Risk Assessment of The Use of Colistin Sulfate In Broiler Due To Escherichia coli Resistance In Broiler Flocks

The risk assessment of antimicrobial resistance is very important to determine the risk of decreasing antimicrobial efficacy can be used as a basis for policymakers in allowing or prohibiting the use of an antimicrobial. This study aims to assess the risk of using colistin against E. coli resistance in the broiler flock. Risk assessment is carried out qualitatively using primary data, interviews, and secondary data. To obtain primary data various studies were carried out including monitoring the prevalence of colistin-resistant E. coli and mcr-1 also mcr-2 genes in broiler flocks, mcr-1 gene transfer from E. coli to Salmonella Enteritidis, mcr-1 gene sequencing, mutant selection windows of colistin against E. coli, and also multiresistant of E. coli colistin-resistant. Assessment of the risk of E. coli colistin-resistant in the broiler flocks through direct contact with live broiler flock environment with the resulting assessment is a medium risk with low uncertainty. Since colistin sulfate is very critically important for humans, the reduced use of colistin sulfate in animal production is necessary to reduce the risk of resistance. Reducing the use of colistin sulfate requires the collaboration of various sectors such as the government, veterinary drugs industries, farmers, and consumers.


INTRODUCTION
The use of colistin sulfate in food production, which is the last drug choice for human as treatment of multiresistant gram-negative bacterial infections, raise a high interest in the world. Especially since the discovery of the mobilized colistin resistant-1 (mcr-1) gene that can be transferred via plasmids in 2015 by Liu et al. (2015). This gene is transmissible to other bacteria and causes many countries to begin to reduce the use of colistin sulfate in animal production. Nevertheless, the prohibition on the use of an antimicrobial must go through scientific studies to produce valid results, such as by risk assessment.
Risk assessment is part of a risk analysis that is very helpful for the government in making policies that are useful for assessing and managing risks to human and animal health regarding the increased antimicrobial resistance used in animals (OIE 2016). Antimicrobial risk assessment based on CODEX (2011) consists of hazard identification, hazard exposure, hazard characterization, and risk characterization. Until the time this research began, studies on the assessment of the risk of bacterial resistance due to the use of antimicrobials in production animals qualitatively and quantitatively have never been done in Indonesia.
We consider, along the supply chain of broiler meat, there are four main pathways of resistantcolistin E. coli exposes to humans. The first pathway is in broiler flocks, the second pathway is at a small slaughterhouse, the third pathway is in traditional markets through broiler fresh meat, and the last pathway is through broiler cooked meat products. In this paper, we only present an assessment of the risk of exposure to colistin-resistant E. coli in broiler flocks. The purpose of this study was to conduct a qualitative risk assessment of colistin sulfate on the emergence of colistin resistance in E. coli in broilers regarding the risk of human exposure to colistinresistant E. coli in broiler flocks. It is hoped, this risk assessment can be used as consideration for determining the continued use of colistin sulfate in food animals, especially broilers in Indonesia.

MATERIAL AND METHODS
The study was conducted from November 2016 until January 2019 and principally generate primary and secondary data. Primary data collection was carried out by in vivo and in vitro effect of colistin sulfate exposure on E. coli resistance, conducting broiler supply chain modeling experiments in the laboratory laboratory, sampling in 47 flocks that use colistin sulfate (cloacal swabs, drinking water, and litters) to obtain the prevalence of colistin-resistant E. coli as well as mcr-1 and mcr-2 genes in 5 districts in Bogor-Indonesia, serotyping of E. coli O157:H7, questionnaires, expert opinions, gene transfer mcr-1 to S. Enteritidis, sequencing of the mcr-1 gene, multiresistance of E. coli resistance colistin isolates, and mutant prevention concentration (MPC) research. The collection of secondary data was conducted thorough studies of scientific publications, unpublished data, and expert opinions. The risk assessment was made by using the data above to generate a qualitative risk assessment that developed from CODEX (2011) dan EMA (2018. Some parts of those researches have been published separately Palupi et al. 2018 b , andPalupi et al. 2019).

Hazard Identification, Exposure Assessment, and Hazard Characterization
To conduct hazard identification, various information about colistin sulfate and E. coli are needed, such as the prevalence of colistin-resistant E. coli, determination of resistance, the occurrence of cross-resistance or co-resistance, and minimum inhibitory concentration (MIC) data of colistin sulfate against E. coli. The information needed in the exposure assessment of colistin-resistant E. coli includes the description of colistin veterinary drug products circulating in Indonesia, the use of colistin in broilers, suppression of colistin resistance selection, the occurrence and rate of transfer of resistance, colistin concentration in the intestine lumen, selective windows of colistin sulfate, the prevalence of colistin-resistant E. coli from living and environmental broilers, and personnel characterization that can be directly exposed to colistin-resistant E. coli (Codex 2011, EMA 2018 The information needed in assessing the hazard characterization of colistin-resistant E. coli from broilers to humans is the use of colistin sulfate for humans and the consequences if humans are exposed to colistin-resistant E. coli. The required details include the presence of colistin sulfate and its alternatives, the prevalence of colistin-resistant bacterial infection in humans, and the horizontal spread of resistance.

The Likelihood Categorization of Exposure Assessment and Risk Characterization
Qualitative risk assessment was analyzed according to the stages in the CODEX CAC / GL 77-2011 document: Guidelines for Analysis of Foodborne Antimicrobial Resistance. Explanation or interpretation of hazard assessment is presented in Table 1.
The results of the exposure assessment are obtained by making a likelihood assessment of each node in the pathway of exposure. The exposure assessment of each pathway was carried out using a combination of the likelihood combination matrix in Table 2 that developed from AFFA (2001).
If multiple exposures are found in the pathway, the overall qualitative risk scoring for the exposure assessment is as follows: (1) if one of the partial risks is high, then the overall risk is also high; (2) if more than one partial risk is medium, overall risk is high; (3) if one partial risk is medium and the other partial risk (more than one) is low, the overall risk is high; (4) if there is one medium partial risk and the other partial risk is not medium, then the overall risk is medium; (5) if all partial risks are low, overall risk is medium; (6) if one or more partial risks is low, overall risk is low; and (7) if all partial risks can be neglected, the overall risk is negligible.
After conducting exposure assessment, the next step is to conduct a hazard characterization assessment.
Interpretations of hazard characterization assessments are listed in Table 3. In a qualitative risk assessment is important to understand the level of uncertainty of the information used. Categorization of information uncertainty using EFSA (2006), which are low, medium, and high uncertainty. The final step in assessing risk is to assess risk characterization. Risk characterization assessment is done by combining the results of exposure assessment and hazard characterization is presented in Table 4 (CODEX 2011).

Hazard Identification: Colistin sulfate and Colistinresistant Escherichia coli
Colistin sulfate is a polymixin antibiotic used in animals and humans. Since 2017, polymyxin is categorized as the Highest Priority Critically Important Antimicrobials for Human Medicine (WHO 2017). Colistin sulfate has been used for decades in food animals as a therapy, prevention of infection, and as growth promotor. Colistin is very difficult to absorb by the digestive tract of broiler chickens (Lashev and Haritova 2003). Some commercial colistin sulfate veterinary drugs contain a single colistin sulfate and some are combined with other antimicrobials (EMA 2013;DGLAHS 2016).
Escherichia coli is a commensal bacterium that very important in monitoring and surveillance antimicrobial resistance in food animals and their products. The commensal bacterium is considered as a reservoir of antimicrobial resistance genes, which can transfer these genes to pathogenic bacteria (OIE 2016). Meanwhile, Escherichia coli serotype O157: H7 is a pathogenic zoonotic bacterium for humans (Riemann and Cliver 2006;Ferens and Hovde 2011). Food animals along with their products and their environments also considered a factor in the increasing or spreading of resistant bacteria. Table 1 The qualitative interpretation of likelihood human exposure to colistin-resistant Escherichia coli The probability of exposure for susceptible people is extremely low so it can be negligible Low The probability of exposure for susceptible people is low but possible Medium The probability of exposure for susceptible people is likely High The probability of exposure for susceptible people is certain or very high The mechanism of resistance of E. coli to colistin is known through (1) mutations in specific regions, such as pmrA / B and phoP / Q, (2) mutations in the structure of lipopolysaccharide in the cytosol (ParR-ParS system), and (3) addition of phosphoethanolamine to lipid A that mediated by the mobilized colistin-resistant (mcr) genes (Fernández et al. 2010;Moskowitz et al. 2012;Liu et al. 2015). Co-resistance can occur with other polymyxin groups and several cationic peptides (Napier et al. 2013;Catry et al. 2015).
The spread of the mcr-1 gene can be through plasmids or conjugation, transposon composites, transformation, and chromosomes (Hadjaj et al. 2017;Lima Barbieri et al. 2017;Tada et al. 2017 a ;Sun et al. 2018). Our study also succeeded in transferring this gene from colistin-resistant E. coli to S. Enteritidis ATCC 13076 through conjugation . Escherichia coli is the most common bacteria found to have mcr-1 gene (Poirel et al. 2016).

Exposure Assessment Colistin-resistant Escherichia coli to Humans in Broiler Flocks
Food animals and their environment are considered as one reservoir of resistant bacteria that can transfer directly or indirectly to humans (Marshall and Levy 2011;WHO 2016). We consider two major exposure branches pathway in the pathway exposure of colistin-resistant E. coli to humans in broiler flocks. First branches are through direct contact with live broilers and second branches are exposure through flock environment that contaminated with colistin-resistant E. coli (Figure 1). Assessment of exposure through direct contact with live broilers involves two likelihood nodes, the first node is colistin-resistant E. coli in broilers due to the usage of colistin sulfate in the broiler (L1) and the likelihood of the process of human get exposed to colistinresistant E. coli from live broilers (L3). The exposure pathway through the flock environment involved 3 likelihood nodes which are colistin-resistant E. coli derived from broilers (L1), the likelihood of flock environment get contaminated with colistinresistant E. coli (L2), then the likelihood of colistinresistant E. coli exposes humans through contact with the flock environment (L3).

Likelihood Assessment Broiler with Colistin-Resistant Escherichia coli Due The Usage of Colistin Sulfate (L1)
Based on observations and questionnaires, personnel who can contact with chickens and the flocks environment are flock workers, health technical services (eg veterinarians), one-day-old chicken (DOC) senders, broiler collectors, broiler catchers, and litter collectors. Direct contact between broilers and humans in the flock, intensively occurs when the chick in, checking the weight (once a week), and the harvest period.
Commercial E. coli is very rarely found in DOC. In our study (Palupi et al. 2018a), only 5.56% DOC was found with commensal E. coli and none were resistant to colistin. At the time of harvest, the personnel most frequently contacted with broilers are flock workers, bird collectors, and bird catchers. Other visitors who can contact the broiler in the flock are technical health service (25.5%) and DOC sender (25.5%).
Broiler farmworkers generally do not wear personal protective equipment (PPE) when working, eg. gloves, boots, wear packs, or masks ( Figure 2). Visitors are also not provided with personal protective equipment. After working, 42.6% of workers simply cleaned themselves with water, 42.6% cleaned themselves with soap, and only 14.8% cleaned themselves with soap and with disinfectants, such as 70% alcohol.
The personnel most often in contact with litter and drinking water are flock workers. There is a litter collector that takes the litter when the broiler harvest is finished. When collecting litters, they do not use adequate PPE. Information on the use of PPE is crucial in taking litter because litter is a good medium for the development of pathogenic microbes. Escherichia coli O157: H7 can survive in the litter for 42-49 days at 37ºC, 46-56 days at 22ºC, and 63-70 days at 5ºC (Solimanet et al. 2018).
Based on the assessment of the characteristics of E. coli, the prevalence of colistin-resistant E. coli with mcr-1 gene, resistance distribution patterns, raising management of broiler, and biosecurity practices in broiler flocks, colistin-resistant E. coli with mcr-1 gene may expose humans. Research conducted by Trung et al. (2017), showed that farmers who were exposed to broiler positive mcr-1 showed a higher risk of colonization of bacteria carrying mcr-1 than those that were not directly exposed to chickens or with farmers whose chickens did not have the mcr-1 gene.
Humans who are most likely to be exposed are flockworkers, then bird catchers, and litter collectors. Based on the evaluation of the information, the L3 exposure assessment is medium with low uncertainty. The uncertainty of information in making this exposure assessment is low because based on references, observations that give the same results, and interviews with experts also give the same opinion.

Results of Likelihood Exposure Assessment of Colistin-resistant Escherichia coli to Humans in Broiler Flocks
Calculation of likelihood for human exposure by colistin-resistant E. coli from broilers flocks is obtained using a combination matrix of Table 2. The results of the likelihood exposure assessment of colistin-resistant E. coli to humans through direct contact with broilers (first branch pathway) are low with low uncertainty. This low assessment is obtained from the possibility of L1 (medium) x L3 (medium).
The likelihood of human exposure assessment by colistin-resistant E. coli through the flock environment is low, this likelihood exposure assessment involves L1 (medium) x L2 (low) x L3 (medium). The uncertainty through this pathway is also low. Low uncertainty assessment based on the majority of information sources obtained based on primary data, references that are very supportive and not contradictory, questionnaire results, and expert opinions that do not conflict with each other. The overall exposure assessment of colistin-resistant E. coli to humans at the broiler flock level uses the principle of multiple exposures in that the values are medium (low + low) with low uncertainty (Table 5).

Hazard Characterization of Human Exposure by Colistin-Resistant Escherichia coli in Broiler Flock
Information for hazard characterization is obtained through scientific publications and direct communication with Dr. Harry Parathon, SpOG (K). The use of colistin in Indonesia is very limited because it is used as the last drug choice for the treatment of pan-resistance. Colistin for the treatment of panresistant cases is difficult to obtain but in some large hospitals, it is provided as alternative medicine. Based on communication in July 2018, colistin methanosulfonate (injection) has not yet included in the health system and is likely to be held in the coming year. Based on communication with experts, it was mentioned that the prevalence of cases of panresistant human infection in Indonesia is still very low.
The prevalence of colistin-resistant E. colipositive mcr-1 gene infections in hospitalized patients has been studied in several countries and ranges from 0% -1.4% (Kim et al. 2017;Terveer et al. 2017;Principe et al. 2018). The higher prevalence is indicated by Eiamphungporn et al. (2018) in Thailand, reaching 27.7%. In the hazard characterization assessment of E. coli zoonosis, our study used E. coli serotype O157: H7. Colistin-resistant E. coli O157: H7 isolates with mcr-1 gene were only found in one isolate out of a total of 380 E. coli isolates from live broiler and flock environment (0.31%) or the prevalence was very low. Several studies have shown that colistin-resistant E. coli with mcr-1 from food animals and pets can move to humans (Olaitan et al. 2015;Zhang et al. 2016;Tada et al. 2017 b ).
In our study, 95.59% of colistin-resistant E. coli isolates were also found to be multiresistant (Palupi 2019). Colistin-resistant E. coli infections can still be treated with other antibiotics as long as the infecting bacteria are not multiresistant to the antimicrobial used. Therefore, based on the information obtained, the hazard characterization assessment of human exposure by colistin-resistant E. coli in broiler flocks is medium with low uncertainty.

Risk Characterization of Colistin-Resistant Escherichia coli in Broiler Flock to Humans
The final step in risk assessment is to carry out risk characterization based on exposure assessment pathway and hazard characterization assessment. The risk characterization assessment uses a combination of exposure assessment and hazard characterization matrix as in Table 4. The results of the risk characterization exposure assessment through the broiler flock are medium (medium x medium) with low uncertainty. The results of the risk characterization assessment are in Table 6.

Risk Mitigation of Colistin-Resistant Escherichia Coli from Broiler Flocks to Humans Health
The veterinary medicine industry plays an important role in the circulation of colistin sulfate. Fulfillment of pharmacological data and colistin sulfate resistance from animal drugs to be registered must be tightened based on scientific data per product. In 2016 the European Committee for Medicinal Products for Veterinary Use (CVMP) requested that all colistin sulfate combinations with other oral antimicrobials be withdrawn from the European Union (EMA 2016). Approval for the registration of animal drug colistin sulfate combined with other antimicrobials must go through in-depth scientific evaluation.
Good farm management will reduce the risk of pathogenic bacterial infection. This will reduce the dependence of farmers on antimicrobials to prevent infection. The use of proper PPE to reduce the risk of direct contact with colistin-resistant E. coli also needs to be done when handling chickens, working, and taking litter in the farm. The use of PPE will greatly help reduce risks, especially for small scale broiler farms which are difficult to implement in three zones of livestock.
Education about the position of colistin sulfate to farmers or health managers on broiler farms is also very important. Farmers and health managers need to understand the importance of colistin for human health so as not to choose colistin sulfate as the first choice in handling cases of gram-negative bacterial infections in broilers.

CONCLUSION
Risk assessment of colistin-resistant E. coli risk in broiler flock through direct contact with live broilers and the enclosure environment to humans is medium with low uncertainty. This is due to the use of colistin sulfate which is the highest priority critically important antimicrobials for humans in broilers, the presence of the mcr-1 gene that is easily transferred between bacteria, low biosecurity at the farm, contamination in the farm environment, and low use of PPE in the farm. Therefore, reducing the use of colistin sulfate in production animals is a necessity that cannot be avoided.