of healthy animals and scanning surveillance, through veterinary diagnostic submissions.

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Antimicrobial resistance: Phenotype

• Characterised by using phenotypic methods such as disc diffusion or through microbroth dilution assays to determine the mínimum inhibitory concentrations (MIC) of a range of antibiotic compounds

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Periplasm

DNA present in Bacterial Cell

Cytoplasmic membrane

Methods for acquisition of MDR in AMR plasmids:

Conjugation Or transformation

Integron

Ravi et al Pathogens 2014

Transposon

IS element

Antimicrobial resistance: Genotype

• Molecular methods are often used in adjunct to phenotypic methods, but are set to replace them in many laboratories due to the greater speed and accuracy they provide in detecting the underlying genetic mechanism(s) for antimicrobial resistance (AMR).

• Some common methods*:

• Polymerase Chain Reaction (PCR)

• DNA microarrays

• Whole Genome Sequencing (WGS)

*Anjum et al, 2017 (in press): Molecular Methods for Detection of Antimicrobial Resistance. In Antimicrobial Resistance in Bacteria from Livestock and Companion Animals; ASM Press.

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Use of PCR for genotyping

• PCR was first developed in the 1980s by Kary Mullis and revolutionised molecular biology enabling rapid and exponential amplification of target DNA sequence.

• PCR is used routinely in microbiology laboratories for detection of any genes that may be present within bacteria, as long as a DNA sequence is available for the whole or partial gene which can be used to design the PCR primers.

• RT-PCR/qPCR or LAMP* is an advancement as not gel based and latter at constant temperature.

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*Anjum et al, 2013, J Food Science

*Kirchner et al, 2017, Veterinary Records

Occurrence of mcr-1 in animals 2015-16 in GB:

• Following the reporting of the transferable colistin resistance gene mcr-1 on a plasmid reported by Liu et al. 2015 in The Lancet Infectious Disease, APHA used RT-PCR for surveillance of mcr-1 in E. coli and caecal samples collected from pigs.

• The mcr-1 E. coli was detected on two pig farms in GB through anonymised surveillance of 387 caecal samples collected in 2015 from pigs at slaughter from 313 different herds using RT-PCR, thus 0.6% of those pig herds sampled were positive1.

• mcr-1 E. coli was also detected on 2/105 (1.9%) of pig farms using RT-PCR from which archived E. coli isolates from veterinary diagnostic investigations in 2015/16 were available1.

1(Duggett et al, 2017, Journal of Antimicrobial Chemotherapy 72:691)

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Use of nanoarrays for genotyping

Antimicrobial oligonucleotide nanoarray

• Genes coding for resistance to:

sulphonamides, trimethoprim, tetracyclines, streptomycin, carbenicillinases, chloramphenicol, florphenicol, aminoglycosides, ß-lactamases, integrase, quinolones.

• Published literature, database search for marker genes.

• Multiple sequence alignment for subgroups within antimicrobial gene family.

• Clustal X to design oligonucleotides (22-30mers) of probes and primers.

~ 100 different target genes and positive controls (ihfA and gapA)

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Distribution of AMR genes in E. coli strains: 50 E. coli and 25 Salmonella clinical isolates of human and animal origin tested.

90%

64% 58%

48%

66%

40%

46% 44%

36% 40%

22%

Batchelor et al, International Journal of Antimicrobial Agents: 2008 vol 35(5).

Whole genome sequencing

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This process sequences the complete DNA of an organism at a single time

APHA SeqFinder reference databases

• AMR reference database consists of 2050 genes

• Virulence reference database consists of 3688 genes

• Custom databases with any set of genes can be curated and utilised

Class Number of genes Class Number of genes

Aminoglycosides 160 Mupirocin 2

Beta-lactams 1444 Nitrofurans 6

Chloramphenicol 34 Quinolones 119

Clindamycin 1 Rifampicin 6

Colistin 23 Streptomycin 3

Fosfomycin 20 Sulphonomides 5

Fusidic acid 3 Tetracycline 43

Glycopeptides 64 Trimethoprim 42

Macrolides 75

Colistin outbreak investigation*

• Three diarrhoeic pigs aged five weeks were examined post mortem from a farm with history of post-weaning diarrhoea and mortality in several batches of pigs.

• The pigs examined had been treated with zinc oxide and florfenicol in-feed, and colistin in-water with a poor response to treatment and 50% mortality reported.

• Post-mortem examination of the diarrhoeic pigs revealed dehydration and enteritis.

*(Anjum et al, 2016, Journal of Antimicrobial Chemotherapy 71:2306)

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Whole genome sequencing and MICs

• Bacteriological cultures of small intestine were performed at 37ºC on sheep blood and MacConkey agars. E. coli were isolated from the intestines of pigs 2 and 3, as part of mixed flora. Salmonella enterica serovar Typhimurium variant Copenhagen phage type U302 was recovered from the intestines of pig 3.

• Representative E. coli from pigs 2 and 3, and Salmonella from pig 3 selected for WGS, which was run through APHA SeqFinder to look for AMR (>2000) and virulence genes (3688).

• MICs performed on ampicillin; cefotaxime; ceftazidime; chloramphenicol; ciprofloxacin; colistin; gentamicin; meropenem; nalidixic acid; sulfamethoxazole; tetracycline; tigecycline; and trimethoprim. Interpretted according to EUCAST and ECOFF.

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Conclusion

• Molecular methods for determining the underlying mechanisms encoding AMR are used routinely in reference diagnostic laboratories due to the greater speed and accuracy they provide.

• Methods such as PCR are still useful today as they are inexpensive and enable rapid high through-put detection of genes (simplex or mulitplex).

• Although methods such as microarrays have been used for detection of large numbers of genes simultaneously, WGS has largely replaced it due to but greater number and felxibility of this approach.

• However a thorough validation and in-depth analysis is required before WGS based genotyping can be applied routinely for reference laboratories.

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Acknowledgement APHA colleagues:

Manal AbuOun Camilla Brena

Nick Duggett Richard Ellis

Miranda Kirchner Francesca Martelli

Rod Card Javier Nunez-Garcia

Emma Stubberfield Robert Horton

Chris Teale Fabrizio Lemma

Sarah Evans Jon Rogers

Luke Randall Louisa Dormer

Heather O’Conner University of Oxford:

Rob Davies Derrick Crook group

Susanna Williamson

Richard Smith

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