Determining the clinical and environmental impact, burden and cost of Extensively Drug Resistant Enterobacteriaceae in China (DETER-XDRE-CHINA-HUB)

Antimicrobial resistance (AMR) is now deemed to be the biggest global threat facing humanity in the 21C. AMR has taken center 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 the treatment of infections back to the "dark ages". 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. Two years ago, 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. This article reached global acclaim and has been cited over 1000 times reflecting its broad impact.

Following on from this discovery and using the same network of Chinese colleagues, hospitals, farming and environmental sectors, we intend to use MCR (MCRPE) and carbapenem-resistant Enterobacteriaceae (E. coli and E. coli-like bacteria)(CRE) as markers to understand how AMR has spread throughout the Chinese human and animal populations (colistin is used in animal feed in China). Firstly, we have a comprehensive sampling platform: work package (WP) 1, normal flora carriage; WP2, primary and secondary care infections; WP3, chicken, duck and pigs including slaughter houses; WP4, flies and wild birds; WP5, Water, soil and waste; WP6, Aquaculture; and WP7, Domestic animals. Pending the type of sample, we will analyze at least 100 samples every 3/6 months to examine seasonal variation and sample from three distinct provinces in China: Shandong, Guangdong and Jiangsu; additionally, we will also chose Qinghai as a control region. 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 bacteria in the human gut (called the microbiome) 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 has recently (2017) withdrawn the antibiotic colistin 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 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, duck 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, high successful and has a proven track record of working together in China and expertise to undertake this exciting and challenging proposal.

To meet our objectives we have devised three technical platforms supported by comprehensive workpackages (WP) as describe:

Sampling platform. We will sample in Shandong, Guangdong and Jiangsu and Qinghai, a control region. WPs to support these platforms are: WP1, normal flora carriage ; WP2, primary and secondary care infections; WP3, chicken, duck and pigs including slaughter houses; WP4 flies and wild birds; WP5, Water, soil and waste; WP6, Aquaculture; and WP7, Domestic animals. We will sample every 3-6 months pending on the type and accessibility of the sample and collect and analyze at least 27,720 from Shandong, Guangdong and Jiangsu. From Qinghai we will collect flies, environmental and human normal flora 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 isolated from these samples. Microbiology analysis includes bacterial identification by MALDI-TOF MS and antimicrobial susceptibility testing. Samples and bacteria will be stored frozen until further required. We will also screen for resistance genes and MLST by PCR and sequencing. This platform will also address specific research questions such as: Why is NDM-5 so prevalent in the Chinese agriculture when carbapenems are not used?

Molecular Platform will include sequencing platforms (WP12) and microbiome analysis (WP13). Sequencing will be primarily done on MiSeq sequencing approx. 2000 isolates. We will use: 1) whole genome mapping analysis: FASTQC, BWA, SAMTOOLS, PICARD, GATK, MUMER, MAUVE, etc. 2) whole genome assembly analysis: VELVET, SOAPdenovo, SPAdes, etc. 3) whole genome annotation: NCBI BLAST, MetaGeneMark, ARDB, CARD, etc. 4) Phylogenetic analysis: Bionumerics, FastTree, BEAST2 etc. Microbe bioinformatics analysis will include SEED, eggNOG, and NCBI non-redundant nucleotide database. For CR genes we will use Kraken - a custom database based on MEGARes.

Our established UK-China network published the first report on MCR-1 (Liu YY, Wang Y, Walsh TR, et al. Lancet Infect Dis. 2016;16 (2):161-8) and follow up studies of its impact on humans (Wang Y, Tian GB, Zhang R et al., 2017. Lancet infect Dis 17:390-399) and its One-Health dissemination in Chinese communities (Wang Y, Zhang R, Li J et al. Nat Microbiol. 2017. 6;2:16260). Our first article on MCR-1 has been cited over 1000 times and has made a huge impact globally. This article was also the catalyst for in-depth discussions with the Chinese Ministry of Health and, based on our evidence, the Chinese officially banned colistin as a growth-promoter effectively removing 8000 tons of colistin from Chinese agriculture (Walsh TR and Wu Y. Lancet Infect Dis. 2016. 16(10):1102-3). As part of our "pathways to impact" we will continue to have direct links with the Chinese government and thus the impact of DXC-HUB will continue to 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 DXC-HUB will enable the Chinese government to make increasingly informed decisions to aid the Chinese population in preventing an unstoppable contagion of AMR.
2. Chinese Ministry of Agriculture. Since 2017, China as withdrawn the use 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 over the next 3 years to examine the concept of "consumption driving resistance".
3. Chinese Ministry of Health. As China has now has introduced 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 etc.).
4. Chinese Ministry of Education. Our key Co-Is in China (CAU, ZJU, CCDC, CAS) are academically very strong and our network brings together a wealth of synergistic ideas and 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, UoOx and SI, Cambridge) will act as secondary supervisors enhancing their approach to tackling scientific problems.
5. DXC-HUB hospital network. As part of our clinical sampling we have enrolled 9 hospitals - 3 each from Shandong, Guangdong and Jiangsu. 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 Food production Community. Through our chicken, duck and pig sampling (WP3), flies and birds (WP4), environmental sampling (WP5), and aquaculture (WP6) we will be able to affirm the importance of AMR connectivity in spreading CRE and MCRPE throughout Chinese farming communities.
7. High-impact Journals, World Press and International AMR Bodies. Since our network was established in 2015, we have published over 18 articles - 5 in Lancet and 3 in Nature journals. We will continue to aspire to scientific excellence generating local and international impact.