Determining the clinical and environmental impact, burden and cost of extensively drug resistant Enterobacteriaceae in China (DETER-XDRE-CHINA)

Antimicrobial resistance (AMR) is now deemed to be the biggest global threat facing humanity in the 21C. AMR has taken centre stage as a global health issue yet most non-specialists are unaware the impact AMR will have on global populations and the potential it has of taking humanity back to the "dark ages". Recent announcements from Prime Minister David Cameron, the UK Chief Medical Officer (Professor Sally Davies), and the US president, Barak Obama, is clear evidence that we are loosing one of medicines precious resources. Therefore, in keeping with the general notion of "One World Health", there clearly needs to a better alignment of thinking and closer co-operation between countries synergizing activities, knowledge and skills to better understand and prevent AMR.

Hitherto, most studies around the world studying the impact of AMR have been small, one dimensional and often biased -too focused on AMR bacteria as oppose to studying the whole bacterial population. Very recently, we published an article with our Chinese colleagues heralding the breech of the last antibiotic, colisitin, that is used to treat the very serious infections caused by already resistant bacteria such as Escherichia coli (commonly known as E. coli). The difference with this new discovery is that the mechanism of colistin resistance (named MCR-1) is mobile i.e. can be readily passed around from one bacteria to another - even between distantly related bacteria.

Following on from this discovery and using the same network of Chinese colleagues, hospitals and faming sectors, we intend to use MCR-1 (MCRPE) and carbapenem-resistant Enterobacteriaceae (E. coli and E. coli-like bacteria)(CRE) as markers to understand how it has spread throughout the Chinese animal population (colistin is used in animal feed in China). Firstly, we have a comprehensive sampling platform: work package (WP) 1, primary and secondary care infections; WP2, normal flora carriage; WP3, chicken farms and slaughter houses; WP4, retail meat; WP5 Environmental sampling. We will analyze at least 100 samples every 3 months to examine seasonal variation and sample from three distinct provinces in China: Shandong, Zhejiang and Guangdong. Bacteria will be analyzed by basic microbiology techniques and selected to be whole genome sequenced where we can interrogate the bacteria's whole DNA and compare it to other bacteria to see if they have spread from one sector (e.g. flies) to another (e.g. humans). This study will also sequence all the bacteria in the human gut to understand the dynamics of AMR bacterial populations. We will also undertake controlled experiments in chicken farms to monitor the spread on CRE and MRCPE and use mathematical models to understand how AMR spreads in animals. Importantly, the Chinese government is likely to withdraw colistin from animal feed so this study is very timely in measuring that effect i.e. will withdrawing colistin impact on MCRPE rates in the environment, human gut levels and MCRPE causing human infections?

Unlike previous studies, this study is deliberately holistic in its approach so as to understand the dynamics and transmission of AMR across a broad range of environmental and human sectors. This study will let us understand the impact of CRE and MCRPE on human populations and the burden and cost to the Chinese health system. It will also help us understand the impact AMR on the chicken and pig trade by using mathematical models. The impact of this study will have immense consequences for the animal, human and economic sectors in China. Our network is well established, has a proven track record of working together in China and expertise to undertake this exciting and challenging proposal.

To meet the objectives we have devised four technical platforms as describe:

Sampling platform. We are sampling in three regions (Shandong, Zhejiang and Guangdong) from five sectors. These workpackages (WP) are: primary and secondary clinical samples (WP1), normal flora fecal samples (WP2), farm animals (WP3), retail meat and (WP4), environment. We will study carbapenem resistant Enterobacteriaceae (CRE) and Enterobacteriaceae possessing the mobile colistin resistance mechanism, MCR-1 (MCRPE). Our statistical advisor (Hood, CU) has powered our study based on our pilot data and predicted positive samples. We will sample every three months and collect and analyze at least 17,280 samples.

Microbiology Platform. Samples will be processed to assess 1. The prevalence of CRE and MCRPE in platform 1 samples and 2, the dominant Enterobacteriaceae. Microbiology analysis includes bacterial identification by MALDI-TOF MS and antimicrobial susceptibility testing according to EUCAST guidelines. Bacteria will be stored frozen until further required. We will also screen for resistance genes and MLST by PCR and sequencing.

Molecular Platform. Data from platform 2 will determine the isolates to be whole genome sequenced. Sequencing will be primarily done on MiSeq sequencers based in Beijing and Cardiff. We aim to sequence between 2200-2400 throughout the study. We will use several software packages including, 1) whole genome mapping analysis: FASTQC, BWA, SAMTOOLS, PICARD, GATK, MUMER, MAUVE, etc; 2) whole genome assembly analysis: VELVET, SOAPdenovo, SPAdes, CA Assembler etc; 3) whole genome annotation and antibiotic resistance analysis tools and database: NCBI BLAST, MetaGeneMark, ARDB, CARD, ARG-Annot, SRST etc; 4) Phylogenetic analysis: Bionumerics, FastTree, Harvest suite, etc.

Mathematical Modeling. We will apply ARIMA models on our three different sample areas with different sample sources and examining CRE and MCRPE variations in different seasons.

Our very recently published work (Liu YY, Wang Y, Walsh TR et al., Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16 (2):161-8) from Chinese animal and clinical sectors has already made an immense impact and the work described in this application will continue that level of public engagement. As part of our "pathways to impact" we have direct links with the Chinese government and thus the impact of DETER-XDRE-CHINA will be considerable.

1. Change of Ministerial policies in China. Our previous studies have already highlighted the need to review the use of antibiotics in China - particularly in agriculture. The evidence provided from DETER-XDRE-CHINA will enable the Chinese government to make informed decisions as to aid the Chinese population in preventing an antibiotic resistance and impact on the treatment of infections.
2. Chinese Ministry of Agriculture. China will soon announce the withdrawal of colistin from animal feed in China. Our data will be used by the Chinese government to examine the stability of MCRPE in the Chinese animal sector (farm animals and retail meat), and examine the impact of this on the normal human fecal flora and possibly cause for endogenous infections.
3. Chinese Ministry of Health. As China is soon to incorporate colistin into therapeutic regimes in hospitals, our data on the incidence of CRE (key indicator for colistin therapy) and the prevalence of MCRPE will be used to examine the impact of MCRPE and whether these bacteria are associated with worse outcomes (prolonged hospital stay, severity of infections, morbidity).
4. Chinese Ministry of Education. Our key Co-Is in China (CA, SCAU, ZJU and CCDC) are academically very strong and or network bring them together and the synergy of ideas, skills and will have an immense impact on these institutions. Chinese students will benefit from UK study periods not only further there scientific expertise but also enhancing cultural experiences. Workshops and our AMR symposium will also be an engaging and positive experience for them. Through the NSFC funding, post-graduate students will be employed and the UK team (CU, UoB, UoOx) will act as secondary supervisors enhancing their knowledge and approach to tackling scientific problems.
5. DETER-XDRE-CHINA hospital network. As part of our clinical sampling we have enrolled 9 hospitals - 3 each from Shandong, Zhejiang and Guangdong. Our data will be fed back to the hospitals to allow them to make informed decisions on antimicrobial therapy and infection control practices.
6. Chinese Farming Community. Through our farm animal sampling (WP3) and environmental sampling (WP5) we will be able to affirm the importance of flies in spreading CRE and MCRPE throughout the farming community and can suggest simple intervention measures to prevent further "flock/herd contamination"
7. High-impact Journals, World Press and International AMR Bodies. Our data will be published in high-impact peer review journals and according attract media attention (both UK and global - see "pathways to impact') and will be able to apply pressure on the WHO and EMEA to examine policies and international remits on AMR as we have already done so with our first publication on MCRPE.